Adèle Par

Dramé-Maigné

Professeur Philippe Minard

Professeur Patrice Soumillion

Directrice Aleksandra Walczak

André Estevez-Torres

Compartmentalized directed evolution of enzymes using molecular programs as self-selection function Développement d'une méthode d'évolution

dirigée d'enzymes semi in vitro dans des microgouttes à l'aide de programmes moléculaires.

1

Résumé :

Les méthodes d'évolution dirigée basées sur les techniques de sélection se sont révélées être très efficaces dans plusieurs travaux mais reposent sur la possibilité de trouver un outil de sélection au cas par cas qui permet de lier l'activité d'une enzyme à la propagation de son gène.

Dans une approche inspirée de la CSR (Compartmentalized-Self-Replication) de Ghadessy et al. (1) nous proposons d'utiliser des réseaux moléculaires artificiels pour créer des fonctions de sélection adaptables à des enzymes de natures variées. Notre processus d'auto-sélection implique l'utilisation de programmes moléculaires relativement simples en tant que boucle de rétrocontrôle liant directement l'activité de l'enzyme avec le rendement de la PCR de son propre gène.

Les programmes moléculaires mis à l'oeuvre dans cette méthode d'évolution utilisent la PEN DNA Toolbox (2). Basés sur l'interaction de courts brins d'ADN et d'un ensemble restreint de 3 types d'enzymes (polymérases, exonucléases et nickases) ils permettent de générer de courts oligonucléotides de séquences prédéterminées à partir d'une variété de signaux d'entrée comme un autre brin d'ADN ou une activité enzymatique. Il est donc possible d'utiliser ces programmes moléculaires pour générer des amorces de PCR ciblant le gène à évoluer et ce en quantité dépendante de l'activité enzymatique. De cette manière le rendement en produit de PCR du gène est directement lié à son activité. L'utilisation de programmes moléculaires permet entre autres de pouvoir contrôler cette relation de manière assez précise et d'influencer la fonction de sélection. La relative simplicité de design et d'utilisation des programmes moléculaires permet aussi de pouvoir agir aisément sur la sélection à chaque cycle et de changer les paramètres assez facilement. Enfin le caractère extracellulaire de ces programmes permet de pouvoir tester les enzymes dans des conditions non-naturelles in vitro.

Nous parlons alors de Programmable External Network Compartmentalized Self-Replication (PEN CSR).

Pour pouvoir réaliser le nombre important de tests requis par les expériences d'évolution dirigée, nous utilisons des gouttes d'eau-dans-l'huile générées par microfluidique afin de créer des compartiments de taille homogène pouvant chacun accueillir un mutant et les réactifs nécessaires. Après génération d'une banque de mutants par PCR error prone, des bactéries contenant et exprimant les mutants sont séparées individuellement dans ces gouttes contenant le programme moléculaire. Le processus est ensuite simplement initié en augmentant la

Abstract:

Selection-based strategies used in directed evolution methods proved to be very efficient but rely on the possibility of finding a case-specific selection tool that links the enzyme activity to the survival of its gene. In an approach inspired by the Compartmentalized Self-Replication (CSR) of Ghadessy et al. (1) we aim at using artificial networks to implement selection functions, adaptive to different nature of enzymes. Our self-selection process involves the use of a simple artificial molecular network introduced as a feedback loop. It links directly the activity of the enzyme with the PCR amplification of its own gene.

Molecular networks, such as the one using the PEN DNA toolbox (2), are designed to produce short DNA strands interacting within each other's thanks to a set of 3 different kind of enzymes: a polymerase, a nickase and an exonuclease. Taking short oligonucleotides or enzyme activity at the input, they can generate short oligonucleotides of arbitrary sequence at the output.

Enzyme activity can be assessed by these molecular networks thus allowing to produce a correlated amount of primers at the output. These primers can then be used to run the PCR of the enzyme's gene. Therefore, the artificial network will be able to link the enzymatic activity to a PCR amplification yield. Molecular networks can be modulated by changing their parameters and using them as a selection function can allow to finely tune this step of the directed evolution process. Also, the relative simplicity of molecular programs design and use allows to change easily the selection process parameters at each cycle. Finally, the programs being added externally to the cells enable to test the enzymes in unnatural in vitro conditions.

We decided to name our method the Programmable External Network Compartmentalized Self-Replication (PEN CSR).

The number of parallel selection tests that can be run in one evolution cycle is one of the major criteria of efficiency. Microfluidics allows us to generate a large amount of monodisperse water-in-oil microdroplets. Up to 10 8 parallel self-selections can be run simultaneously on individual copy of the gene with a small quantity of reagents. Practically, after generation of a mutant's library by error prone PCR, bacteria carrying and expressing the mutants are separated in individual droplets containing the molecular program. The process is next initiated in the droplets simply by raising the temperature to at least 45°C. After running the program for some time, each droplet contains an amount of the enzyme's gene forward and reverse primers corresponding to the activity of the enzyme expressed by the bacterium. A PCR is then launched in the droplets. Its yield in each droplet will then depend on the amount of primers, therefore on the enzyme activity. Droplets are merged afterwards to retrieve a library enriched in the best mutant genes and a new evolution cycle can be run.

The nicking enzyme Nt.Bst.NBI, one of the nicking enzymes used in the PEN DNA Toolbox, was used as a first model with this adaptive self-selection tool and gives promising results. The several development steps of this new method allowed to optimize bacteria culture conditions and co-encapsulation with the molecular program and PCR reagents. Once the selfselection feedback loop was set up, it was able to successfully select the wild-type gene of Nt.Bst.NBI among a large excess of an inactive version of the enzyme going from an initial faction of 10% to 90% in only one round on selection thus acknowledging for the high selection capacity of the method. Selection cycles on a mutant library of this same enzyme are still running but the analysis of NGS sequencing of the mutants selected in a first cycle already attests an efficient selection process.

(1) F. J. Ghadessy and al. Proc. Natl. Acad. Sci. U.S.A., vol. 98, Apr. 2001. (2) K. Montagne and al. Mol. Syst Biol., vol. 7, Feb. 2011.

À mes grands-parents Augustine et Théodore Maigné ainsi que Fatou

Diagne qui ne m'auront connue que comme éternelle étudiante.

Aknowledgements/Remerciements

First, I would like to thank my supervisor Yannick Rondelez who trusted me and gave me the opportunity to work on this very interesting project. I also thank him for his attentive reading of my manuscript and his corrections. My thanks also go to the members of the jury who found the time to read and assess my long manuscript: Philippe Minard, Patrice Soumillion, Aleksandra Walczak, and especially André Estevez-Torres who lent me his annotated version to ease my corrections. I am very grateful for the useful feedback I got in the reports and during my defense.

I also would like to express my gratitude to all the members of both the Fujii laboratory and the Gulliver lab, especially in the SPM team. They helped me in the different aspects of my project and made both labs nice environments to work in. Thanks to the heads of labs, Elie Raphael and Fujii sensei, for welcoming our team.

A warm thank you to Rémi Sieskind for setting up the lab and installing all the new machines in Paris when I was still in Tokyo, allowing me to transfer smoothly. Thank you also for the fruitful discussions about our research, your support in the despair of not-workingexperiments, your help fixing the bugs of the different machines, your handling of the ordering process so that we never run out of consumables, and lending me your strength to open firmly closed PDMS (or else) bottles. A special thank you to Guillaume Gines, who masters the PEN DNA Toolbox modules and oligos design better than anyone and gave me a lot of useful advice in molecular programming but also in microscopy image capture and in powerpoint arts. A big thank you to my sempai, Alexandre Baccouche, who not only taught me how to use the fluigent pressure pumps with my capricious first microfluidic devices in Tokyo but also helped me discover most of the Japanese food and drinks, the best eating places around the lab in Tokyo and a lot of the ordering process vocabulary. I would like to thank Anthony Genot for his advice in image analysis and his accessibility. Thanks to Ruochi Hsu, Daisuke Kiga and Kazuaki Amikura for initiating the project with Yannick, to Kim Soo Hyeon for his help with my first microfluidic device, to Shohei Kaneda for teaching me how to use the Fujii lab facilities to make my devices and to Nathanaël Aubert-Kato for his modeling of the oligonucleotides production by the PEN toolbox. Thanks also to the Sakai lab which let me use their DNA nanodrop and the Takeuchi lab for letting us use their flow cytometer and sharing the 8 th floor space peacefully with us despite the fact that we were growing bacteria.

I also would like to thank Anton Zadorin for his work on the CSR replication project and our long but always very interesting and instructive discussions about any scientific points. Big thanks to Rocio Espada, for collecting and analyzing the MinION data, for teaching me a bit of R programming, and for her everyday positivity. Thanks to the interns who worked with me on my project: Ellie, Yasya, Nicolas, Léon. Thanks also to Thomas, Roberta, Anne-Sophie, Vasily, Antoine, Vincent and Thibault for your help in preparing my defense and your presence in the lab every day. Thanks to all the former SPM members that also contributed to the working atmosphere: Valentin, Pierre-Aurélien, Neha, Elia, Kaori, Gabriel, Laeticia, François, Fillipo. A particular thank you to Solange Rogue for her support, her precious help with administrative hassles, and for always making sure our orders were made correctly and efficiently.

I would like to express my sincere thanks to all the Fujii lab master students who organized very nice lab events and made me discover some Japanese traditions. Thanks also to all the LIMMS members and foreigner friends for sharing lunches, Izakayas nights, karaoke sessions, and hikes: Bertrand, Pierre, Nicolas, Guillaume P, Wenjin, Nathalie, Dennis, Mathieu, Florian, Olivier, Maurizio, Yannick T, Jaja, and all that I may have forgotten.

Thanks to the ENS Lyon and the ERC for their financial support. And again, a special thank you to Fujii sensei for finding a bit of extra money for my stay in japan to complete my French grant and for his accessibility despite his high hierarchy position.

I must thank the Laboratory of Biochemistry and its members for their material and precious scientific support. In particular, I would like to thank Dany Chauvin, Marco Ribezzi, Clément Nizak, Roberta Menafra. I am also very grateful for Valérie Taly's laboratory that provided us with surfactant, without which I could not have run my experiments.

Je voudrais bien évidemment aussi remercier ma famille et mes amis pour leur soutien pendant ces quatre longues années. Je tiens à remercier en particulier ma mère Annick Maigné, pour son soutien moral, le temps passé à écouter mes plaintes et mes colères et tous les bons conseils qu'elle me donne. Je la remercie aussi pour m'avoir aidé à gérer à distance des problèmes administratifs et autres mais aussi dans les allers-retours France-Japon et les déménagements. Merci à ma soeur et mon frère, Sophie et Charles Dramé-Maigné, pour leurs encouragements, leurs conseils, et les moments de partage qui permettent de s'aérer l'esprit. Je remercie aussi pour leur soutien, ma marraine Annie, Patrick, Marie-France et Pascal grâce à qui j'ai pu emménager à Paris près du laboratoire. Je voudrais aussi remercier mon père Khaly Dramé qui me soutient de loin. Enfin mes amis de longues dates, Malika pour les sorties running qui m'ont permis de me maintenir en forme, Karen pour les sorties fitness, Pauline, Cécile, Julie-Anne, Ninon, Nadège, Nesrine, Lara, Paul, Flore, Nathalie, pour votre présence en continu depuis toutes ces années malgré l'éloignement. I would also like to thank Ruselle who I wish I could have brought back to France with me and who really embellish my stay in Japan.日本への歓迎と日本文化の発見に私の友達、美香 と 道代、に感謝します。Je me dois de remercier Monkeydou qui me suit partout depuis maintenant de nombreuses années et qui est toujours là pour me réconforter.

J'aimerais également remercier Mme Van Der Rest qui a su me donner confiance en mes capacités pendant ma première année de prépa, quand je n'osais à peine écrire sur la fiche de renseignement l'école que je visais et sans qui je n'en serai probablement pas là aujourd'hui.

Avant de terminer je voudrais remercier encore une fois Rémi, cette fois pour son soutien au quotidien et la grande aide qu'il m'a apporté pendant la rédaction en sacrifiant une plus grande part de son temps aux tâches du quotidien malgré le travail que lui demande sa propre thèse. Merci de m'avoir supporté pendant cette période de rédaction difficile.

Enfin, je voudrais me remercier pour tous les efforts que j'ai fait pour tenter de mener à bien ce projet. Namaste!

Notes and Abbreviations

Notes

Bacteria concentration are given in bact/nL and were estimated by OD600 cuv measurement (see Annex iiG.ii, p.325). As final experiments are to be run in droplets, Table 1 represents correspondence between droplets diameter or volume and the bacteria concentration.

Table 1. Correspondence between droplet diameter, volume, bacteria concentration in bulk and bacteria optical density at 600nm

Introduction: Protein Engineering & Molecular programming

Proteins play critical roles in biology. By their huge diversity, they assume vital structural and dynamic functions in the cells. Enzymes especially are great technological achievement of nature. They necessitate very little energy to catalyze reactions very efficiently.

Outside of their natural context, catalytic capacities of enzymes have been used for millennia by humans, for fermentation process. But it is only since the end of 20 th century that the potential of biocatalysts started being unveiled. First, the invention of the polymerase chain reaction (PCR) in the eighties and then the development of directed mutagenesis techniques in the nineties paved the way of using proteins and more specifically enzymes for biotechnology and therapeutic applications.

The main issue that scientists face when they want to use proteins in a non-natural fashion or environment is the extreme sensibility of these molecules to environmental factors such as temperature, pH, ions concentration and so on. Natural proteins have been selected

through billions of years of evolution to work in very specific condition with high efficiency, adapting to their environment. That's why one often needs to redesign an enzyme to get it work in new conditions. This domain of repurposing or creating new proteins is called protein engineering.

The protein engineering field aims at having proteins or enzymes with greater activity, altered substrate specificity, improved enantioselectivity and increased stability mainly for industrial or therapeutic applications. Using enzymes adjusted for industrial process can reduce energy costs while specific molecular recognition is very valuable in therapeutics. On another aspect, protein engineering contributes to a better understanding of structure-function relationship, as well as physical and chemical properties of enzymes or proteins. It can also serve to question the evolution process.

Important technical steps have been developed to allow protein engineering, benefiting from improvements in biochemistry and biophysics. X-ray crystallography, NMR spectroscopy, clever mechanistic studies by enzymologists, appreciation for protein dynamics, single-molecule studies along reaction coordinate, PCR and recombinant gene technology, combinatorial mutations studies, in vitro expression systems and lastly predictive computer algorithms complementing experimental data, all contributed to the development of the field.

Nowadays, complete computational de novo construction of proteins can be achieved.

Two approaches are mainly used in the field, sometimes concomitantly, rational design based on structural information or directed evolution based on evolution mimicry. In this work, we proposed to introduce some rationality in the directed evolution method by using molecular programs to design selection function. But first, let me introduce proteins particularity more in detail.

A.

What is a protein ?

Proteins were first discovered in the 1830s by Gerardus Johannes Mudler and named wortelstof (root fabric) before, in 1838, Jöns Jacob Berzelius suggested to call them proteins.

Protein comes from the ancient Greek protos which means first or essential. This is possibly because they represent 60% of the dry weight of cells and are indispensable to life. Another theory sees the name "protein" as a reference to the Greek god Proteus who could change his shape at will. Proteins have multiple functions and can indeed recover many forms, but this was discovered later in the course of the 20th century.

a. Roles of protein in biology

Proteins have a huge variety of roles in biology. Some are involved in the structural organization of the cells, like the tubulin protein. Tubulins assemble and form long chains called microtubules that are the backbone of cell's tridimensional architecture.

Membrane proteins insert in cell's membrane and form for example, pores or receptors.

Pores allow exchange of molecules between the cells or between the cells and their environment. Such canals like the sodium potassium pump are essential to fight against osmotic pressure equilibration inside the cell and generate a resting electrical potential in neurons. The resting potential of neuron membranes is necessary to induce electric signal transfer in neuronal cells. Other membrane proteins like the ATP synthase can use gradients of protons to generate energy in the form of ATP. Receptors can be canals, while others rely to intracellular proteins to transduce the signal and activate downstream cascade reactions in the cells. The neuronal GABA receptor opens under fixation of the GABA neurotransmitter to let chloride enter the cell and inhibit the neural cell. The protein G receptor and its cognate protein G initiate a cascade of reactions. Extracellular binding of the target on the receptor triggers the intracellular protein G, which then starts interacting with other proteins, activating their catalytic activities.

Protein with catalytic properties are called enzymes. Their catalytic power allows to accelerate chemical reactions by several orders of magnitudes and render them compatible with life. Without the enzymes, the catalysis of glucose metabolism would require several months, instead of just a few seconds.

Proteins still have other functions. They can also act in the transport of molecules, like the hemoglobin in blood cells carrying and delivering oxygen to the whole body. Some proteins like kinesin or dynein actively travel on use the cells' vast network of structural proteins to transport vesicles between the different parts. Movement at a larger scale is also managed by proteins. Muscular fibers are made of myofibrils, an assembly of myosin and actin filaments sliding one over the other to create contractions.

The high protean capacities of proteins allow them to adapt to perform a lot of interactions and functions. Antibodies are defending proteins that use these properties to bind specifically to various foreign molecules or cells and protect the organism integrity. Even if most hormones are generally lipid molecules some are proteins, such as insulin, involved in glucose level regulation. Energy storage can also be made in the form of protein, as an alternative to the usually long sugar chain or lipids that are used to store energy. The ovalbumin found in egg's white, which allowed the discovery of proteins, is an energy reserve used by the developing organism. Information storage is possibly the only role that proteins have not been shown to carry, but they are involved in its processing and regulation.

Dealing with a variability of crucial roles in living cells and organisms, proteins are of first importance and are aptly named. The secret of protein's versatility in form and action lies in their composition and structural organization.

b. Structural organization of a protein

One could think, reading this thesis, that proteins are cool round-shaped molecules very easy to apprehend. But it is not the case. Proteins have very complex structures that researchers still struggle to understand. They are made of amino acids building blocks. 20 different amino acids are commonly used in living systems. They are presented in Figure 2. The rarely found selenocysteine and pyrrolysine can sometimes be added as the 21 rst and 22 nd amino acids. In humans, some amino acids are synthesized in situ, but many need to be obtained from the diet and are called essential amino acids. Amino acids found in living organisms are all Lstereoisomer. They are composed of a central carbon, named carbon α, attached to an amino group, a carboxyl group, a hydrogen and finally the side chain.

A sequence of amino acids linked by peptide bond forms the primary structure of proteins. This sequence is encoded in DNA, with an RNA molecule serving as intermediary between the DNA sequence and the protein sequence. The central dogma of molecular biology states that messenger RNA, transcribed from DNA by an RNA polymerase is then translated into a chain of amino acids by the ribosome. The ribosome reads the RNA and produce the corresponding protein. Every amino acid corresponds to one or more triplets of nucleotide bases called a codon. t-RNA recognizing the 3 bases triplet and carrying a corresponding amino acid sits in the ribosome until the peptide bond between the growing chain and the new amino acid is made. Thus, by knowing the DNA or RNA sequence coding for a protein you can determine the amino acid primary sequence, following the so-called genetic code. Chains made of less than 50 amino acids residues are called peptides, while longer chains constitute proteins. The peptide bond is made between the carboxyl group of a first amino acid and the amino group of the next one. This organization leads to an orientation of the molecule from the N-terminal to the C-terminal end.

Side chains of amino acids are very diverse, as shown in Figure 2. It is this chemical richness that allow proteins to be able to create an important range of different interactions and endorse many functions. Amino acids or protein residues can be grouped according to the chemical properties of their side chain; polar, cyclic apolar, hydrophilic, hydrophobic aliphatic, negatively or positively charged. Amino acids can undergo post-translational modifications such as glycosylation or phosphorylation. Interactions between the different amino acids will allow the polypeptide chain to fold in a tridimensional structure that is unique and specific to the amino acid content. Each amino acid's side contributes to the chemical, physical and structural properties of the final peptide or protein.

The secondary structure of proteins consists of repetitive motifs. The peptide bond between two amino acids is rigid because of electron delocalization, but rotations can happen around the α carbon. These rotations allow folding and the emergence of the secondary structure. Because of steric clashes, some conformations are favored, and similar motifs are found in proteins. Four types of motifs can be observed. The α-helix, the β-sheets, the β-turns and less defined regions called random coils. The four motifs are represented in Figure 3. These These secondary structure motifs can combine with each other thanks to a variety of interactions. In addition to hydrogen bonds, ionic bonds, hydrophobic interactions and cis-cis disulfide bonds also take place between the different amino acids. These four types of bonds and interactions allow to further fold the peptide chain and cre²ate a tridimensional structure called the tertiary structure. Ionic bonds can form between charged residues, hydrogen bonds between polar residues and disulfide bonds are covalent bonds formed by a redox reaction between two cysteine residues. Hydrophobic interactions are the results of assembly of several hydrophobic residues to expel water molecules and reduce contacts with hydrophilic residues.

The tertiary structure gives the protein its final shape and function. Cavities created inside the protein allow other molecules to enter the structure and interact with the different amino acids it contains. The accurate shape and organization lead to very specific interactions.

Proximity of some amino acid groups with ions and/or cofactors near a molecular bond can allow to catalyze some chemical reactions. Surface exposed residues can also interact with molecules or surfaces present in the environment.

For some proteins, folding into the tertiary structure is not the final stage of assembly.

Proteins can also be constituted of several subunits that need to interact to be functional.

Myoglobin, presented on Figure 3, resembles closely to the hemoglobin subunit illustrated below. While the myoglobin is storing oxygen in muscles, hemoglobin, roughly organized as a combination of four myoglobin is transporting it through the blood. Indeed, the coupling between proteins subunits can allow to perform more complex functions thanks to conformational changes in the different subunits. This can result in modification of binding kinetics. For instance, the hemoglobin displays an allosteric effect. When an oxygen is bound to one of the subunits it is easier for the other subunit to bind an oxygen allowing for non-linear and fast uptake of oxygen. The types of bonds stabilizing the quaternary structures are the same than the one found in the tertiary structure. Quaternary assembly of the same subunits constitute homopolymers whereas assembly of different subunits are named heteropolymers. 1 Several ways exist to study proteins at various level of its structure. The primary structure can easily be determined thanks to the encoding DNA sequence using the genetic code. In the absence of genetic information, several methods exist to determine protein sequences. Historically, researchers were able to determine small peptide or protein sequences by using proteases able to cut the peptide chains at specific positions. Full hydrolysis of the protein and chromatography and spectrophotometry analysis of the obtained residues allow to have the composition of the protein. Subsequently, the short fragments obtained with the proteases are analyzed thanks to chemical reactions allowing to remove one by one the final residue, whose nature is then determined with chromatography. Using several proteases, it is possible to assemble the different pieces of the puzzle and determine the full sequence.

Nowadays mass spectroscopy techniques follow the same principle to determine the sequence of thousands of predigested small peptides in an automatized process.

Secondary structure motifs can be characterized with circular dichroism spectroscopy.

Circular dichroism is based on the absorbance difference of a material according to the light polarization. α-helix, ß-sheet and random coil have a characteristic spectrum in the far-UV region. Analyzing the spectrum of a protein allows to estimate the fraction of each motif in the structure 2 .

Finally, the tertiary or quaternary structures can be determined using X-ray crystallography, Nuclear Magnetic Resonance (NMR) or cryogenic Electon Microscopy (CryoEM) 3,4 . In the X-ray crystallography techniques, a large and well-ordered crystal of pure protein is required. Obtaining this crystal is the main challenge of the method as proteins crystallization conditions vary from one to another and are very difficult to find. When crystallization is a success, X-rays are diffracted through it and the recovered diffraction pattern allows to measure the short distance between atoms and gives a three-dimensional electron density map. Software can then allow to build a model from the obtained map. This technique can yield very high resolution, down to the Å level, but the obtained structure does not necessarily represent the protein active structure, as crystallization conditions can modify its conformation. Also, the structure is observed in a fixed form. If proteins can have several conformations only one will typically be observed. With NMR the protein is in solution and free from the crystal lattice's constraints. Two-dimensional NMR analysis allows to retrieve the protein structure by analysis of atoms proximity. NMR resolution is generally lower than crystallography but can be quite high for small molecules. As NMR analysis techniques improve, bigger protein structures can be determined with higher resolution. cryo-EM was initially limited to large complexes or low-resolution models but recent advances in electron detection and image processing now allow it to rival X-ray crystallography in some cases [START_REF] Bai | How cryo-EM is revolutionizing structural biology[END_REF] .

Computer prediction of the tridimensional structure of a protein can also be used. When a protein sequence is close to a protein with known structure, the scaffold of the known protein can be used to build a model of the target one. Using chemico-physical restriction parameters software can give a putative model. In the absence of similar pre-existing modeled proteins, ab initio modeling software can try to do some predictions, but some conceptual challenges remain and these kinds of prediction are very complicated, often not accurate and usually not verifiable.

Proteins are very fragile molecules. As their structures rely mostly on weak interactions, various stress factors like the pH, temperature, desiccation, metal ions, high shear forces or the presence of hydrophobic surfaces can disrupt the protein native structural state. Chemical factors including oxidation, deamination, peptide-bond hydrolysis, disulfide-bond reshuffling and cross-linking can also act on the structural organization. This denaturation process results in unfolding of the protein into a fully random coil structure or misfolded shape, leading to a loss of the natural function. Aggregation also produces aggregates with denatured shape and impaired function. In 1950, Anfinsen added urea and mercapto-ethanol in a solution of RNAse [START_REF] Kresge | The Thermodynamic Hypothesis of Protein Folding: the Work of Christian Anfinsen[END_REF] .

The enzyme lost its structure and function because of the denaturation process but when urea and mercapto-ethanol were removed, the enzyme was able to take back its native state and activity. This demonstrated that protein folding was, in some case, a reversible process.

However, not all proteins can take back their original shape and not all denaturing conditions allow this reversible process. Still, this experiment shows that the primary structure of a protein contains the necessary information for it to take its natural tridimensional configuration.

The folding of a protein in its final structure is a complex phenomenon not fully understood. The protein-folding problem was first asked one half-century ago, and expressed as a set of questions presented by Ken A. Dill and Justin L. MacCallum in their review about protein folding. "What is the physical code by which an amino acid sequence dictates a protein's native structure? How can proteins fold so fast? Can we devise a computer algorithm to predict protein structures from their sequences?" [START_REF] Dill | The Protein-Folding Problem, 50 Years On[END_REF] . Thermodynamic effects are a first answer.

Proteins, as every molecule, try to minimize their energetical state. Some advances in the studies of foldable polymers showed that compact, and low-energy conformational assemblies have fewer possible conformations. This suggested that protein-folding energy landscapes are narrower at the bottom, resulting in few low-energy, native-like conformations and many open and unfolded structures. Proteins fold by taking random incremental downhill steps in the energy landscape. This discovery did not allow to predict the folded structure on the computer as the smooth paths to equilibrium are not easy to find with computational tools and local scales.

A complete explanation of the folding mechanism, allowing to understand the general principle applicable to a broad range of proteins and explaining how the folding pathway derives from its amino acid sequences, is still missing. But, some general rules have been set. The folding operates by assembly of units of secondary structures. Local secondary structures are formed before they assemble into a more global structure. The stability increases as partial structures are formed. This splitting of the process into nested local folds explains the rapidity of protein folding. Energetic funnel landscape of protein conformation suggested that same proteins with the same sequence do not all follow the same path to reach their final conformation [START_REF] Dill | The Protein-Folding Problem, 50 Years On[END_REF] .

The folding problem has some impact in therapeutics, because diseases such as Creutzfeldt-Jakob, type II diabetes or neurodegenerative diseases are linked to protein misfolding. But it also limits our ability to understand protein structure-function relationship and predict the structure of a protein from its primary sequence. This is of first importance for many reasons. First, methods to monitor the tridimensional structure of proteins exist but they require a very long process to obtain the information. Furthermore, they just give a fixed view of a protein that can sometimes not be its native shape, because of the unnatural conditions in which it was obtained. Therefore, with a better understanding of protein folding, computer simulations could help to have a more complete view of the structure and therefore more insight of the function. With the advent of genome sequencing it would be interesting to predict the structure of encoded proteins, instead of having to test them one by one. On another side, proteins are very interesting targets for engineering. Being able to fully understand the folding mechanism would help the design of proteins with desired function from scratch or to modify existing proteins precisely to improve or change their functions.

A lot of work in the protein-folding domain gave better understanding of the folding process but some questions are remaining, and some challenging cases need further investigations. Membrane proteins are more difficult to study and little is known about them on the structural point of view. Intrinsically disordered proteins or random coiled regions in structured proteins, are also still mysterious despite their large occurrence in proteins. This interrogation led to the "protein nonfolding problem" or "unstructural biology" field. Viruses are often relying on more flexible protein's structures, which appear to perform as well as the structured ones [START_REF] Uversky | Flexible Viruses: Structural Disorder in Viral Proteins[END_REF] .

c. The particularity of enzymes

Among all proteins, enzymes play a crucial role and have particularly interesting properties. A living organism needs to be able to execute a lot of chemical reactions efficiently and with specificity to interact with its environment and fuel its metabolism. Enzymes accelerate chemical reactions. As catalysts, they are not consumed during the reaction and do not modify the thermodynamic equilibrium. Enzymes are specific for a substrate or a category of molecules with a well-defined molecular structure. But they are also specific in the type of reactions they catalyze. This unique specificity is related due to the 3D structure of the enzyme, which provides a highly structured micro-environment for the reaction to take place and gives it stereospecific discrimination capacities. Even if some enzymes were given particular names, they are generally named after the name of their substrate and/or sometimes the type of reaction they catalyze added with the suffix "ase": DNase act on DNA, various hydrolases catalyze hydrolysis reactions. Six classes of enzymes can be determined according to the type of catalyzed reactions 1 .

• Oxydoreductases: They catalyze redox reactions. Many of them are named dehydrogenases but some can be called oxydases, peroxydases, oxygenases or reductase. • Transferases: They catalyze the transfer of molecular groups from one molecule to another. Kinases, transferring phosphate groups, are part of this category.

• Hydrolases: They catalyze hydrolytic cleavage.

• Ligases: Ligases or synthetases catalyze ligation reactions where covalent bonds are created between two molecules. They usually require the use of ATP.

• Isomerases: They catalyze isomerization or intramolecular rearrangements.

• Lyases: They catalyze substrate elimination reactions that are neither oxidative or hydrolytic or lysis reactions generating double bonds. They are therefore also catalyzing the addition of a substrate to the double bond of another molecule.

The later enzymes are also called synthases.

This catalytic versatility implies that enzymes are involved in lots of distinct functions.

They can work on various substrates like DNA, RNA, sugars, lipids or other proteins. They can have mechanic roles, serve as motors, or just perform chemical reactions necessary for the metabolism. Diversity can also be involved in tissue specificity. Isoenzymes for instance, are specific of a tissue. They catalyze the same reactions but in different types of cells.

Enzymes accelerate reactions by decreasing the energy activation barrier necessary to reach the transition state of a chemical reaction (Figure 5). The interactions they create with the substrate provide new routes to reach and stabilize this transition state. The first model proposed for the interaction of the substrate with the enzymes was the key-lock model. In this model the enzyme catalytic site shape perfectly matches the shape of the substrate. Nowadays models are adapted and consider the flexibility of the enzyme, which is not a completely rigid body. In the induced adaptation model, the enzyme shape is modified when it binds to the substrate or more probably its transition state. These conformational changes may affect all the enzyme, or only the catalytic part. According to structural data obtained with X-ray crystallography, the enzyme's catalytic site is generally a cavity in the protein structure, delimited by hydrophobic amino acids. The hydrophobic environment increases the importance of electrostatic interactions thanks to the weak local dielectric constant. Active sites also typically possess a few polar or charged amino acids as well as water molecules able to strongly interact with the substrate. Enzymes can also leverage the reactivity of metallic cofactors or coenzyme molecules. Metallic cofactors will bring some extra charge in the catalytic site that can be used to stabilize interactions as well as redox properties. Coenzymes are modified during the reaction, hydrolysis of their bonds allow to furnish energy to the reaction.

Enzymatic reactions can be described with several parameters. These parameters can be used to describe their efficiency and compare them. The Michaelis-Menten equation characterize the kinetics of an irreversible reaction catalyzed by an enzyme on a unique substrate following the scheme:

𝐸 + 𝑆 𝑘 1 → 𝑘 -1 ← 𝐸𝑆 𝑘 2
→ 𝐸 + 𝑃 with E the enzyme, S the substrate, ES the transition state, P the product and kx the kinetic constants of the different steps.

It links the stationary initial reaction speed to the initial concentration of substrate and some characteristic enzyme parameters. The equation usually used to extract these parameters is:

𝑣 𝑖 = 𝑣 𝑚𝑎𝑥 ×[𝑆 0 ] 𝐾 𝑀 +[𝑆 0 ]
with vi the initial speed, vmax the initial speed when the substrate concentration saturates the enzyme, [S0] the initial substrate concentration and KM the Michaelis constant of the enzyme (corresponding to the concentration of substrate where vi= vmax/2. KM values are often close to the physiological concentrations of their substrates. Therefore, a slight change in the substrate concentration can generate a large effect in a reaction cascade.

To measure the catalytic efficiency of an enzyme, the catalytic constant kcat or the "turnover number" determines the number of substrate molecules turned into product by unit of time when the enzyme is saturated.

𝑘 𝑐𝑎𝑡 = 𝑣 𝑚𝑎𝑥 [𝐸 𝑇 ]
with [ET] the total concentration of enzyme. kcat is the maximum rate of the reaction at saturating substrate concentration. This limit corresponds to the point were the only limiting step is the diffusion of substrates.

The Michaelis-Menten equation does not describe perfectly all enzymatic reactions and requires performing experiments in special conditions to be able to measure the different parameters. Enzyme concentration should be much lower than the sum of the substrate's concentration and the KM. Since the KM value is a priori not known, very low concentration of enzymes regarding to the substrate concentration are used. It can therefore not allow to describe in vivo enzyme catalytic process. That is why some researchers try to better model enzymatic reaction and propose new tools to describe enzymatic reactions [START_REF] Choi | Beyond the Michaelis-Menten equation: Accurate and efficient estimation of enzyme kinetic parameters[END_REF] . But, the experimentally measured KM and kcat values are usually the one used to assess an enzyme efficiency.

Beside the enzyme's catalytic speed and efficiency, many other parameters can be important to describe an enzyme. First, related to the kinetics of the reaction, the binding parameters, the association and dissociation constants of the enzyme with the substrate or the product can be studied. The level of specificity is also important. Some enzymes can be specific to a substrate but also bind slightly to others. Some enzymes can perform enantioselective reactions, meaning that they can be specific of a certain isomer of a substrate, or always generate of type of stereoisomer as a product. The enantioselectivity can be partial, with a bias toward one isomer but not a perfect selectivity. The sensitivity of the enzymes to external factors like the pH, that can change amino acids state, the cofactors concentrations, the buffers composition, additives or living organisms can also influence catalytic properties. Finally, the protein stability and sensitivity to temperature can vary according to the type of host from where it comes. Thermophilic organisms possess enzymes with more rigid structures and higher thermoresistivity. Degradation of enzymes is not only linked to the temperature. In living organisms some systems allow to control the number of enzymes and their duration. Proteins and therefore enzymes can be tagged to be degraded by the cell's proteasome. Besides degradation several regulation paths exist to regulate enzymes activity in cells. The expression can be enhanced or decreased, regulatory molecules can bind on the enzymes, phosphorylation and dephosphorylation mechanisms, as well as the proteolytic cleavage, can allow to activate and deactivate an enzyme. The enzyme conformation can also be self-regulating when binding of the substrate generates allosteric effects.

Inhibitors can also be found to inactivate enzymes. Diverse types of inhibitors exist and do not have the same effect on the kinetics of the reaction. Competitive inhibitors bind in the catalytic sites but cannot be processed by the enzyme, they prevent the binding of the substrate.

Non-competitive inhibitors bind on another site that the substrate binding site while uncompetitive inhibitors bind the enzyme+substrate complex preventing the release of the product. These two types of inhibitor do not prevent substrate binding but prevent the catalysis to happen.

d. Interest of proteins for biotechnologies and therapeutics

Proteins are essential in living organisms, but they can also be used as tools. Two properties of enzymes are typically of technological interest: specific binding and catalysis.

Specific binding capacities of enzymes are of particular interest in therapeutics.

Antibodies are natural agents in host defense against infection and disease. Their importance in therapeutics to target some infectious or pathogenic agents is self-evident. Other applications of specific binding can be found in molecular biology. For example, the biotin-streptavidin interaction can be used to attach biotinylated modified DNA strands on streptavidin-covered surfaces.

Enzymatic catalysis is used in various field. Enzymes, as catalysts, can act at low concentration as they are not consumed in the reaction. They can transform thousands of substrate molecules in a second and can do it in mild chemical conditions, meaning at relatively low temperatures, in aqueous solution and at near neutral pH conditions. Non-biological catalyzers usually necessitate higher temperatures and therefore more energy consumption to accelerate chemical reactions. The specificity of enzymes both for their substrate and for the type of reaction they catalyze also make them very convenient for practical use as only the desired reaction will be accelerated. All these advantages make them interesting in therapeutics as well but also in research, industry, such as food processing or biofuel production, in agriculture, environmental protection, biomaterials, biotechnologies and obviously in chemistry [START_REF] Romero | Dissecting enzyme function with microfluidicbased deep mutational scanning[END_REF] . For instance, in molecular biology several enzymes like ligases, endonucleases and polymerases are used for DNA manipulation. Lysozyme can be used for cells lysis. Some laundry detergents contain proteases and lipases improving their efficiency at low temperatures.

Using enzymes in chemistry can allow to reduce the number of toxic agents required for reactions and decrease the necessary amount of energy thanks to lower working temperature, leading to a greener chemistry. For environmental protection, enzymes able to degrade toxics components or accelerate waste degradation can be very useful.

In many cases, appropriate enzymes or proteins can be found in nature. They can be either extracted from the producing organism or produced in recombinant form by transferring their genetic sequence in model organisms tuned to overexpress the protein. After purification the enzyme can be added in the reaction with all the desired component. But as proteins are the results of billion years of evolution, they have been shaped to work in a particular cellular environment. In most of the cases some adaptations are required to use them properly in artificial settings. Typically, pH modification, compared to its original working environment, temperature change or substrate concentration variation can completely change a protein behavior or shape and render it completely inefficient. Two solutions can be envisioned. The first one is to adapt the reaction condition so that they fit the enzyme requirement. The other one is to modify the protein so that it can work in the desired conditions. This second option is the more convenient but require the use of protein engineering. Beside the adaptation of a protein to its new environment, protein engineering can be used to try to modify a protein target or an enzyme activity. This can allow to catalyze new reactions on compounds or conditions unlikely to have been encountered in nature.

Protein engineering relies on the modification of the protein amino acid sequence to create a new modified protein with desired function or properties. It was first made possible by the apparition of gene directed mutagenesis techniques in 1978 (see Figure 6). Using directed mutagenesis, it is possible to mutate a base in the DNA sequence at the desired position. The codon for an amino acid can therefore be modified to code for another one. Using this technique mutations were introduced in the protein sequence of tyrosyl-transfer RNA synthetase (TyrRS) in 1984. Mutation on the position Thr51 gave birth to the first engineered enzyme with improved activity [START_REF] Brannigan | Protein engineering 20 years on[END_REF] . The protein structure of TyrRS was known and, based on simulations, the effect of this mutation was partly anticipated. In the following years, directed mutagenesis, cloning and oligonucleotides synthesis became widely spread in laboratories and many mutagenesis experiments were done. They allowed to confirm the importance of specific residues, clarified the mechanism of enzymes but also revealed some wrong hypothesis about some protein functioning [START_REF] Brannigan | Protein engineering 20 years on[END_REF] . As explained earlier, even if a lot of data have been accumulated on protein structure, the prediction of the tertiary or quaternary structure from the primary sequence is still challenging. Despite the various available computational tools, researchers discovered that it was not easy to engineer a protein by rational design. A new technique for protein engineering appeared in 1993 [START_REF] Chen | Tuning the activity of an enzyme for unusual environments: sequential random mutagenesis of subtilisin E for catalysis in dimethylformamide[END_REF] . Called directed evolution and based on the exploration of randomly mutated sequences, this technique allowed to discover proteins with new properties without requiring previous structural or functional information.

Either by rational design or directed evolution, several proteins have been engineered in the last 30 years. As for therapeutic applications, Haemoglobin was engineered to make a better cell free oxygen carrier or blood substitute by lowering its oxygen affinity and improving the stability of its quaternary structure. Monomeric insulin with faster uptake by the blood system and longer resilience in the plasma could also be designed. More recently, a recombinase able to excise the provirus HIV-1 with high specificity was evolved by Karpinski et al. [START_REF] Karpinski | Directed evolution of a recombinase that excises the provirus of most HIV-1 primary isolates with high specificity[END_REF] . Several antibodies specific to antigens of interest were also designed for many targets. In other fields, carbon-silicon bonds formation could be catalyzed for the first time in living organism thanks to an engineered version of the cytochrome c in the laboratory of Frances Arnold [START_REF] Arnold | Evolution: Bringing New Chemistry to Life[END_REF][START_REF] Kan | Directed evolution of cytochrome c for carbon-silicon bond formation: Bringing silicon to life[END_REF] .

Polyhydroxyalkanoate, a biopolyester, production systems based on enzymes could also be improved and made more efficient thanks to protein engineering [START_REF] Taguchi | Evolution of Polyhydroxyalkanoate (PHA) Production System by "Enzyme Evolution": Successful Case Studies of Directed Evolution[END_REF] . In biofuel industry, a tailored enzyme was introduced into crop plants to produce stearate and increase the proportion of unsaturated fatty acids in seed oil. Enzymes are also more and more used in animal food to help cellulose digestion and improve animals' wealth but also decrease risks of bovine spongiform encephalopathy [START_REF] Brannigan | Protein engineering 20 years on[END_REF] . Some enzymes have also been designed to degrade environmental pollutants such as the Methyl-parathion hydrolase which can be used for pesticide detoxification [START_REF] Liu | Improved efficiency of a novel methyl parathion hydrolase using consensus approach[END_REF][START_REF] Tian | Enhanced thermostability of methyl parathion hydrolase from Ochrobactrum sp. M231 by rational engineering of a glycine to proline mutation[END_REF] .

In the following part we will see the available tools to design and engineer proteins based on accumulated knowledge.

There are diverse ways to engineer a protein. The two main approaches are rational design and directed evolution, but they can also be used together. Rational design is based on the knowledge and understanding we currently have of the relation between the sequence, structure and function of a protein, whereas directed evolution relies on an artificial implementation of the process of Darwinian evolution.

B. The limit of rational design for enzyme engineering

In the 1980s, with the apparition of DNA mutagenesis techniques, oligo synthesis, improvement in E. coli strains handling and the increasing ease of use of genetic DNA manipulation techniques and gene cloning it became increasingly easy to generate mutants.

Based on the knowledge that scientists had acquired on proteins, experiments first used sitedirected mutagenesis to introduce mutations at positions thought to have an impact on the protein function. Changes in single amino acids, then complete loops and finally complete domain exchange were used to modify proteins and enzymes properties, creating the new field of protein engineering. Despite some encouraging achievements, these experiments mostly revealed the lack of clear understanding of protein functioning. The often-disastrous effect of rationally introduced mutations prompted some scientists to rename the protein engineering field as « protein terrorism » [START_REF] Brannigan | Protein engineering 20 years on[END_REF] . These studies suggested that the structure-function relationship in proteins should not be studied locally but rather taken at the level of the whole structure.

Armed with the acquired experience and further accumulation of protein structures, researchers today have many improved tools at their disposal to help them in the rational design of protein.

These improvements are due to great progresses in protein design algorithms, molecular force fields, structural bioinformatics and computational strategies.

a. Acquiring the necessary information

While we have easy access to DNA sequences and to the corresponding amino-acid chains, we cannot directly link them to tridimensional structures and even less to functions (this process has generally to be done by analogy with already annotated genes). Protein engineering by rational design addresses the reverse problem of finding a sequence, given a fold or a function. Given the difficulty of the task, most projects start from an existing protein, and try to alter its properties. Collecting experimental information on these start-point proteins can help to better understand it and give insight on how to modify it.

A lot of information can be gathered through different methods of protein study. X-rays crystallography, RMN, cryoEM and circular dichroism experiments presented in A. b. can all give important information. A tridimensional model of the protein or information about the secondary structures (α-helix and/or β-sheets) can be obtained. But these experiments are not straightforward. Obtaining a protein crystal requires the screening of many crystallographic conditions, and sometimes never works. It also requires the ability to produce or collect the protein in a large amount with high purity. This can be very challenging for many reasons, such as the eventual toxicity of the overexpressed protein, or the misfolding of the target protein in the model organisms.

If it is not possible to get direct structural information from experimental data, computational modeling can be used. Because of the difficulties explained above, computational modeling is based on sequence homology between the target protein and available tridimensional structures in the protein data bank (PDB). Sequence alignment allow to find protein with high similarities. BLAST [START_REF] Altschul | Gapped BLAST and PSI-BLAST: a new generation of protein database search programs[END_REF] or FASTA [START_REF] Pearson | Improved tools for biological sequence comparison[END_REF] programs perform comparison of the sequence with all available sequences or can be used for alignment. PSI-BLAST [START_REF] Altschul | Gapped BLAST and PSI-BLAST: a new generation of protein database search programs[END_REF] uses a 2step process to compare multiple protein sequence with an increased accuracy of the alignment.

When no related sequence can be found for a query, threading or fold recognition one can use the HMMER [START_REF] Finn | HMMER web server: interactive sequence similarity searching[END_REF] software based on pairwise comparison of Hidden Markov Models i . Once a homolog is found, template-target alignment needs to be performed. CLUSTAL W [START_REF] Chenna | Multiple sequence alignment with the Clustal series of programs[END_REF] or PRALINE [START_REF] Simossis | PRALINE: a multiple sequence alignment toolbox that integrates homology-extended and secondary structure information[END_REF] methods based on secondary structure element analysis are the best. After this, structure prediction can be run. MODELLER [START_REF] Sali | Comparative protein modelling by satisfaction of spatial restraints[END_REF] or SWISS-MODEL [START_REF] Arnold | The SWISS-MODEL workspace: a webbased environment for protein structure homology modelling[END_REF] can be used for this purpose. Sometimes several templates can be used to model the different part of a protein.

Reproduction of the perfectly matching pieces or small identic or similar fragments can be assembled to create the backbone of the new model. The template spatial constraints will determine the position of the atoms. Loop modeling is used to predict structure of unmatching parts and finally, side chain modeling and evaluation and refinement of the model by computing energy level complete the process. Even if it is rarely possible to check the veracity of a model, those generated from proteins with more than 70% of identity are usually of good quality.

Homology modeling could potentially be applied to 30% of sequences. For a given protein, there are 20 to 50% of chances to find an associated protein structure. When less than 25% of sequence identity is found between the target protein sequence and any protein in the PDB, ab initio prediction software like I-TASSER [START_REF] Yang | The I-TASSER Suite: protein structure and function prediction[END_REF] or ROBETTA [START_REF] Kim | Protein structure prediction and analysis using the Robetta server[END_REF] can be used.

Tridimensional structure discovery of proteins is ongoing, but researchers are finding less and less new 3D folds. From these observations, it seems that almost all existing or most adopted folds have been discovered. It also suggests that general folds available in nature are restricted.

Therefore, it seems possible to estimate a protein folding based on the accumulated data. I-TASSER modeling uses alignment of small parts of the sequence on the database of known structures and repeated compilation of the pieces. Structural simulation allows then to assess the global model and its reliability. ROBETTA cuts the sequence into small predictable parts. Some part will be aligned and modeled by homology if enough similarity is found, others will be modeled from scratch in very little pieces via loop modeling. Then fragments are combined, and the global structure is evaluated.

b. Protein design from acquired information

Starting from structural information, several software can be used to predict the effect of amino acids substitution on the protein structure and/or function. According to the purpose of the protein engineering, different software can be used. The CAVER program for instance was developed to identify tunnels in protein with buried active sites [START_REF] Damborsky | Computational tools for designing and engineering biocatalysts[END_REF] . After the lock-key model and the induced-fit model for substrate binding to enzymes, the more recent 'keyhole-lock-key' model insists on the importance of the substrate entry in the protein cavity. These entry tunnel can be more or less selective and need to be modified simultaneously to the active site when substrate adaptation is targeted. To study protein-protein interactions, FOLD-X, ORBIT or COMBINE algorithms can help. The COMBINE algorithm for instance is based on alanine scanning, a method that can also be used in wet lab to determine the importance of an amino acid in the protein function. The alanine amino acid is a relatively passive amino acid that cannot have much interactions with the other or stabilize interaction with other molecules. If mutation of an amino acid into alanine has no strong effect, it suggests that the nature of this amino acid is not determinant in the protein. Here, systematic estimation of the alanine substitution is addressed in silico and can be used to determine which amino acid is interacting in the protein-protein interface. For thermostability, tools like VOLSURF can be applied.

VOLSURF identifies structural requirements for increased thermal stability by analyzing data on 22 thermophilic and hyperthermophilic organisms and calculate interaction energy grid to apply them on the target protein.

Simulation of the effect of the mutations in silico can allow to calculate a theoretical protein fitness landscape [START_REF] Au | Direct Calculation of Protein Fitness Landscapes through Computational Protein Design[END_REF] and determine the best protein sequence. Programs can use molecular dynamics simulations using coarse-grained or more refined models, free energy calculations, quantum mechanical calculations, amino acids contacts or distance predictions to have an idea of the positive or negative effect of a mutation. They can facilitate the complex calculation on protein flexibility and energetics. Calculating the free energy of a structure with different mutations can also allow to predict stabilizing and destabilizing effects. In silico mutations are interesting because they can reach a portion of the fitness landscape that is not reachable by experiment and can allow to define new rules for protein building [START_REF] Au | Direct Calculation of Protein Fitness Landscapes through Computational Protein Design[END_REF] . Computation enables the search over huge configuration spaces. Simulation can also be run in variable conditions like for instance with pH modifications or in presence of some specific compounds and even unnatural amino acids. Molecular docking can also help to predict interactions between the target protein and small molecules, nucleic acids or other proteins.

Data-driven approaches

Quantitative Structure-Activity Relationship (QSAR) [START_REF] Fersht | Quantitative analysis of structureactivity relationships in engineered proteins by linear free-energy relationships[END_REF] analysis establishes a quantitative relationship between structure and biological activity by correlating residues with functional data for a set of proteins carrying systematic substitutions at some fixed positions.

This approach requires experimental data but enables quantitative interpretation of mutations effects and anticipated predictions of the variant's properties. Other approaches based on machine learning can also infer, from a relatively small set of mutants and their characterization, what mutations should be introduced to improve an enzyme.

Multiple sequence alignment can also give some insight to guide the design of a protein.

Consensus design presented in Box 1 (p.33) uses most conserved residues in a consensus sequence to improve protein stability [START_REF] Porebski | Consensus protein design[END_REF][START_REF] Bershtein | Intense Neutral Drifts Yield Robust and Evolvable Consensus Proteins[END_REF] . Sequence alignments and evolutionary analysis can also give information on activity parameters when similar sequences with different functions are analyzed or when they are combined with functional data [START_REF] Damborsky | Computational tools for designing and engineering enzymes[END_REF] . Here it is not the consensus position but more the differences that can help determining the role of each amino acid.

c. Completely ab initio design

All the previous methods are based on availability of structural information and preexisting proteins, but some researchers try to design enzymes from scratch. Some remarkable achievements have been made using rational design of proteins like the first de novo design of an enzyme catalyzing a non-natural reaction in 2008 with the Kemp elimination [START_REF] Röthlisberger | Kemp elimination catalysts by computational enzyme design[END_REF] . The engineered enzyme could accelerate 10 5 times the speed of the reaction.

De novo design of proteins usually still uses a scaffold issued from structural data available in the PDB. As explained earlier, very few new folds are discovered in the new obtained proteins structures [START_REF] Dawson | CATH: an expanded resource to predict protein function through structure and sequence[END_REF][START_REF] Andreeva | Data growth and its impact on the SCOP database: new developments[END_REF] . This can be because the other folds are not crystallizable or that they simply do not exist. Based on this second assumption it seems that almost all naturally available scaffold sampled by nature are more or less known. Thus, it appears as a good idea to use scaffolds issued from natural proteins to design new ones as others would potentially not be nature-proof. Another way of constructing scaffolds is to use a symmetric pattern with repetitive motifs. Repetitions of similar motifs and presence of symmetric axes in existing protein sequences are frequently observed. It is even thought that proteins could have originated from the replication and assembly of an ancestor motif [START_REF] Smock | De Novo Evolutionary Emergence of a Symmetrical Protein Is Shaped by Folding Constraints[END_REF] . Some researchers therefore try to build scaffolds based on motifs repeat like the α-rep [START_REF] Urvoas | Production and Molecular Structure of a New Family of Artificial Alpha-helicoidal Repeat Proteins (αRep) Based on Thermostable HEAT-like Repeats[END_REF] or symmetrical layout of helices and βsheets [START_REF] Huang | De novo design of a four-fold symmetric TIM-barrel protein with atomic-level accuracy[END_REF] .

To design a new catalytic activity, a general approach is to try to stabilize the transition state of the desired reaction by positioning putative catalytic residues around it. Then, scaffolds of known protein structures are screened to find the ones where these amino acids' position could fit. Either a fixed position for the catalytic residues or different positions inside the catalytic site can be tested (Figure 7). De novo enzyme design is more and more used, with ROSETTA and ORBIT being the most widely used software suites. Programs are always improving using more robust, accurate, and reliable algorithms. Throughout the years, initial scaffold libraries refinement has been improved as well as flexibility of the protein backbones in simulations and local amino acids substitution effects. Such improvements allow to better apprehend the flexibility of real proteins.

A main target of this engineering approach is to catalyze non-biological reactions like the proton transfer in the Kemp Elimination. Another success was the breaking of a carboncarbon bond in a non-natural substrate with the retro-aldolase [START_REF] Jiang | De Novo Computational Design of Retro-Aldol Enzymes[END_REF] . These artificial enzymes managed to accelerate unnatural reactions but current de novo designed enzymes do not meet natural enzymes efficiency. Further optimization using directed evolution is always needed. It also happened that a protein design project produced the correct fluorescent properties, but with a completely unexpected structure -despite the fact that the design was all based on the structure [START_REF] Figueroa | The unexpected structure of the designed protein Octarellin V.1 forms a challenge for protein structure prediction tools[END_REF] . These observations show the limit of our structural understanding and therefore the limit of rational design used alone to create new enzymes. Another drawback of the rational design approach is the requirement of a lot of computational power just to design one protein.

However, designing new proteins is a perfect way of testing our understanding of biomolecular systems.

Box 1: Consensus design Information to guide protein engineering can be obtained from alignments of homologous sequences. Multiple sequence alignment of proteins can allow to infer ancestral relationship between proteins but also to create consensus sequences from the most frequently observed amino acid identified at each position in the alignment. Sequence alignment databases such as Pfam can be used to initiate the process. Most conserved positions of the consensus sequence that are not matching in the target protein sequence can be spiked back either separately or in various combination. This consensus design approach can increase stability with melting temperatures gaining 10 to 32°C for both functional and non-functional proteins. Among the conserved residues 50% are generally associated with an improved stability and 10% are neutral. The remaining 40% can be deleterious, therefore consensus positions and their combination need to be tested in order to find the interesting ones. Another solution can be to directly use the consensus sequence to dilute the destabilizing effects of these mutations. Yet, this approach can give a good idea of positions to target and mutations to insert and if combined with directed evolution approach can reduce the number of mutants to study. Explanations on this effect of consensus mutations are not clear but could be due to the conditions in which ancestral proteins appeared.

Acquisition of the consensus sequence is one of the main concerns with this approach. Too little or too much diversity in the aligned sequence can have an important impact. Exiting from a taxonomic domain can also negatively affect the results. When constructing the sequence alignment set, a balance needs to be found between sequence similarity, helping to create a good quality alignment, and diversity, which allows for a greater coverage. Using as few as 4 sequences can already help determining important positions. Addition of statistical analysis can help improve the consensus sequence. Consensus design cannot be considered completely as being part of rational design or directed evolution but rather as a complementary method for both. Usage of consensus sequences have shown that they can stabilize the native state of a protein and increase its evolvability. Conserved positions seem to remodel the folding landscape. Thus, it can be useful to introduce them in directed evolution as a starting point or in rational design to start from a more stable protein limiting the destabilizing effect of introduced mutations. One striking example of the power of consensus design is the evolution assays performed on the ab initio designed Kemp Eliminase. First rounds of directed evolution did not yield to any functional or improved mutants. After spiking of the scaffold protein consensus mutations in the Kemp Eliminase, directed evolution could be run and the enzyme improved.

Another interesting aspect of de novo rational design, which is not much exploited yet is the possibility to free itself from the restriction of natural proteins. It is possible that the conformation founds in nature all originate from a subsample of possibilities that happened to be tested since life apparition and do not reflect bio-physico-chemical restrictions.

C.

The power of directed evolution b. In vivo/ in vitro, linking activity to gene selection

As they are focusing on molecules and not on organisms, implementations of DE techniques are not always easy. The principal concern in DE is to find a way to measure the "fitness" of the proteins or to select proteins with the highest "fitness". A first step is to correctly define the parameter one wants to improve in for the target protein. Then, it is critical to design the process to select for that property, and not something else. The statement "you get what you selected for" emphasis the importance of this design. When the protein being targeted is just a part of a complex system (for example a full living organism), one has to make this protein the most critical part, or the limiting factor of the selection process. In many cases, DE design must change the "role" of the protein in an organism or amplify its importance. Because of the huge diversity in protein roles and functions it is difficult to find a regular/standard protocol. Many different ingenious ideas have emerged, specific to one protein or one class of proteins. No universal directed evolution method has yet been established, and efficient methods with high adaptability are still missing. In Darwinian evolution, the phenotype-genotype link is an essential element since only the genotype, carrying the information, is transmitted to the next generation. In a DE method, if the artificial "fitness" can be linked to reproduction or cell survival, we come back to the natural evolution case. In other situations, maintaining the phenotype-genotype linkage during the course of the experiment is essential to enable the artificial evolutionary loop.

Different techniques support the retrieval of the encoding gene with the selected protein.

When an organism like yeast or bacteria is used to express the target protein, the cell membrane or cell wall already constitute an initial compartment where the protein and its encoding gene are co-localized. Unfortunately, in many cases a functional assay to test the mutant's activity requires the lysis of the cell to release the target protein in the test mixture. Thus, the compartment is broken, and, if this is done for a population of different mutants, phenotype and genotype would be mixed and averaged. Separating the cultures for each mutant is the first evident solution, but growing large numbers of mutant bacteria in tubes or 96 well plates is not very convenient, and can quickly become very costly. Testing 10 2 to 10 4 mutants in microtiter plates have been used successfully in the beginning of directed evolution to engineer proteins with desirable properties but experimental evidence show that larger libraries can yield better proteins.

To increase the throughput of experiments, micro-compartmentalization techniques were developed to be able to study cells in picoliter volume of reagents. The main compartmentalization system used in directed evolution is the encapsulation of cells in waterin-oil droplets. Droplets are used as microreactors where each individual mutant cell can be tested. In these microreactors, the phenotype-genotype link is preserved even after the cells are lysed. Everything happening in or to the droplet will concern both the gene and the corresponding protein. Emulsions can be generated by various methods (see III, p.209), the simplest of which involves only mixing oil with a cell suspension and shaking the reaction tube.

This however generates droplet of various sizes. To obtain a uniform droplet size, microfluidic systems ii allow to generate monodisperse droplets at the micrometer range.

Compared to strategies based on host cell survival, the use of droplets as microreactors can allow the direct observation and measurement of individual library members, but also decrease or even remove the complexities linked to the intracellular environment and bias associated with the organism's maintenance [START_REF] Taly | Droplets as microreactors for high-throughput biology[END_REF] . Once the cell is diluted in the droplet buffer,

ii Microfluidics: Microfluidics deals with precise control and manipulation of fluids that are geometrically constrained to a small microliter scale. See p.xxx for more explanation on this fields.

selection tests can be performed in unnatural, in vitro conditions. For example, classical fluorescent activity assay can be used. Droplets are then sorted according to their fluorescence intensity, either into microfluidic devices or Fluorescent Activated Cells Sorting (FACS) systems. For the later, a double emulsion of water-oil-water is generally necessary due to the design of the machine. Alternatively, beads can be introduced in droplets and capture the DNA as well as the expressed protein and/or the fluorescent reaction product, giving functionalized beads with phenotype-genotype-reporter linkage that can then be sorted. Similarly, gel beads can be formed into droplets to encapsulate the reaction reagents before FACS [START_REF] Fischlechner | Evolution of enzyme catalysts caged in biomimetic gel-shell beads[END_REF] . With simple droplet reaction, only one parameter of the protein can be tested. But using these display systems, beads can undergo several reactions and sorting. Another way to perform complex protocols using droplets is the microfluidic fusion of each droplet with another one bringing fresh buffer and new reagents. It is also possible to produce the protein using cell-free systems based on in vitro transcription-translation (IVTT) mix. Griffiths and Tawfik used microreactors, IVTT protein expression and display of all the component of beads to evolve an extremely fast phosphotriesterase [START_REF] Griffiths | Directed evolution of an extremely fast phosphotriesterase by in vitro compartmentalization[END_REF] .

In vivo expression of proteins can be very convenient, as model organisms are easy to handle and can produce large amounts of overexpressed target protein. However in vitro expression systems provide other interesting advantages. In such systems, cell viability and growth are not relevant, therefore proteins that are usually toxic for the host cells can be expressed. For the same reason, IVTT mix can be optimized toward the exclusive production of the target protein. These systems provide an open environment with direct monitoring, sampling and reaction manipulation [START_REF] Carlson | Cell-free protein synthesis: applications come of age[END_REF] , devoid of membrane permeability issues. In vitro expression systems can also allow to introduce unnatural components like for instance unnatural amino acids that can extend the scope of possible reaction catalysis [START_REF] Packer | Methods for the directed evolution of proteins[END_REF] . Introduction of unnatural amino acids in vivo is possible but less straightforward [START_REF] Santoro | An efficient system for the evolution of aminoacyl-tRNA synthetase specificity[END_REF] . Unnatural reactions can be tested with toxic substrates or products that cells would not support. Furthermore, cloning steps, which are a major bottleneck in DE experiments, are avoided. It is even possible to use linear DNA, directly from the PCR [START_REF] Sun | Linear DNA for Rapid Prototyping of Synthetic Biological Circuits in an Escherichia coli Based TX-TL Cell-Free System[END_REF] . Two kind of in vitro systems are predominantly used by researchers, either cell extract-based IVTT, or the PURE system, based on purified expression components. The PURE system 52 has the advantage of containing only the protein necessary for protein expression but has relatively low production yield compared to cell-extract. Cellextract is made from bacteria lysate. Bacteria are lysed and centrifuged to retrieve the inner cell components [START_REF] Shin | Efficient cell-free expression with the endogenous E. Coli RNA polymerase and sigma factor 70[END_REF] . In this case, various other proteins, notably exonucleases, are present, and can interfere with the selection. Also, some activity assays cannot be run in the IVTT mix, in which case secondary encapsulation to dilute the first droplet or to encapsulate the functionalized bead are necessary.

Lots of different display techniques have been developed [START_REF] Roberts | RNA-peptide fusions for the in vitro selection of peptides and proteins[END_REF][START_REF] Odegrip | CIS display: In vitro selection of peptides from libraries of protein-DNA complexes[END_REF][START_REF] Skirgaila | Compartmentalization of destabilized enzyme-mRNA-ribosome complexes generated by ribosome display: a novel tool for the directed evolution of enzymes[END_REF][START_REF] Naimuddin | A High Performance Platform Based on cDNA Display for Efficient Synthesis of Protein Fusions and Accelerated Directed Evolution[END_REF] , not only for in vitro expression but also as a way to increase experimental throughput. Phage display, ribosome display, mRNA-peptide fusion via puromycin, or CIS display [START_REF] Taly | Droplets as microreactors for high-throughput biology[END_REF] all allow robust attachment of the DNA or RNA coding sequence to one or multiple copies of the corresponding protein. In phage display, the pII protein of the phage containing the target gene is modified to express the target protein on the phage surface [START_REF] Fernandez-Gacio | Phage display as a tool for the directed evolution of enzymes[END_REF] . In ribosome display, the lack of stop codon prevents the release of the ribosome, which stays attached to both the protein and its encoding RNA [START_REF] Amstutz | In Vitro Selection for Catalytic Activity with Ribosome Display[END_REF] . In the mRNA-peptide fusion [START_REF] Roberts | RNA-peptide fusions for the in vitro selection of peptides and proteins[END_REF] , covalent fusions between an mRNA and the encoded peptide or protein is generated by in vitro translation of mRNAs carrying puromycin, a peptidyl acceptor antibiotic, at their 3′ end. Finally, CIS display exploits the ability of a DNA replication initiator protein to bind exclusively to the template DNA from which it has been expressed [START_REF] Odegrip | CIS display: In vitro selection of peptides from libraries of protein-DNA complexes[END_REF] .

These display systems are particularly well suited for affinity selection. Proteins with affinity for the target attach it and are automatically selected from the pool. It is smore difficult to adapt these approaches to catalytic activity.

Once c. An efficient method with many outcomes [START_REF] Yano | Directed evolution of ampicillin-resistant activity from a functionally unrelated DNA fragment: A laboratory model of molecular evolution[END_REF] . This shows the power of this method. High number of improvements have been achieved not only in catalytic activities but also in other properties such as stability, enzyme selectivity, specificity, protease susceptibility, allosteric control, ligand binding, solubility of transcriptional activation [START_REF] Brannigan | Protein engineering 20 years on[END_REF][START_REF] Acevedo-Rocha | Directed evolution of stereoselective enzymes based on genetic selection as opposed to screening systems[END_REF] . DE first success inspired a lot of scientists. The simplicity of the concept, the absence of any special instrumentation requirement and the frequent discovery of useful solutions unanticipated by rational thinking makes it accessible to a broad audience of scientists and engineers.

First targets were individual proteins, in which random or position-specific mutations were introduced, evolved with the goal of obtaining more stable or active biocatalysts. Such enzymes were particularly interesting for increased compatibility with industrial processes [START_REF] Cobb | Directed Evolution: Past, Present and Future[END_REF] .

Nowadays, proteins are more and more evolved in the context of metabolic pathways, to create whole cell biocatalysts with applications in chemicals, biofuels or pharmaceuticals. Another trend is represented by the efforts to design enzymes with novel functionalities not found in nature. Even if industrial applications stay the main motivator of most DE experiments, it is also an invaluable tool to study the evolutionary principles and test evolution hypotheses.

In therapeutics, apparition of DE led to alternatives to natural antibodies. In the case of antibodies, one major problem is their immunogenicity. As they are often produced in rodents, they can generate defense reactions from the host organism. Chimeric or humanized antibodies can be made by changing the antigen recognition domain but other approaches using other kinds of proteins can also help solving this issue. Thanks to combinatorial protein design, new kinds of antibody-like molecules could be envisioned using small and robust scaffolds equipped with prescribed binding functions. Adnectins, Affibodies, Anticalins, DARPins and Kunitz-type inhibitors are part of this next generation of antibodies [START_REF] Gebauer | Engineered protein scaffolds as next-generation antibody therapeutics[END_REF] and can help to solve some issues linked to the use of antibodies. The obtained specific binding proteins can be used in therapy or for in vivo diagnosis. Some have been developed to target VEGF and inhibit tumor angiogenesis [START_REF] Ng | Pegaptanib, a targeted anti-VEGF aptamer for ocular vascular disease[END_REF] , others allow the monitoring of cancer treatment [START_REF] Lee | Affibody Molecules for In vivo Characterization of HER2-Positive Tumors by Near-Infrared Imaging[END_REF] or are developed for cancer immunotherapy [START_REF] Skerra | Anticalins as alternative binding proteins for therapeutic use[END_REF] or cystic fibrosis treatment [START_REF] Attucci | EPI-hNE4, a Proteolysis-Resistant Inhibitor of Human Neutrophil Elastase and Potential Anti-Inflammatory Drug for Treating Cystic Fibrosis[END_REF] . Improvements of insulin for diabetes treatments and haemoglobin presented earlier were also achieved by DE, as was the recent discovery of an enzyme able to extrude specifically HIV-1 provirus.

In chemistry, after the first evolution of an enzyme working in organic solvents, Frances

Arnold laboratory evolved several enzymes to perform unnatural reactions. For instance, they managed to pair chromophore substitution and DE to obtain a bright near infra-red fluorescent rhodopsin [START_REF] Arnold | Evolution: Bringing New Chemistry to Life[END_REF][START_REF] Herwig | Directed evolution of a bright near-infrared fluorescent rhodopsin using a synthetic chromophore[END_REF] . Using cytochromes as the starting point they also managed to evolve a Carbon-Silicon bond forming enzyme as well as an enzyme able to catalyze nitrogen-transfer reaction [START_REF] Arnold | Evolution: Bringing New Chemistry to Life[END_REF] .

Directed evolution now represents a centerpiece of industrial biocatalyst development.

Detergents, biorefinery, food industry, especially cheese, beer, and wine, as well as textile industry and paper industry are all using various kind on enzymes like hydrolases, proteases and carbohydrases. As they are often used at elevated temperatures, DE has often been used to increase their thermostability [START_REF] Rigoldi | Review: Engineering of thermostable enzymes for industrial applications[END_REF] . Other issues, like increasing the reaction rate or the ability to work in organic solvent were also addressed by DE [START_REF] Jemli | Biocatalysts: application and engineering for industrial purposes[END_REF] . DE has also been intensively used in the field of synthetic biology and supported the rapid development of the field over the last decade.

In a more fundamental context, evolution experiments allowed to discover the existence of local minima in fitness landscapes, where enzymes are trapped and cannot evolve anymore.

Neutral drift was also used to study structure-function relationship. And DE experiments revealed that some enzymes can evolve towards a new activity while keeping their original one [START_REF] Cobb | Directed Evolution: Past, Present and Future[END_REF] . The growing number of DE experiments combined with sequence data analysis or deep mutational scanning will also help to have more insight on proteins functions.

Despite the many successes and methodological progresses of DE, the solution space that needs to be explored is too large for exhaustive coverage. More and more works have focused on the reduction of this sequence space by designing smart libraries (see part II). These smart libraries often use structural information or rational knowledge to decide which part of the target protein should be targeted. Such libraries, smaller and with more active variants, reduce the need for high-throughput capabilities through the synergistic application of rational design with directed evolution. This kind of work is at the interface between rational design and DE and is part of the semi-rational design field [START_REF] Rosenfeld | Protein Engineering by Combined Computational and In Vitro Evolution Approaches[END_REF] . Among the biggest accomplishment of DE in combination with rational approaches, we can cite the Kemp Eliminase. This protein was first designed completely in silico to catalyze the unnatural Kemp elimination [START_REF] Röthlisberger | Kemp elimination catalysts by computational enzyme design[END_REF] . Several rounds of directed evolution greatly improved its stability and proficiency [START_REF] Khersonsky | Bridging the gaps in design methodologies by evolutionary optimization of the stability and proficiency of designed Kemp eliminase KE59[END_REF] Interestingly, despite being practiced by many academic and industrial scientists around the world, relatively few publications can be found on DE, compared to rational design in general. In Figure 9, we can see that they represent only 3.9% of the global publication on protein engineering.

D. Screening versus Selection Methods

In directed evolution, two main methods can be used for "selecting" the mutants matching the desired criterium. They are generally referred as selection or screening methods [START_REF]Directed Enzyme Evolution: Screening and Selection Methods[END_REF] .

Screening methods are based on biochemical assays run to characterize each mutant activity or capacity and the physical sorting of the variants matching a given criterion. The throughput of these methods is highly dependent on the nature of the biochemical assay, for example if it is based on fluorescent readings, or mass spectroscopy. Selection methods are based either on the survival of the host cell carrying the mutant in vivo, or the replication or retrieval of the mutant gene in vitro. Higher throughputs can be reached with selection methods, but they require casespecific adaptation that makes them less versatile. Both methods have some pros and cons that will be detailed in this section.

a. Screening methods

Various screening methods have been designed and are schematized in Figure 10.

According to the method, the throughput of the evolution experiment can change drastically.

The simplest implementation to screen mutant recombinant libraries is to simply grow the cultures in separate tubes and test them one by one with classical biochemistry protocols. Here, the throughput will be low and highly dependent on the nature of the assay and level of automation. From tubes to 96 well plates and eventually automatization of the plate's analysis throughputs from 10 2 to 10 4 can be reached.

At a smaller scale, screening of colonies on agar plates can be implemented for fluorescent or colored proteins. Fluorogenic or chromogenic substrates can also be used for detection of enzyme catalysis. Picking the colonies by hand can be extremely laborious and results in very low screened library size at each round of evolution. Nowadays, armed robots have been designed to analyze agar plates and automatically pick the interesting clones.

In order to increase the throughput, one can work at a still lower level, using Fluorescent Activated Cell Sorting (FACS), or assimilated techniques like the Fluorescent Activated Droplets Sorting, FADS [START_REF] Baret | Fluorescence-activated droplet sorting (FADS): efficient microfluidic cell sorting based on enzymatic activity[END_REF] , to sort individual cells, particles or droplets. This usually, but not always 77 requires a fluorescent output, which is measured when the cell passes through a laser beam. Using this technique, up to 10 8 entities can be screened at each evolution cycle. Display systems or encapsulation techniques allow to perform in vitro and binding assays, as well as using cell free expression systems.

In general, screening experiments have the advantage of low complexity and are relatively straightforward to implement, when they do not require the use of droplet compartmentalization. Colonies screening on solid media, isolated liquid cultures or screening of fluorescent cells with FACS do not require specific lab equipment. They can be adapted for almost any protein or enzyme as soon as a measurable feature of the protein activity matching the measuring device is found. Using liquid cultures of host cells and testing them one by one can be time-consuming but allow to perform several tests on the same mutant. It is therefore possible to test an enzyme for its activity on different substrates, allowing to screen for specificity or, on the contrary, for less specific enzymes.

Figure 10. Screening methods in directed evolution. Different methods can be used to sort the mutants : Picking colonies on solid media, using isolated liquid cultures, using display methods (gel beads, droplets, yeast display) and Fluorescence Activated Cell Sorting to sort the active variants.

The main disadvantages of screening methods are that they are generally more timeconsuming, laborious and have generally low throughput, except for FACS or FADS based screening. Low throughput screenings evolution campaigns are often coupled with rational library design based on structural information. In all cases, the selection of mutants is based on measured parameters that are highly dependent on the errors than can be made by the machine, the experimentator or the intrinsic noise of the assay (e.g. for single cells).

b. Selection methods

Selection methods, presented on Figure 11, can be more difficult to implement. As they rely on automatic gene selection by replication or host survival, a biomolecular way to link the target protein or enzyme activity to the gene survival or replication needs to be found. To do so, genetic circuits can be engineered inside host cells for in vivo complementation. For instance, the growing medium can be depleted from an essential nutrient that can only be synthesized with the target gene. One can also use the modification in an antibiotic resistance gene, which needs to be compensated by the target enzyme. Santoro et al. introduced a stop codon in the chloramphenicol resistance gene to select aminoacyl-tRNA synthetase able to introduce an unnatural amino acid in place of amber stop codon [START_REF] Santoro | An efficient system for the evolution of aminoacyl-tRNA synthetase specificity[END_REF] . These methods can have very high throughput, generally only limited by the host transformation step.

In vitro selection of proteins for specific binding capacities is also relatively straightforward. Mutant proteins can be passed in a column containing the target, or mixed with beads coated with the target, so that only proteins with affinity for the target are retained. But the retained proteins are not enough to close the evolutionary loop, as their genotype is also required, one of the single molecule display method introduced above (C.b, p.36) is necessary.

These methods generally have very high throughput of 10 10 to 10 12 limited by transformation and/or the selection experiment throughput.

Selection of enzymes based on their catalytic activity with displays is more challenging than selection based on affinity. A few reports still exist. Substrates or modified substrates can be used to capture the enzymes in conditions in which no reaction is possible. Then changing the buffer composition can allow the enzyme to catalyze the reaction on its substrate and detach [START_REF] Fernandez-Gacio | Phage display as a tool for the directed evolution of enzymes[END_REF] . Binding affinity is clearly an element of enzymatic activity, but the catalytic efficiency and the product release rate can strongly determine the global enzyme efficiency. These properties are not necessarily maintained or improved during these modified binding assay.

Because they do not require human intervention, selection methods also open the way to a continuous evolution system, which increase the potential of directed evolution experiments. A system based on bacteria phage infection was presented in 2011. In the Phage Assisted Continuous Evolution (PACE) [START_REF] Esvelt | A system for the continuous directed evolution of biomolecules[END_REF] , phages' replication is linked to the activity of the T7 RNA polymerase. The gene encoding the pIII protein necessary for the phage to grow is only present in the bacteria under a T7 promoter. The gene of the T7 polymerase is brought by the phages and would only be able to be selected and infect other cells if they can synthesize the pIII protein. Fresh bacteria are continuously inserted in the medium, which is continuously diluted to eliminate non-infectious phages. Due to the high mutation frequency of phages, mutations are continuously introduced in the DNA sequences. With this system, due to the short phage life cycle, 24 to 30 generations can be tested in a day, as progeny production start 10 min after phage infection.

Organism survival or PACE are based on host organism replication in vivo, while display techniques use the retrieval of the DNA sequence of good binders. Some examples of successful in vitro direct linkage between the gene amplification by PCR and the enzyme activity have been published. In one approach, Ghadessy et al. proposed the compartmentalized selfreplication (CSR) [START_REF] Ghadessy | Directed evolution of polymerase function by compartmentalized self-replication[END_REF] technique where the target gene is replicated in vitro in relation to the activity of the protein it encodes. This system was first proposed for evolution of the Taq polymerases. A bacterial library of the Taq polymerase gene is encapsulated in droplet with PCR components and then lysed thanks to high temperature. Encapsulation in droplets allows to separate bacteria expressing the mutant and carrying its encoding gene to perform separate PCR reactions using Tac-specific primers. Polymerase with higher activity will replicate faster than less active ones and when the droplets are broken, the retrieved genetic population is enriched in the best mutants. This method allows the directed evolution of enzymes in unnatural conditions, as the test is not done inside the cell. But, its utilization is restricted to polymerases.

In a modification of this process, Ellefson et al. developed the Compartmentalized Partnered Replication (CPR) [START_REF] Ellefson | Directed evolution of genetic parts and circuits by compartmentalized partnered replication[END_REF] where the expression of the Taq polymerase is linked to the activity of another target enzymes, here the T7 polymerase, or to the suppression of the amber stop codon in the Taq gene thanks to modified tRNA synthetase. The mutants leading to more expressed Taq polymerase are selected via a preferential gene replication in droplets. CPR allows to extend the possible targets. This process resembles natural selection and avoids bias often induced by the screening procedure, but loses the versatility on test conditions, as the target enzyme needs to perform inside the cell, before its lysis. In the CPR a 1500-fold improvement of the incorporation rate of an unnatural amino acid by a modified tRNA synthetase has been reached.

The main advantage of selection methods is that it is an automatic process that does not rely on measurement or individual manipulation of the variants. It also eliminates the error possibly brought by the measurement device. Selection methods are closer to the natural selection process. In the case of the PACE or organism survival, the selection, or evolution, provided that enough mutations can be introduced, can run continuously without genetic manipulation or transformations at each step. Generally, all of these methods have relatively high throughput of at least 10 8 , even for the simpler one relying only on host survival.

In short, well-designed selection offers unparalleled throughput providing that a way to link the activity to host survival or gene replication can be found. This step can be difficult to design, and simple targets are restricted to enzymes essential in metabolism/survival or involved in DNA replication or protein expression. Thus, selection experiments are generally less adaptable than screening assays. In addition to this limitation, one of the main disadvantages of selection strategy can be the complexity of protocols. Many methods require technical and or experimental expertise like the use of cell-free extracts or microfluidics.

c. Comparison of the two methods

Directed evolution experiments require high throughput to be very efficient and to explore the highest possible number of variants. The design of the selection or screening experiment is therefore very important and different parameters should be taken into account to choose the best strategy. In both screening or selection, methods with highest throughput often require expertise as the techniques were developed relatively recently (see Table 2). They are therefore not so easy to implement in a standard laboratory. This maybe explains why DE is not used that much in protein engineering, despite being a really powerful tool. The different specificities of each DE methods are compiled in the Figure 12. It ranks the different methods according to a few important parameters: the complexity of the method, the library size that can usually be tested in one cycle of evolution and the adaptability of the method. We can see that selection methods have on average a higher throughput than screening methods. But, they are also more complex. Screening methods can be adapted to a bigger variety of enzymes. If we compare the duration of one evolution cycle in the different methods (Table 2, last column) we can see that they are more or less the same except for the PACE, which performs several cycles per day (albeit with less control).

The method that I started developing during my PhD is inserted in the table and the graph as PEN CSR (standing for Programmable External Network for Compartmentalized Self-Replication). PEN CSR is related to the CSR protocol and aims at increasing its versatility concerning activities that can be evolved, while also keeping the possibility to perform selection assays in vitro. In that sense it should be well adapted to search for enzymes that will be used in purified form, outside of a living organism. This method is based on the use of molecular programs based on short DNA strands and enzymes able to link an enzyme activity with the replication of its own gene. Due to the nature of the molecular program, enzymes with DNA substrates can all be targeted but, theoretically, any activity that can be linked to the molecular program can be selected. Using molecular programs would also provide the selection process with control over the selection function. In such programs, different modules can be combined to test several parameters of an enzyme simultaneously but also to adapt the stringency of the selection. Contrary to most available selection techniques, molecular programs can allow to select for the speed of the enzyme and dynamic phenomenon, not only its presence or absence.

The program used in the PEN CSR are created using a well characterized toolbox and should not require a high level of expertise in order to use them. We will see in the next part how molecular programs can be used in directed evolution. It is therefore not surprising if, following the discovery of the mesmerizing capacities of living cells and the molecular basis behind them, researchers started wondering if such exquisite molecular systems could be used for non-biological computation. As the limits of Moore's law on increasing power of computer processors are being reached and improvements slow down, alternatives like quantum computers are sought. Contrary to the binary system of 0 and 1 used nowadays in silicon computers, the quantum bits can have a variable number of states greater than 2. This allows more information to be encoded in one bit. The DNA molecule carrying the genetic information also possesses some higher bit state capacity. As it is composed of four different bases, one can hope to greatly improve the capacity of a bit using DNA as computing material. Not only does DNA possess interesting encoding capacities, but it is also very small and uses 50 atoms, a cubic nanometer, to code 1 bit [START_REF] Adleman | Molecular computation of solutions to combinatorial problems[END_REF][START_REF]Richard Feynman Introduces the World to Nanotechnology with Two Seminal Lectures[END_REF] . This has to be compared with the physical limit of silicon transistors -which cannot go below 7nm because of quantum tunneling effects iii . In contrast to attempts to build computers at the molecular level with designed artificial electronic molecules, DNA is a versatile material that has many advantages.

It can easily self-assemble, can be used as a scaffold, and even has catalytic activity. Handling tools are also well developed. It can easily be synthesized, read with sequencing, stained with fluorescent molecules or manipulated with various types of enzymes able to cut, ligate, extend or chemically modify the DNA.

In 1994, Adleman decided to encode a famous NP-complete problem iv using DNA strands, the Hamiltonian path problem (Figure 13). The problem can be described as follows:

Find the shortest path through a set of different cities while travelling to each city exactly once.

Conventional computers handle this problem by brute force algorithms that try all the possibilities one by one. In his molecular implementation, Adleman encoded 7 cities with short DNA strands of 20 nt and the available paths with others 20 nt strands matching the end of a strand and the beginning of another. When mixed together with a ligase, strands can anneal, and many possible paths are created, testing all solutions in parallel. Using molecular biology tools and carefully designed steps he could retrieve the right path after 7 days of experiments.

This was a great demonstration of the parallel capacity of the DNA computing: if ligations are

iii The quantum mechanical phenomenon where a subatomic particle passes through a potential barrier that it should not surmount under the provision of classical mechanics. iv Computational problems for which no efficient solution algorithm has been found. counted as operations, 10 14 operations were realized per second in such DNA computer versus a maximum of 10 12 for a supercomputer at that time. Scaling up the process, 10 20 operations could theoretically be run per second, rivaling with today's supercomputers that hardly reach 10 17 operations per second. More interestingly, such systems use an extremely small amount of energy, at least 10 10 times less than computers at the time. This work basically launched the field of DNA computation [START_REF] Nangreave | DNA origami: a history and current perspective[END_REF] . A lot of work was then realized to push the possibilities forward.

But the many drawbacks of using DNA to replace silicon computers, already perceived at the beginning, are not yet overcome. A molecular computer would require regular refueling and cleaning, and many of the current demonstrations are based on design that are not re-usable.

DNA strands need to be designed according to each performed operation, therefore components would be highly specialized. The error-rate of DNA replication further increases in vitro and may not be compatible with the high accuracy requirements of computation. And finally, the time scale associated with chemical operations in solution is much larger than with siliconbased computers.

With respect to information storage, the cheap -and ever decreasing-cost of DNA compared to silicon chips synthesis and sequencing, its stability over robustness in time and its high information density make it a very promising medium. In a human cell, 3 billion base pairs Even if molecular computers cannot compete with current silicon computers on calculation speed, convenience, or physical systems control, they have a clear advantage when it comes to interact with biological or molecular matter. That is why the molecular programming community is now targeting applications involving interactions with biology.

Conversely, one interesting aspect of building molecular programs is to better understand the underlying biological reaction networks, to create artificial models of these architectures. This corresponds to a learning-by-doing approach to better apprehend biological functions.

a. DNA hybridization is a reversible phenomenon sensitive to temperature changes, chemical agents or mechanical forces. Since the binding energy is different between the two base pairs, and considering the additivity of these energetic contributions, the dehybridization temperature of two single strands is highly dependent on its composition and the length of the paired sequences. The melting temperature of a duplex, corresponding to the temperature at which half of the DNA bases are hybridized and the other half is not, can be estimated for each sequence combination. This temperature is dependent on salt concentration, as salts interact with DNA and stabilize the duplex. Since the DNA backbone is negatively charged because of the many phosphates, it is highly sensitive to positive ions, especially Na + and Mg 2+ . Overall, in solution, a single strand DNA can recognize and bind to a complementary or partially complementary sequence, with various but predictable affinity. small DNA-based immobile junction structures using simple sticky end base-pairing to build complex multi-dimensional objects [START_REF] Seeman | Nucleic acid junctions and lattices[END_REF] . Many researchers then tried to construct specific architectures. Multiple-crossover motifs were discovered to give the necessary rigidity to assemble large objects and were used to build quadrilateral, cubes, and truncated octahedron.

Researchers then used branched DNA molecules (or 'tiles') to increase the size of the objects and construct one or two-dimensional periodic networks. Such networks can be used to precisely position proteins, nucleic acid strands, metal particles or quantum dots v on a DNA lattice. But these types of constructs do not possess finite sizes, so researchers then attempted to make finite structures using DNA scaffold strands [START_REF] Nangreave | DNA origami: a history and current perspective[END_REF] . In 2006 Paul Rothemund [START_REF] Rothemund | Folding DNA to create nanoscale shapes and patterns[END_REF] provided a new and relatively easy way to produce nanostructures of desired shapes using a long 'scaffold' strand and short 'staple' strands. Figure 16 illustrates the principle. Such scaffolded DNAorigami have high yields, few defects, fixed dimensions, precise shape and precisely defined positions in the lattice. Some computer-aided design tools have been designed for easy v Quantum dots are very small semiconductor particles, whose nanometer size confers specific optical and electronic properties. They can emit light of specific frequencies if electricity or light is applied to them.

production of these DNA origami with various shapes [START_REF] Douglas | Rapid prototyping of 3D DNA-origami shapes with caDNAno[END_REF] .

Outside of these structural capacities, nucleic acids, similarly to proteins, can bind specifically to other molecules or act as catalytic agents. The first observation of specific binding capacities was made in viruses. Naturally occurring small adenoviral RNA produced during a cell infection can bind and inhibit a cellular kinase involved in the cell's antiviral defenses. The human immunodeficiency virus-1 possesses RNA elements able to enhance interactions between a viral protein and a cellular one, thus improving viral transcription. [START_REF] Ng | Pegaptanib, a targeted anti-VEGF aptamer for ocular vascular disease[END_REF] The scientific community realized that DNA and RNA strands with specific binding capacities could be of great interest for many applications, especially in the therapeutic domain, because they are easier to produce and modify than proteins, could address a wide range of targets, not only the immunogenic molecules, and should not create humoral response [START_REF] Zhuo | Recent Advances in SELEX Technology and Aptamer Applications in Biomedicine[END_REF] .

Aptamers are nucleic acid oligonucleotides obtained by SELEX (see C.a, p.34 ) and selected for their high affinity and high specificity for various kind of other molecules, or even cells [START_REF] Wu | Aptamers: The "evolution" of SELEX[END_REF] .

Combining origami and aptamers, some researchers constructed a smart nanocage locked by aptamers interactions with a DNA strand that can be open thanks to antigen binding 89 (Figure 16). Thanks to SELEX and aptamers discovery, a new drug against ocular vascular diseases was developed and approved by US FDA in 2004 [START_REF] Ng | Pegaptanib, a targeted anti-VEGF aptamer for ocular vascular disease[END_REF] . SELEX also led to the discovery of DNAzymes or RNAzymes, catalytic oligonucleotides able, for some of them, to cleave substrate strands [START_REF] Fu | DNA-Based Dynamic Reaction Networks[END_REF] . Besides, because of the ubiquity of nucleic acids, some researchers believe that life could originally have relied only on these molecules. But many of these applications require some form of information processing. Therefore, embarked, molecular computation systems are required to allow sensible interaction between life and these DNA nanostructures. First proposed computation systems were exclusively made with DNA strands but then hybrid molecular programing system including RNA and few proteins appeared to solve some issues related to the exclusive use of DNA.

a.2 Molecular programs exclusively based on DNA

The major drawback of Adleman's demonstration of high parallel computation capacity of the DNA was the high number of laboratory steps required to access the result. Sakamoto et al. proposed an all-in-one protocol where they used DNA hairpins to solve an instance of the satisfiability problem vi 91 but hybridization fidelity issues and reaction kinetics were limiting.

In 2000, the team of Yurke created a DNA tweezer able to be closed and opened again by a strand displacement mechanism [START_REF] Yurke | A DNA-fuelled molecular machine made of DNA[END_REF] . The system is illustrated on Figure 17. Strand displacement is a competitive binding of a DNA strand with higher affinity to a target strand against another lower affinity strand. This mechanism is made possible by the base-pair breathing phenomenon. Indeed, stabilization of the hybridization process is only done by weak interactions (hydrogen bonds and stacking). So, each base-pair is continuously undergoing an vi A SAT problem is a logical propositional formula with n variables and the requirement to find a value (true or false) for each variable that makes the formula true. (Image borrowed from A logic-gated nanorobot for targeted transport of molecular payloads 89 .) equilibrium between the bound and unbound state. Therefore, some bases are temporally unbound, especially at the strand's extremities, and can be replaced by bases from a concurrent strand. This strategy led to the apparition of toehold-mediated strand displacement systems presented on Figure 17. In these systems, a strand with high affinity for a target strand is irreversibly displacing a partially complementary strand with lower affinity for the target [START_REF] Zhang | Control of DNA Strand Displacement Kinetics Using Toehold Exchange[END_REF][START_REF] Simmel | A DNA-based molecular device switchable between three distinct mechanical states[END_REF] .

The addition of one strand can thus liberate another one. We thus have a system where an input strand releases an output. The common region on the single strand target is called a toehold.

Researchers mainly use this system to recreate logic gates found in silicon-based computers.

AND, NOT and NOR gates were created, culminating in system able to play the Tic Tac Toe game [START_REF] Srinivas | Enzyme-free nucleic acid dynamical systems[END_REF][START_REF] Stojanovic | A deoxyribozyme-based molecular automaton[END_REF][START_REF] Stojanovic | Deoxyribozyme-Based Logic Gates[END_REF] . However, since the competitor strand must be longer than the released strand, it is difficult to use this approach to construct multilayer networks.

This implementation was then followed by a variation called the toehold-exchange strand displacement presented on Figure 17 [START_REF] Zhang | Control of DNA Strand Displacement Kinetics Using Toehold Exchange[END_REF] . This time the affinities of the two exchanged strands are similar and the process becomes reversible. The first strand replaces a second strand but can be then replaced by this second strand. This system allows an easier construction of downstream layer and quicker strand displacement reactions. Using this system, AND, NOT and NOR gates were also built [START_REF] Seelig | Enzyme-Free Nucleic Acid Logic Circuits[END_REF] . These DNA gates were then used in cascade to create more complex networks.

To allow proper functioning of cascades, restoration and thresholding systems avoiding non-specific or unwanted behavior were introduced, increasing the complexity of the system.

To address signal restoration issues, Zhang et al., proposed an entropy-driven toehold exchange system able to generate several output strands from a single input strand [START_REF] Zhang | Control of DNA Strand Displacement Kinetics Using Toehold Exchange[END_REF] . Leakage phenomenon, where part of the system starts in the absence of input signal, are often happening in molecular reaction networks because of reversible equilibrium, defects in strands synthesis, or imperfect hybridization. Several systems of toehold protection can be used to avoid this selfstart. One can also introduce mismatch in the sequences, or artificial bases can be used to generate toehold activation under photon exposure. A Stem-loop system preventing reactions to happen before any initiation was also presented [START_REF] Turberfield | DNA Fuel for Free-Running Nanomachines[END_REF] . An adaptation using hairpins with hidden complementary sequences allowed a chain reaction process 100 . This system called HCR for Hybridization Chain Reaction is triggered by an initiating strand (Figure 17). It can be used to detect molecules when combined with aptamers.

Using the toehold exchange tool, in order to free some hidden gates when the initially active ones can no longer be used. But keeping dynamic behaviors and time-responsiveness would require an energy source or constant degradation, removal, or renewal of the strands. This can only be achieved either by

working in an open system with constant input of new components, or relying on an internal source and sink based on a precise catalytic control of reaction rates. At present, this last option seems only feasible in the context of enzymatic systems.

In order to combine structural DNA nanotechnology and molecular computing, an approach using spatial organization of DNA strands as a mean of controlling circuit behavior (very much like in electronic devices) was recently published 106 . In this work, a DNA origami chip is used to immobilize hairpins. Distance between the hairpins controls the possibility of strand migration. When the distance is short enough migration can occur, but when hairpins are too far from each other the reaction is inhibited.

vii Artificial neural networks are computing systems inspired by the biological neural networks. Such systems "learn" to perform tasks by considering examples, without being programmed with any task-specific rules.

a.3 Hybrid molecular programs inspired from biology

All the above-mentioned methods were designed exclusively with DNA molecules and are principally used to design logic-gates or other silicon-computer-like computation systems.

Another branch of the molecular programming community is taking its inspiration from cell biology. This approach can have two goals. One is to better understand biological networks by having a bottom-up approach allowing to fully control the network architecture. The second one is the design of useful networks able to interact with biological or molecular matter. This approach is a bit different from synthetic biology, but the two fields can sometimes overlap. In the growing field of synthetic biology, researchers engineer paths and reaction networks using genes and expression regulation systems inside cells or in cell-free extracts. This field helped to give new functions to cells and created logic gates and computer-like circuits within cells.

However, using a living cell as a computing hardware is not as straightforward as it may seem.

The cell machinery is very difficult to harness and, because of interoperable regulatory elements, load issues or cross talk with housekeeping functions, non-linear effects and unintended behaviors are often observed.

Cell-free extracts contain all the necessary machinery for protein expression but are not constrained by the processes of life and growth. They can be either built by combining the different proteins and factors required for protein synthesis or by using bacteria (or other cells)

lysates. Advantages of using cell-free extracts is the absence of wall or membranes allowing to introduce any kind on desired molecules and the absence of necessity to keep the host cell alive while performing the computation. RNA strands corresponding to the encoded sequence. An RNase is also introduced to continuously degrade the RNA products and keep the system dynamic and out of equilibrium.

The genelets system developed by Kim et al. in 2006 (and also presented in Figure 18) is a closer analogy to in vivo genetic regulation system 109 , except that no protein is expressed and regulation takes place at the transcriptional level. Input and output strands are RNA oligomers, and only two types of enzymes, RNAse and T7 polymerases are used. As in the RTRACS system, the RNAse is used to degrade output RNA strands and keep the system out of equilibrium. DNA duplexes called genelets encode an output sequence downstrean of a T7

promoter. DNA activator strands can bind the genelet and complete the T7 promoter sequence, switching it from an OFF state to an ON state, from which the T7 RNA polymerase can start producing output RNA strands. To close the regulatory pathway, RNA strands dynamically produced by genelets interact with other nodes by binding and inhibiting their activator strand. This system allows to generate several RNA output strands from an initial input RNA using various templates and several enzymes; a reverse transcriptase, a RNAseH, a DNA polymerase and a T7 polymerase. At the bottom, the genelets systems is schematized. In this system, an activator strand completes a T7 promoter sequence enabling the production of several output RNA strands. Produced RNA strands can interact with other nodes by binding and inhibiting their activator strand.

The Polymerase Exonuclease Nickase Dynamic Network Assembly (PEN DNA) toolbox, first presented in 2011 110 by the team of my PhD supervisor Yannick Rondelez, is very similar to these systems but is only based on DNA and enzymes without RNA intermediates.

Further explanation concerning the PEN toolbox are given in part 2.

RTRACS, genelets, and PEN DNA systems use dNTPs or NTPs as an energy source.

Nucleotides can be seen as a fuel and the reactions are stopped once they are exhausted. One of the main drawbacks of these systems, according to the DNA computing community, is the very dependence on enzymes. They see it as a black box that cannot be fully understood or controlled as, contrary to DNA, there is no simple sequence-function predictions available for enzymes.

Therefore, a large part of the system is not directly amenable to rational engineering. In practice, this does not preclude modeling and predicting, as one routinely models the kinetics of enzymatic processes involving an enzyme and a substrate using Michaelis-Menten equations.

The situation can become more complex, but still numerically manageable, when enzymatic activities are shared between many substrates and inhibited by various products. and 'output' oligo strands are also more likely to just dehybridize dynamically by themselves and not requiring strand displacement activity of the polymerase that would generally slow down the reaction. This dynamic behavior also ensures that the output production rate is linked to changes in input concentration. All reactions with the PEN DNA Toolbox are run at constant temperature between 42 and 55°C, close to the melting temperature of the DNA duplexes so that both inputs, outputs and inhibitors are dynamically hybridizing and separating, given their melting temperature are lower than the working temperature. It is an isothermal process that doesn't require temperature change and can therefore be performed in a simple thermoregulated machine. The set of enzymes used is made of thermophilic enzymes and the length and melting temperature of the strands are adapted for this range of temperature. Using this relatively elevated temperatures allow to have manageable reactions time-scale.

Combining modules to create networks

Using these presented tools, various modules can be built with reactions following the pattern input→ input + output. The Figure 20 gives an overview of the different reactions we can combined in a molecular program that are presented below:

• Autocatalysis: On the autocatalytic template, the produced strand (output) has the same sequence than the trigger (input). Thus, cycles of polymerisation/nicking lead to the exponential amplification of the trigger. This mechanism can be referred as an EXPAR (EXPonential Amplification Reaction) in some other works 114 .

• Activation: On an activation template, the produced strand has a different sequence than the trigger. A new strand is therefore produced thanks to the binding of the trigger. This mechanism can be referred as an NEAR (Nicking Enzyme Amplification Reaction) in some other works 116 .

• Inhibition: The inhibitor strand of generally 15 nt is partly complementary to the template. It can bind on it but its 3' side is not matching, preventing from polymerase elongation. Its sequence is determined so that it can form stable duplex with the template with enough binding affinity to displace the normal input trigger. Inhibitors do not have recognition site for the nicking enzyme and cannot be cut. This inhibitor strand therefore hinder strand production and is repressing the production of output strands.

• Deactivation: The deactivation module is based on a pseudotemplate 117 . The pseudotemplate, upon binding of the cognate trigger, drive the addition of a few bases, thus preventing its use by other templates (i.e. the deactivated trigger can bind to its cognate template but can no more be used as a primer because of the additional mismatching nucleotides). The nickase cannot cut the added tail, thanks to modification on the template recognition site changing a T by a U. The spontaneous release of the deactivated trigger restores the pseudotemplate.

Hence, the pseudotemplate can be seen as a catalytic sink that is able to absorb a certain flux of trigger. It provides an easy way to adjust the linearities of the degradation process of a specific DNA strands (by opposition to the exonuclease that degrades unspecifically any unprotected single strand of DNA). This deactivation module is particularly suited for preventing nonspecific amplification occurring with an autocatalytic module. Indeed, it has been shown an autocatalytic template in presence of the polymerase and the nickase eventually exponentially amplifies triggers, even in absence of the initial input 118 .This is due to spurious reactions that initiate the production of a few output strands, enough to trigger the -uncontrolled -amplification. By absorbing these undesired triggers, the pseudotemplate delayseven cancelledthe selfstart of the autocatalytic node and allows for good prediction of the molecular outcomes of the DNA circuits.

• Degradation: This module corresponds to the degradation of produced strands by the exonuclease. This module can be removed if one does not need a program that stay out of equilibrium.

• Detection: The detection module is variable and can be used to detect molecules or enzymes activities external to the molecular program. Theoretically every activity that would directly or indirectly act on DNA should be detectable with a strand build to produce a trigger or turning into a trigger when modified. On a same manner it is also easy to detect DNA or RNA strands by simple complementarity with the starting part of a template. For all other types of molecules or activity chemical modification of the template can be done as well as using aptamers that are short DNA strands able to bind to any kind of molecules. In this module, a recognition element of the target molecule is introduced to convert the activity or possible interactions into a trigger production.

All the reaction's incoming and outgoing products are short oligonucleotides, so these components can be arbitrarily connected by choosing the right sequences. The output of one template can simply become the input or the inhibitor of another template. By combining all these different modules as nodes, various networks can be built. One can easily design a set of template sequences that would encode the different modules without having any interaction between the templates. The short size of the templates limits the number of nodes you can add in a program built with the PEN DNA toolbox but gives enough possibility to make quite complex systems. Some basic rules need also to be respected, e.g. for the melting temperature of the strand, or the nickase recognition and restriction sites. According to the type of nickase the restriction site can be outside or inside of the recognition site, modifying the template design. Secondary structures should be avoided on the designed strands. This can be checked on DNA/RNA secondary prediction software like NUPACK 119 . Thermodynamics parameters adjusting length or GC content can be computed and tuned using well-known algorithms.

Kinetic parameters are controlled by enzymes concentrations or adding toehold sequences 120 .

dNTPs are used as fuel in the networks, they are the initial material used by the polymerases to produce the oligo strands. If the reactions run out of dNTPs the programs cannot continue to work therefore in a close tube, there is a limited amount of time to run the molecular program.

Computer tools have been developed to assist network building

Software like the DNA Artificial Circuits Computer-Assisted Design (DACCAD) 121 have been developed to provide support in the design of the networks. It can assist the design by predicting what will be the outcome of a given network. This is made possible by building on the experimental knowledge available on the DNA toolbox (enzymatic kinetics, thermodynamics of hybridization reactions…). The mathematical models implemented automatically by the software DACCAD provide a mathematical description of the system that is as accurate as possible. One can therefore use DACCAD to check if a set of nodes gives the right output. On the contrary, if one has no clue about the network structure that gives the targeted behavior, an in silico method 122,123 has been developed to generate a network matching the output function. In this approach 122 an evolutionary algorithm search for a network matching a desired function. It thus predicts the diverse types of nodes one will need. In another method 123 , using network topology and targeted behavior, a biochemical network of arbitrary complexity can be screened for parameter values that display the desired function. Moreover, this program can generate optimal DNA sequences with key characteristics extracted from mathematical analysis of the network. After using all these tools, the modules must be tested and assembled in a bottom up manner to check that they are providing the expected output 124 .

Figure 21 gives a global scheme of how these different computational approaches can be combined to design a PEN DNA Toolbox network.

Chemical modifications of template strands

Besides the sequence of the templates, chemical modifications may or must be added.

Generally, templates have to be protected against exonuclease degradation so that the encoded program continue during the time of the experiment. A few phosphorothioate bond modifications on the 5' extremity of the template is enough to prevent degradation by exonuclease. Other modification such as fluorophore, quenchers, spacers can also be added to allow the follow up of the reactions. Figure 22 presents a list of the possible and generally useful modifications.

Monitoring of networks reaction by fluorescence

Reporter modules can also be added to the molecular program to assess strand production. Mainly, three techniques can be used to monitor strand productions and networks functioning.

• Intercalating dyes: Introducing fluorescent DNA intercalating dyes like EvaGreen® or SybrGreen® allow to follow the global quantity of produced single-stranded and double-stranded DNA. With increasing concentration of DNA, the dyes fluorescence will increase and can be followed on real-time PCR machine but just provide sequenceunspecific monitoring.

• Reporter strands: To have a follow-up of the concentration of one species in the reaction reporters can be designed to emit fluorescence when bound to a specific strand. Diverse types of probes can be designed but they all consist of oligonucleotides dual-labeled with a fluorophore and a quencher, whose quenching efficiency relies on the distance between the two chromophores. Any binding event that will induce a change in the Spacers can be added between the sequence and added element to avoid steric clashes that could prevent strand polymerization completion. It can also be used to concatenate sequences. Biotin at the extremity, in combination with streptavidin in the medium. Biotin streptavidin interaction will prevent strand degradation by polymerases. It can also be used to attach template on a streptavidincoated support.

distance will therefore modify the fluorescence intensity. Binding of the specific species on the reporter is generally pulling apart the quencher and fluorophore provoking an increase in fluorescence intensity. Depending on the goal of the study, one system will be preferred over another. But since they are capturing some of the produced strands and sometimes require the action of some enzymes used by the molecular programs, these reporting techniques can interfer with the general network.

o Random-coil probe: It refers to a randomly folded sequence that brings together the quencher and the fluorophore attached to their extremities.

Upon binding of the targeted strand, the duplex become stiffer, results the separation of the two dyes.

o Bimolecular quenched probe: Similarly to template/trigger design, two strands can be designed to hybridize with one containing a fluorophore and the other one a quencher. Binding of the target will generate the departure of one of the strand because of a better affinity, or after displacement by the polymerase.

o Hairpins quenched probe: Finally, hairpin loops maintaining quencher and fluorophore together can be used and will be opened by the binding/extension of the target strand. According to the sequences design and enzymes used all these reporting systems can be either reversible, meaning the fluorescence signal will go back to its original state when the species is disappearing from the reaction, or irreversible, meaning that the fluorophore and quencher are permanently dissociated after binding of the target.

• N-quenching: The last option is to follow the loading of the template strands by the input strands. For this purposes nucleobase quenching 125 can be used. It relies on the quenching or enhancing effect of nucleotides bases on proximal fluorophores. In this approach, the template carries a fluorophore on its 3' extremity. Hybridization of the input strand will produce a change in the fluorescence level because of the interaction between the 5' base of the input and the fluorophore. An increase or a decrease in fluorescence is observed depending of the fluorophore and its surrounding nucleaobase.

We can therefore follow the input loading on the template. This technique minimally affects the network, but still has some detectable effect on the kinetics.

The Figure 23 summarize all the different type of reporting system that can be used to monitor strands production in the network reaction.

Achievements of the PEN DNA Toolbox

The PEN DNA Toolbox have already been used to produce or study various networks.

The first program built with the PEN DNA Toolbox, nicknamed Oligator 110 , was encoded with or hairpin loop reporters (blue box) can detect target strands in the reaction. All these methods rely on separating a quencher and a fluorophore in presence of the target strand. According to the design the reporting can be made irreversible by for instance adding restriction endonucleases like in the irreversible reporting method describe in the dark blue box. Similar design can be done for annealed strand. These kinds of reporting are adding a load to the network because they use or capture transiently a part of the produced input. Furthermore, when stabilization by the polymerase is required, additional load appears due to enzyme activity sharing.

3 templates and produces sustainable oscillations (up to 40 cycles) for several hours in a closed system. This remarkable achievement was also supported by a convenient way to mathematically simulate the outcome of the network because of the relatively small number of implied DNA strands and enzymes as well as the ease to characterize the different involved kinetic and thermodynamic parameters. In a second implementation 115 , the PEN DNA Toolbox was successfully used to create a bistable system able to be in state A or B according to the initial amount of input strands α or β introduced in the system. It was successively transformed in a two-input switchable memory element where the circuit could be change from state A to B by addition of some input strand by adding extra nodes. Finally, a computer system-like singleinput push-push memory circuit was built, able to switch consecutively from state A to state B state using the same input strand. The toolbox was then used to mimic naturally occurring phenomenon and proved to be a very efficient modeling tool. In 2013 the classical Lokta mathematical equations of predator-prey ecosystem was encoded in molecular templates 126 to study the specific dynamics of ecological communities. Typical ecosystem behaviors were observed for the first time at the molecular scale like predator-prey oscillations, competition induced chaos and symbiotic synchronization. More recently the toolbox proved to be a powerful tool for mimicking the process of embryo development pattern formation by morphogens controlling cellular fate 127 . It was even possible to observe physically differential change in the various stripes thanks to beads aggregating because of staple production. This work is using bistable circuits designs similar to the ones used in the switchable memories and relies on the creation of a gradient of pseudotemplates and reaction-diffusion principles. Indeed spatio-temporal waves and spiral of strands production related to the aforementioned predatorprey system was already observed in 2013 128 .

The visualization of differential physical modifications along the French flag pattern on beads was made possible by another previously started work also recently published 129 emulsions with dNTPs and primers matching the Taq polymerase gene. Each droplet containing a bacterium is a compartment in which the Taq phenotype and genotype are linked, even if the bacteria is lysed. By performing a PCR in the droplets, Holliger's team ensures that the best mutants will replicate their genotype exponentially and faster than the inactive or less active mutants. Unlike in the selection-by-binding methods, the good genes are not just recovered but will be amplified according to their fitness.

This approach brings two more advantages to the selection by survival techniques. The first one is that the amplification of good genes is really strong compared to the dynamics of reproduction. In just few hours, the genetic population is greatly enriched in favorable mutants.

Of course, this comparison is highly dependent on the type of organism replicating and the selection design. But, CSR techniques or assimilated ones can lead to a 100-fold enrichment per round, leading to substantial narrowing of the initial library pool in few cycles [START_REF] Ellefson | Directed evolution of genetic parts and circuits by compartmentalized partnered replication[END_REF] . On Table 4 we exposed the different amplification yields according to selection techniques.

The second important advantage is that making the link between enzyme activity and gene replication entirely in vitro opens up the scope of possibilities regarding the tests that can be performed before selection. Indeed, once the bacterium is cracked open setting the enzymes free, they can be tested in various environment conditions or using any molecular machinery.

In this way you keep the ability to replicate DNA as in a survival selection, but it does not require the organism to stay alive in the process. This point is very relevant because in all survival selection methods the pressure put on the organism to survive can result in bypass other than using the ability of the carried mutant. And conversely, survival of the organism can apply hidden selection processes on the evolving mutant. Effectively, organisms are complex with many interacting parts and various parameters can alter the general organism fitness. Such cheating behavior are less likely to happen if there is no, or few pressures applied on the organism carrying the library. It is also possible to evolve the enzymes to work in non-natural conditions or towards new functions or catalytic reactions that cannot happen inside of an organism. That's actually what was done in the 2001 paper where Taq polymerase resistant to high concentration of heparin was found [START_REF] Ghadessy | Directed evolution of polymerase function by compartmentalized self-replication[END_REF] but also in a later published paper where polymerases were evolved to incorporate chemically modified fluorescent bases 134 . Finally, the discontinuous nature of the selection process offers an element of control on mutations introductions and selected mutants that limits parasite accumulation and avoids foreign mutations affecting the selection.

Using molecular program as a feedback loop to increase CSR adaptability

CSR has already been declined in several ways like the Compartmantalized Bead

Tagging 135 or the Compartmentalized Ribosome Display 56 to evolve polymerase activity. As interesting as this CSR method can be, a big remaining limit is its specificity on the type of enzymes that can be evolved, mostly the polymerases. Polymerases evolution is a very active domain with a lot of applications in biotechnology, but it would be very nice to adapt this method to other kind of enzymes. This is where the PEN DNA Toolbox comes in. The goal of my PhD project is to provide the first demonstration of the use of molecular programs build with the PEN DNA Toolbox to link a new kind of enzyme activity to a PCR yield in droplets compartments. The toolbox molecular networks are designed to produce short oligonucleotides interacting within each other, so they can generate short oligonucleotides at the output.

Conveniently, genetic amplification and especially PCR require also short oligonucleotides as primers to start the replication of a specific part of DNA. PEN networks can therefore be used to produce primers for a following PCR step. In a PCR the yield should be dependent on the primer's concentration, having more yield for higher primers concentration. So, if, through molecular programming, one links the enzyme activity to the primers production rate one can easily add the feedback loop required for a compartmentalized self-selection of the enzyme. Any enzyme that can directly interact with the molecular program could be evolved with this method, like the nickases for instance. This can also easily be transferred to any enzyme working on DNA substrate. But detection modules can also be designed to allow interactions with other kinds of enzymes, for instance aptamers or hybrid DNA-substrate molecules could allow to select for a various kind of activity, greatly extending selections beyond enzymes acting on DNA.

To put it simply we plan to introduce in the CSR method an additional module comprising a detection and processing block, to adapt it to various kind of enzymes. The idea presented in Figure 24 is to evaluate the enzyme's activity at the input and generate the primers needed for its own gene amplification step at the output. Combined with a PCR step in the presence of a PCR polymerase and dNTPs, such a process will allow to control enzyme's gene amplification by the input activity. The success of our method would promise interesting use of various existing DNA computation networks for directed evolution with the possibility to extend the method to all kind of proteins relatively easily.

Advantages of molecular programs designed with the PEN DNA Toolbox

Using the PEN DNA toolbox to create selection function for directed evolution present many other advantages.

❖ Selection performed completely in vitro

Besides this ongoing work, another attempt based on this same idea to adapt the CSR to other kind of enzyme have been developed in the Ellington Lab and called the Compartmentalized Partnered Replication (CPR) (see D.b, p.45). In this modified version of the CSR genetic circuits or partner genes are introduced in E. Coli bacteria to control the expression rate of the Taq polymerase. The CPR report showed that it is indeed possible to link another kind of enzyme than polymerases to gene replication in vitro but the direct application is still limited to enzymes or proteins related to gene expression. They also didn't take advantage of the capacity offered by the technique to realize in vitro selection tests as the feedback loop is introduced in the bacteria and selection pressure is applied in vivo inside of the bacteria. It is however interesting that using CPR one can select for a gene part in vivo without the need to simultaneously select for organismal fitness. And the short duration of the in vivo functional selection limits fitness effects on the organism, which could disadvantage highly-active genetic circuits. This method is therefore good for any synthetic biology related purpose because the enzyme is evolved in the context in which it will be used. However, designs of enzymes working in non-natural environments or performing xenobiological reactions suited for biotechnological applications or to create new chemistry processes is not possible.

In the PEN DNA Toolbox approach, the molecular program is external to the cells and can be introduced at the droplets generation time. It would allow to perform fully in vitro selection tests and will not require extra bacteria cloning like in the CPR. Like in the CSR a lot of selection parameters can be fixed freely in the droplet supporting evolution of non-natural reactions. Performing the selection reactions outside of the cell is relieving the organism internal selective pressure and limiting interactions with inner cell processes. Also because of the foreign nature of the molecular program and the absence of need to use the cell machinery the risk of having unexcepted interactions that could lead to primers production is very unlikely.

False positives are therefore less likely to be selected in contrary to the CPR method where Taq polymerase production could be triggered by other inner cell components or expression noise.

❖ Adaptation to various enzymes

Adaptation of the CPR to enzymes with no direct influence on the expression can be considered but would require a lot of time-consuming cloning steps that would need to be very precisely designed to avoid host's genes interactions. Comparing to CPR, using molecular programs is increasing again the adaptability of the method in an easier fashion. Many detection modules can be envisioned to detect any biological activity producing short trigger strands for the processing step that would lead to primers production.

❖ Fine tuning of the selection

An in vitro molecular program is accessible and can be tuned by just introducing new template strands which make it mere to program, test and modify compared to the use of genetic constructs in vivo. In the Programmable External Network Compartmentalized Self Replication (PEN CSR) approach, the fitness selection function can be tuned very precisely using the different PEN DNA Toolbox modules. Complex networks can be designed to take into account many aspects of the catalysis. With such ability the PEN CSR method combines one of the main advantages of the screening techniques, which can potentially provide rich informative data, with the fast turnover of the CSR. Indeed, screening methods allow to measure different parameters and to choose the mutants according to many criteria like for instance the catalysis speed, the binding affinity or enantioselectivity. For instance, for the directed evolution of the artificial Kemp eliminase, the authors made several screenings with the enzyme and different substrates to select the mutant that efficiently catalyzes the first reaction but can also use another substrate [START_REF] Khersonsky | Bridging the gaps in design methodologies by evolutionary optimization of the stability and proficiency of designed Kemp eliminase KE59[END_REF] . Using artificial networks, one could build an AND gate for instance to select for an enzyme able to catalyze a reaction over two different substrates or to bind a particular type of substrate but not the other one to increase specificity, thus facilitating the process. Screening acquired data can also help to choose appropriate stringency for subsequent rounds of evolution.

In the same manner a finely designed molecular program could do the same without any external intervention by setting molecular activity threshold and combining imperatives. Using artificial networks also should allow to perform dynamic selection. The method is not restricted to binary approaches, the catalytic rate can be taken into account. It does not necessarily rely on the choice of a specific threshold; any activity level can be selected more or less, according to the chosen network design and the shape of the programmed selection function. A network can be build requiring a minimum activity level to start producing primers or to smoothly select every activity according to enzyme fitness.

❖ Fundamental study on selection pressure effects on evolution

On a more fundamental point of view, tunable selection function can allow to study the effect of the selection function or pressure on the evolution itself. What is the best fitness selection function shape for a desired goal? Is it better to have a high stringency to select for thermostability or a smoother curve that will select also not so improved mutants? What about catalytic efficiency improvement? Is it more efficient to select all the mutants having a minimum efficiency with the same amplification rate or is it better to have an amplification rate directly proportional to the activity?

❖ Size-controlled droplets

Water-in-oil droplets allow to create the compartments isolating library variants contained in bacteria linking the genotype and phenotype. Contrary to the CSR and CPR methods where emulsions are generated by shaking, selection using molecular programs will be run in droplets generated using microfluidics. Droplets generated by microfluidics are monodisperse. Calibrated droplets allow to better control evolution as all reactions are carried in similar compartment so with the same conditions. Nowadays microfluidic devices can generate water-in-oil droplets with relatively high throughput matching with directed evolution requirement.

To conclude on this part, the Compartmentalized-Self-Selection method that we propose to develop here will allow to beneficiate from all the advantages of the CSR method for a wide range of enzymatic activities combined with fine tuning of the selection parameters by molecular programs:

• High throughput method

• Fast high fold enrichment of the most active mutants

• In vitro selection test opening to new chemistry and reducing organism interactions

• Easily tunable, accessible and programmable selection function

Droplet selection, although being a powerful approach, restricts slightly the parameters that can be fixed in the selection and therefore the condition we can test. A consensus buffer must be found to run molecular programs and PCR in the same compartment. Moreover, resistance and well-functioning of the enzymes on which the selection process is relying in nonnatural conditions will also limit the scope of available selection conditions. DNA molecules being quite robust, stability of the PEN DNA Toolbox enzymes in an unfamiliar environment will be the major restraining point. For this reason and also the fact that its activity is already recognition sites are close to each other which is a type of arrangement that appears four times in bacteriophages T7, due to its presence in the T7 promoter sequence. However, sequencing of these nicking enzymes revealed a 100% sequence identity between them, meaning that the different strains could be a unique worldwide spread strain or that they are the results of horizontal gene transfer.

Currently, 5969 nicking endonucleases are referenced on REBASE, a database collecting all the information about restriction enzymes. Out of the 5969 nickases, only 36 are not putative and have been clearly identified. Only 15 are commercially available 137 . Since 2003, a nomenclature rule has been decided to distinguish between two types of nickases. In addition to the one from the NBI family designed with names starting with a N, a second group named with a V as a first letter corresponds to endonucleases associated inside cells only with m5C DNA methyltransferases. The V letter comes from the best studied enzyme of this type the "very short patch repair" (Vsr). They introduce a nick near mispaired G-T resulting from cytosine deamination. This second type of enzyme is very particular and does not share common features with restriction endonucleases. Yet, they represent the majority of putative Its recognition site is GAGTC and when bound it can cut the top strand 4 bp further. One particularity of NBI is the presence of three negatively charged residues in its catalytic site, contrary to classical restriction enzymes that usually possess one positively charged residues. The origin of NBI was proposed to either i) come from an homodimeric enzyme having lost its dimerization capacity or ii) be the large subunit or a heterodimeric restriction enzyme. The second option seems to be the more probable. Like classical restriction enzymes, NBI is part of a Restriction-Methylation (R-M) system. The related methylase recognize the palindromic site GASTC and methylate the adenosine.

Restriction enzymes with the same recognition site have been used to better understand NBI particularities.

nicking endonucleases discovered so far.

The N type of nicking enzymes might be a particular case in the evolution scheme, as they do not seem to have an identified function. They could as well be a separate type of restriction endonuclease or have evolved from Type IIs endonucleases and lost their ability to cut both strands. Interestingly, NBI has sequence similarities with 2 mesophilic Type IIS The associated restriction endonuclease is not able to cut DNA methylated at the level of its recognition site. This ensures that the DNA of the host cell is protected against restriction, but foreign DNA, newly entering the cell, will be cleaved. R-M systems are dispensable to bacteria that can survive without it but a survey on 10 000 bacterial strains found R-M systems in more than 25% of the strains. Strains can have numerous R-M systems, for example more than 20 putative systems have been found in Helycobacter pilori. showed that the small protein was not binding to DNA, neither forming a complex with the N.BspG6I protein, but when the two enzymes were present in solution, the nickase activity was changed and both strands were cut. This indicated that this small protein was indeed a small subunit of an heterodimeric restriction endonuclease named R.BspD6I. Two other heterodimeric endonucleases, BtsI and BsrDI, having a large subunit exhibiting nicking activities in the absence of the small subunit were discovered in the following. The crystal structure at 1.8 Å resolution of the large subunit N.BspD6I whose sequence has 100% identity with NBI was also acquired during this study and confirmed the previously depicted domains: In could be interesting to target the blue part that seems to interact with DNA to increase the velocity of the reaction. In C, Superposition of NBI, Ple1 and Mly1 modelled structure with active site residues. Blue: NBI, Purple: Mly1, Beige: Ple1. These two enzymes are mesophilic and work at 37°C so we can compare their overall structure to try to find the part responsible for the higher thermostability of NBI and try to enhance it. Molecular dynamics simulation at different temperatures could be used to find the residue that are destabilized in Mly1 and Ple1 but not in NBI to apply site directed mutagenesis. But error prone PCR can also be a simpler option that can be allowed thanks to the high throughput of the method. This structural information can help in the directed evolution process.

a N-terminal (302 aa) binding domain, a linker (303-380 aa) and a C-terminal catalytic domain (381-604 aa). Similarity of this structure with the restriction enzyme FokI that also cleaves DNA aside its recognition site can give more insight in the enzyme function. FokI has been crystallized in complex with DNA and similar interactions can be envisioned for NBI. D456

and, E469, previously predicted to be part of the catalytic site, as well as P455, V470, and E482

were assigned to the catalytic center. The small subunit structure was also determined, this time with a 1.5 Å resolution and displayed high similarity with the C-terminal domain, despite low amino acid sequence homology. These data suggest two possible routes of evolution: either the duplication of the catalytic domain yielding the small subunit, or the deletion of the DNAbinding domain on one of the ancestral subunits 140 . An interesting particularity of this small subunit, and the nickase active site was that all three amino acid residues are negatively charged, unlike in most restriction endonucleases that generally possess one positively charged lysine.

Before this, only BamHI was known to have three negatively charged residues in its active site.

The rich information obtained with this structure can help us to guide the evolution process if needed.

Outside of this structural information, two small different adenine methyltransferases were isolated simultaneously to NBI. One sequence of 301 amino acids is in frame with the enzyme and would correspond to the putative ancient R-M system. The other one is not in frame and is thus not thought to be part of the system. The two methyltransferases have obviously the same target site but are from different families. Their target sequence is also the same as the methyltransferases associated to pleI and MlyI. Contrary to ordinary type IIS restriction systems, which usually require two associated methylases able to methylate one site and its asymmetric counterpart, here the methylase can act on both strand as its target is GASTC, with S = G or C. These methyltransferases are of particular interest for the difficult expression of the enzyme in bacteria, as discussed below.

b. Current usages

• Isothermal amplification

Because they generate 3' free ends available for extension by polymerases with strand displacement activity, nickases can be used for isothermal amplification. Thanks to the elongation by the polymerase, the nicking site is regenerated and the nickase can initiate a new strand polymerization. Thus, one template can be used to produce many strands. The main advantage of such amplification method is that they can be performed at a constant temperature and therefore do not require instruments for cyclic temperature regulation. Another advantage is that they do not require DNA denaturation step that can sometimes degrade the DNA. These isothermal amplification mechanisms can be initiated by an initial DNA melting step and primer annealing or by using two types of primers, one primer type containing the nicking site and another outer primer used to leverage the strand containing the nicking site. Thus, allowing to generate double stranded nicking sites. Another mean to keep amplification going on is through extension around a circular target. In sequences already possessing the nicking sites, a simple nick of the double strand can allow to create a starting point 143 .

The EXPonential Amplification Reaction (EXPAR) is used as the central PEN DNA toolbox output strands production mechanism. A short trigger is used to prime extension followed by nicking on a template strand. The amplification can be linear, sometimes being referred as the linear Nicking Enzyme amplification reaction (NEAR) 144 , or exponential, if the output strand has the same sequence as the trigger. The exponential mode can reach an amplification of more than 100-fold in under 5min. EXPAR is generally used to amplify very short sequences that sometimes do not even necessitate the use of the strand displacement activity of the polymerase. Because of the short strand affinity for the template it can itself unbind from the template at the reaction temperature of 55°C 145 .

Nickase can also be used to initiate Rolling Circle Amplification (RCA) without the need of primers 146 . In the classical RCA process, the template strand is circular so the polymerases keep copying continuously the template by strand displacement, creating concatenated complements of the circular template. Primers can eventually bind on the circular DNA, and then on the produced single strands. Strands amplified by the polymerase will be also strand displaced generating new primers binding sites and therefore exponential

x A single-nucleotide polymorphism (SNP) is a variation in a single nucleotide that occurs at a specific position in the genome, where each variation is present to some appreciable degree within a population.

amplification. The nickase can be used either before the reaction to create a starting nick facilitating the reaction start or during the RCA to generate multiple starts and an exponential amplification mechanism without primers.

Figure 29. Nicking-based molecular biology applications. EXPAR allows exponential amplification of a trigger strand. This reaction can be combined with another allowing to generate the trigger from a target sequence to be used in diagnosis, of generally target detection. NEAR is similar to the EXPAR but the reaction is linear as the produced strand is not the same as the trigger strand. SDA is using two nicking sites at each extremity of the target sequence to amplify the DNA without the need of denaturing. Outer primers are sometimes necessary to generate the starting strands. All these methods are based on nicking and extension of the produce 3' extremity by a strand displacement polymerase. Similarly, RCA is based on a circular template that allows to create sequence concatenates. Detection methods of target sequences containing a nickase site, like NESA, can be done using fluorescent molecular probes. DNA labeling can also be performed by removing a part of a strand thanks to dual nick introduction. The created gaps can be filled with probes, priming strand or filled by polymerases (not represented).

Another exponential isothermal amplification method by strand displacement conveniently named Strand Displacement Amplification (SDA) is also based on nicking and strand displacement but this time on a linear strand 147 . This method uses two primers adding nicking sites ate the strand extremities. After a first heat denaturation step the primers bind the target and are extended by the polymerase. The introduced nicking site are nicked and generate new single strands that can be bound by other primers. The original implementation of this method was using classical restriction enzymes with some practical limitations. A second implementation used modified αSdATP to partially protect the HincII site. HincII can behave like a nickase and is able to introduce a nick in semi-modified sites when only one strand is modified. Use of an authentic nickase eases the process, mainly by avoiding the use of αSdATP.

• Target sequence detection All these amplification methods can be used to amplify the signal of a target sequence and provide rapid and low-cost diagnosis tools.

For instance, EXPAR, which is very sensitive and can achieve over 10 6 times signal amplification, could be used for clinical diagnosis. Some researchers proposed to use a reaction called fingerprinting, involving single-strand nicking at adjacent nicking recognition sites in genomic DNA sequences, to generate short DNA pieces usable as triggers to start the EXPAR 118 . Suppressing the early phase background amplification, they manage to detect trigger strands at attomolar concentrations.

The Nicking Endonuclease Signal Amplification (NESA) method can be used to detect specific single stranded DNA sequences or double stranded DNA sequence with addition of a denaturation step 148 . This signal amplification method relies on the use of a probe matching a target sequence comprising the nicking site and carrying a fluorophore. When the probe anneals to the target, the nickase binds on the probe. The two parts of the probe detach because of affinity reduction, while the target is left intact. Free target can bind another probe and therefore several signals can be generated from one target. Amount of cut and uncut probes are then analyzed by gel electrophoresis. Using a hairpin molecular beacon as Zheleznaya et al. did in a similar method allows to directly observe a fluorescent output. In their method, the hairpin probes contain the target sequence, comprising the nicking site, in its loop and a fluorophore and a quencher at each extremity brought together in the closed conformation. Once the target binds and opens the loop, the nicking site is available and the quencher and the fluorophore are irreversibly separated by the nickase leading to an irreversible enhancement of fluorescence.

These methods require the presence of the nicking site in the target sequence, but due to the relatively frequent appearance of the nicking site (for example NBI site on average every thousand base pairs in random sequence), this is not a strong limitation. Similar mechanisms can be used to detect RNA. Once opened by the RNA target, a hairpin DNA strand can expose a previously hidden sequence that can bind the DNA probes used in above-mentioned methods.

Finally, in the primer generation RCA (PG-RCA) 149 , a circular single stranded probe containing a nicking site can bind partially a target on one part of its sequence. The target plays the role of the primer and the RCA of the circular probe is triggered leading to accumulation of detectable large quantity of DNA. This sensitive system can reportedly detect 60 molecules of a specific genomic DNA type.

• DNA labeling

Nickases can also be used in DNA labeling techniques. Using two nickases whose restriction sites are relatively close (15-25 bp), one can remove a single stranded part of a DNA duplex. This approach can allow to then use oligonucleotides able to bind or not to this region according to sequence specificity. Such method can be combined with the PG-RCA. A team of researchers used oligonucleotides binding to this gap but having a floating 3' extremities used as a primer to perform PG-RCA with a circular probe 150 . This allows to detect labelled DNA strands. Nickase-induced gaps can also be used to introduce labelled oligonucleotides in a DNA sequence. Single nick and incorporation by a polymerase of fluorescent nucleotides can also be used to map chromosomes observed by microscopy 140 .

• Site-directed mutagenesis and cloning

Cloning of PCR fragments into a vector is traditionally performed using restriction enzymes to create cohesive ends but the short size of these overhanging parts require the use of a ligase. Introducing a nicking site in PCR fragments primers and using nickases, cutting aside of their restriction site, overhangs of any length can be generated. This allows a single annealing procedure between PCR fragments and the vector with no need for ligation 142 .

Using nickases to introduce single stranded gaps in the DNA can allow site directed mutagenesis thanks to oligonucleotides insertion.

• Other applications

Strand-specific nicks generated by nickases have been shown to be able to induce c. Nickase as first target for the PEN CSR evolution process

Nicking enzymes are interesting targets because it is difficult to find them in nature

NBI was chosen as the first model for the herein proposed directed evolution process.

As nicking enzymes were discovered only recently, they remain quite mysterious and study their structure-function relationship is of major interest. Besides, as explained earlier, nicking enzymes are very interesting tools for molecular biology. However, one major defect that slows down the widespread use of this class of enzymes is the small number of different enzymes with different specific recognition site. New recognition sites can allow to expand the use of detection methods like NESA, which requires the presence of the nicking site in the target sequence. It can also allow to generate various fingerprints to detect sequence that are now out xi DNA sequencing method based on the "sequencing by synthesis" principle, relying on light detection based on a chain reaction when pyrophosphate is released.

of reach for nickase-based systems. And many other applications would beneficiate from it.

One explanation to this relatively low diversity is the difficulty to isolate such nicking activity from natural sources 142 . Based on the discovery that NBI, or its exactly similar protein Nt.BspD6I, was actually the large subunit of an heterodimeric endonucleases some new nickases have been found by studying the large subunit of some already known heterodimeric endonucleases. But registration of such activity in bacteria lysate is rather complicated.

Traditionally, restriction endonucleases are first detected by incubating substrate DNA with bacterial lysate. Then purification of the different putative targets is done, and their activity tested again against the DNA substrate. In the case of heterodimeric enzymes with unit of different size, the two subunits are separated since they just interact in presence of DNA. Thus, the activity is not recovered, and experiments are usually stopped. Using the gene organization of R-M systems can also help to find restriction enzymes and their composing subunits but as it is not the same in all bacteria it is also a not straightforward mean to discover new nickases.

Furthermore, most naturally occurring Nicking enzymes have been found in bacilli strains, letting researchers wonder if it is not an organism particularity.

For these reasons, researchers decided to engineer nicking enzymes starting from restriction enzymes. The goal is to obtain nicking enzymes with new recognition sites, and thus fully exploit their potential. Nowadays, most commercially available nickases are engineered ones.

New nickases can be obtained by protein engineering

Several methods have been used to engineer nicking enzymes.

• Homodimeric restriction enzymes

The first record of nicking activity engineered from a restriction endonuclease was made in the team of Pingoud. The target enzyme, EcoRV, recognizes a palindromic sequence.

Therefore, even if nicking activity was indeed detected, the mutant could still cut both strand without distinction 154 . The first real engineered nickase resulted from the site directed mutagenesis of MlyI, preventing its dimerization 155 . The obtained N.MlyI enzyme recognize the same site than MlyI, and therefore NBI, and cut the top strand outside of its recognition site, but the distance was not determined. This success did not yield a new recognition site, but showed that mutating dimerization amino acids in a type IIS restriction enzyme could be used as a general process to discover new nicking activity. Based on this same idea of preventing dimerization, a second approach based on a full domain exchange allowed to generate N.AlwI.

This time, the c-terminal domain of the AlwI restriction endonuclease was replaced by the cterm domain of NBI. Nt.AlwI recognizes the site of its original restriction enzyme parent but cut only the top strand, 4bp further. This confirmed the dimerization target strategy but, as this method was successful thanks to a relatively high sequence similarity, it is not easily transferable to other enzymes.

Interestingly, experiments on mutation preventing dimerization of FokI restriction enzyme resulted in a nicking enzyme with very low activity. This suggests that type IIS restriction endonuclease can have various cutting mechanisms.

• A strand-specific engineering method was then designed to engineer SapI nickases. SapI cuts the bottom and top strand at various distance from the recognition site. Therefore, researchers thought it would be possible to isolate a top and a bottom nicking enzyme. They also performed directed evolution experiments but this time adding a selection step able to discriminate between enzymes cutting the top or the bottom strand. In their design, the randomly mutated SapI are cloned and should cut their plasmid accordingly to their specific activity. The plasmids are then recovered, and a second type of known nicking enzyme is used to cut either the top or the bottom strand not far from the SapI restriction site. When the plasmid was properly nicked by SapI, an adaptor could bind the plasmid and start PCR amplification.

The selected genes were then expressed and tested for their nicking activity 157 . Results revealed that SapI must be a monomeric enzyme, but this method should be applicable to other kind of restriction enzymes.

•

Why targeting NBI?

Given the general focus to search for nickases with new target recognition sites from restriction enzymes, one can wonder why we selected NBI (as it already has at least 4 "other"

xii The CRISPR-Cas9 system consists of a restriction enzyme Cas9 and a guide RNA allowing to target precisely a sequence in the genome. . nickases with the same recognition site). Even if broadening the scope of available recognition sequence is of interest, other parameters of nicking endonucleases are worth to be studied. On a fundamental point of view, NBI has the particularity to contain three negatively charged residues in its active center. Before its discovery, BamHI was the only known restriction enzyme with such organization. Despite the high homogeneity in the spatial organization of restriction enzymes, it seems that exceptions exist. Better understanding of the catalytic mechanism of NBI compared to other enzymes is therefore an interesting purpose. But it is not the only one.

One principal issue of the amplification methods based on nicking and strand displacement relies on the production of unexpected products increasing with the time of the experiment. Several studies assessed this problem by studying the best DNA template sequences to avoid such unspecific byproducts 145,162 . Some of them suggested computational programs to smartly design the oligonucleotides that one should use to build a reliable reaction or network. But the origin of the byproducts may also be related to the quite low temperatures used during the isothermal amplification. Contrary to PCR amplification here there is no temperature cycles to facilitate duplexes association/dissociation. By increasing the temperature to 50-60°C the unexpected products are less present 162 . Performing NEAR or EXPAR at higher temperature require the use of enzymes that remain stable and keep their activity at higher temperature during sometimes a prolonged period. NEB website indicates that commercially available NBI (New England Biolabs) works at 55°C for 1 hour but, experiences in the lab showed it could only resist few minutes at this temperature. It is most likely to perform well at 45°C for molecular network utilization. So, one purpose of NBI evolution would be to increase its processing velocity -or its thermo-resistance in order to improve molecular networks or molecular biology protocols.

NBI is neither a DNA polymerase nor is involved in protein expression so current compartmentalized self/partnered-replication protocol cannot be directly applied. It is therefore adapted to the challenge of creating a new selection method to adapt CSR to a larger variety of activities. Yet, as it is already part of the PEN DNA toolbox, it seems relatively straightforward to link NBI to an oligonucleotides output yield in the molecular program. Thus, it reduces the difficulty that could arise from targeting an external enzyme and allows us to focus on the adaptation of the PEN DNA toolbox to perform autonomous selections of enzymatic activities.

The challenge is already huge, as it is the first time the toolbox is used in non-optimal, or reallife, conditions in particular in the presence of bacteria cell lysate.

The PEN DNA Toolbox is the central element used to build the feedback loop required to link the nicking of the enzyme to the replication of its own gene, in order to perform compartmentalized self-selection. But it is also an essential tool in our lab, where it is involved in almost all ongoing projects. An improved nicking enzyme could be extremely useful to boost the library of molecular programs running both in our lab and in other teams using the toolbox.

But, starting the development of the method targeting one of its components can also ensure us to obtain an improved selection tool for the next evolution target.

Using our selection method, it could also be interesting to see if it is possible to change the recognition site of the enzyme. Few nickases are thermostable and cutting aside of their recognition sites. This configuration facilitates the design of DNA sequence in the PEN toolbox.

The team of Ellington used the CPR to produce T7 RNA polymerases recognizing different promoters 163 . There different T7 RNA polymerase can now be used in synthetic biology to have several programs running in parallel. Having NBI-like nickases with various recognition sites could allow us to also have more parallel working nodes and build more complex networks.

Outside the use in the PEN toolbox, other parameters could be studied about nickases.

Nickases engineered from restriction enzyme have been shown to still possess the star activity of their restriction enzyme parent. Starting from a nickase could maybe avoid this. Investigate how star activity arise and how to prevent it using molecular programs could also be done.

Increasing the tolerance of the nicking enzyme for modified nucleotides, RNA or methylated

G.

General methods of the selection process

The complete workflow of the selection method proposed here is depicted in Figure 31.

The different steps are explained below. First, a mutant library is generated by error prone PCR.

The library is then cloned in E. coli bacteria. After growth and induction of the target protein expression the bacteria are washed and mixed with the molecular program and a second solution of molecular program with lysing agents is prepared. A microfluidic device is then used to encapsulate single bacteria in monodisperse picoliter droplets, warrantying that the phenotype and genotype of each bacteria linkage and identical condition during the selection step. Because bacteria encapsulation follows a Poisson law, the concentration of bacteria is adjusted to have the maximum number of droplets containing only one bacterium but not increasing too much the number of empty droplets. The microfluidic device used in this process contains two inlets allowing to have separate flows for the bacteria and the lysing agents so that the bacteria are lysed just after encapsulation in the droplet. This is limiting the leak and activity sharing of enzymes mutants in the droplets and provide us with longer available time for droplets generation. The droplets are then collected and placed in a PCR machine. An isothermal incubation at 45°C is set before PCR amplification protocol. During this incubation time the molecular program will interact with the released enzyme in each droplet and assess its activity taking it as an input, the ouput will be a concentration of PCR primers of the tested enzyme gene directly link to its efficiency. This step of primers production by the molecular program is called isothermal primer amplification (IPA). Selection pressure can be applied just before or during this step. For instance, a heat shock can be added to the protocol before this isothermal incubation time, surviving enzyme will be able to activate the program and produce primers to select their genes. On another side the buffer or molecular program in the droplet can be easily changed to force the enzyme to adapt to some new buffer components or novel activity. The incubation time has also to be set carefully so that the range of produced primers matches with differential PCR amplification of the gene. It can also be used as a selection pressure to look for faster enzymes. So, after running the program for some time, each droplet contains an amount of the enzyme's gene forward and reverse primers corresponding to the activity of the enzyme expressed by the bacterium. The PCR is then launched in the droplets and the yield in each droplet will then depend on the amount of primers, therefore on the enzyme activity.

Droplets are lysed afterwards to retrieve a library enriched in the best mutant genes and a new evolution cycle can be run after recloning. Retrieved amplified DNA can also be used for analysis of the selected mutations and follow up the general evolution process by NGS or recloning, expression and characterization. Droplets are then placed in a PCR machine to undergo a 45°C incubation followed by a classic PCR protocol except for the buffer. This incubation allows production of primers in each droplet according to the encapsulated mutant activity. The PCR yield is dependent on the reach primers concentration and therefore of the activity of the enzyme. Finally, the droplets are broken to retrieve the amplified DNA, corresponding to a library enriched with the best mutants. The selected genes can then be used to start a new cycle of evolution or to analyze the selected mutations and/or selected mutants.

Results

I. Isothermal Primers Amplification

Presentation of the different modules

For the molecular network adopted in this work, only a polymerase with strand displacement activity, Bst 2.0 WS and the nicking enzyme NBI, our evolution target, were used.

Bst 2.0 WS can also be replaced by other polymerases with strand displacement activity.

Different modules have been designed and combined in the course of the project but not necessarily at the same time. Figure 32 shows the four modules, with details given below:

• Primer generating module (yellow box): It is an activation module and the principal component of the selection program. It allows to generate primers using a trigger strand of 11 bases called v11. It is therefore the module that will control the gene amplification reaction downstream. The 2 templates take v11 at the input and generate either the forward or reverse primer at the output, in a reaction that is dependent on NBI activity. Like all activation modules of the PEN toolbox, it is based on a polymerase extension and nicking, after annealing of the v11 trigger on the template. The production of primer strand is therefore directly dependent on the activity of the NBI nickase. Note that, for the purpose of selecting for nickase activity, we could in principle simply use this module to linearly produce primers from a starting concentration of trigger (see below).

• Autocatalytic charging module (pink box): The autocatalytic module allows the exponential amplification of the trigger strand v11. It will generate the many trigger strands necessary to activate the primer-generating module presented above. Once enough v11 have been produced to saturate the input sites of the primer-generating templates, primer production should be linear in time, with a dependence on NBI activity. This exponential module also relies on NBI activity and fulfils a number of purposes:

o Change the shape of the selection function by creating a delay in primers production. If this module is used instead of a high initial concentration of v11 trigger, the primer-generating templates will start producing primers slowly until they are fully charged by v11 triggers.

o Avoid troubles linked to the direct introduction of the v11 trigger like false positive amplification. This problem will be described later.

• Source module (blue box): The source template is composed of a hairpin loop allowing it to prime itself so that no trigger strand is needed. It will start producing the v11 trigger after extension by the polymerase, and again dependent on nickase activity. Introduction of this module instead of v11 trigger is also modifying the shape of the selection function. This module can also be used to initiate v11 production without having to introduce the v11 strand in the initial reaction mix. We will see in the following development of the project why this is particularly important to avoid false positives.

• Pseudotemplate module (purple box): The pseudotemplate module is working as presented in the PEN DNA toolbox presentation. Here it is targeting the trigger strand v11 and mitigate self-start issues associated with the positive feedback loop of autocatalytic templates. Depending on the concentration of the pseudotemplate, this module will also participate in the generation of a delay in the primers amplification process. In other words, a higher nickase activity will be necessary to overcome the activation threshold and launch the autocatalytic v11 production, followed by linear primer amplification.

Figure 33 shows how the different modules can interact in a molecular network.

Possible adaptation for other enzymes

This molecular program design can be easily adapted by changing the primers production module and the source module. Since all the modules are linked with the v11 trigger, just changing the source input activity is sufficient to trigger all the following nodes of the network. Then one can just change the sequence of the produced primers to adapt it to the new gene of interest. But, alternatively many different molecular networks can be imagined the selection of other enzymes.

Shape of the theoretical primer production curve

By combining these different modules, primers production according to NBI activity curves can be obtained by numerical simulation based on a mathematical description of the PEN toolbox reactions (Figure 34). This can be done in an automated way using the software DACCAD 121 . We can use these plots to predict the shape of the selection function. Figure 35 shows the different shapes of the selection function according to various modules combinations.

The two-step amplification scheme, combining the primer-production module and the autocatalytic one, provide a more stringent selection on NBI activity. The initial autocatalytic stage discriminates against very inefficient NBIs, while the linear stage separates between well performing NBI (Figure 34). Note that kinetic parameters were not adjusted, so these simulations should be understood in a qualitative way.

What about the exonuclease of the PEN toolbox?

In this work the exonuclease RecJf was not used but it could be added to the molecular program to set a primer concentration limit or to make the use of the pseudotemplate more effective. Indeed, addition of the exonuclease would add a constant degradation of all the produced strands and therefore a dynamic equilibrium between primers production and degradation will be progressively established until their concentration reaches a steady state. In that case the running time of the IPA does not need to be controlled so much and the activity of the enzyme gets related to a steady state primers concentration.

Design of the strands

Besides network structure, some precautions have been taken on the strands design.

Sequences are presented on the Table 6. All the templates are designed to integrate the NBI recognition site so that NBI cuts at the end of the input strand. For the pseudotemplate, the thymine base is replaced by a uracyl in the recognition site, in order to prevent NBI cutting that would regenerate the targeted trigger. The number of extra bases added by the pseudotemplate is also optimized to have effective inhibitory behavior (a minimum of two bases should be added to have an effect) 117 . avoid evolving an enzyme that would be specific for an extended recognition site. It should also have the side effect of slowing down primer production efficiency, as relative concentrations of perfectly matching primers and templates gets lower.

Primer production rate in vitro

Primer production rate is dependent on enzyme activity

To estimate the primer production rate, we first tried to observe dNTPs consumption by the isothermal primer amplification (IPA). In the following experiments, IPA is run using only the primer producer templates (ppT), the autocatalyst (aT) and the trigger strand v11 (T). IPA were run with various concentration of dNTPs and we followed the amplification on a real-time PCR machine thanks to a fluorescent intercalating dye (EvaGreen). When the curves reach a plateau, we can consider that all the dNTPs have been consumed and we can therefore estimate the amount of primers produced at this time. This assay, whose results are presented in Figure 36, allow us to estimate an average primers production rate of 6.4nM/min. When working with dNTPs concentration higher than 50µM, a phenomenon called divergence, linked to the apparition of replicating parasites and where the fluorescent intensity suddenly grow exponentially, appears before the plateau is reached. Also, the estimation is made considering that the trigger v11 and the reverse and forward primers are produced at the same pace, which is probably not the case. Table 6. Some sequences of the used templates and trigger. In bold you can see the recognition site of the nicking enzyme NBI, in orange the base switch allowing the pseudotemplate pV11 not to be cut and in red the complementary parts of the hairpinloop of the source template.

Later in the project, to gain a better idea of the primer production rate, probes specific to the primers were designed. The development of these probes is detailed in annex iii. The first hairpin probes, MBp17r3 and MBp25f2, were designed and tried by my intern Giselle MacCallum. In an experiment, she used different concentrations of MBp17r3 and MBp25f2 reporters to look at primer production by the IPA network, with either 0.2 or 0.5% of commercial NBI. These percentage refer to dilutions from the commercial stock at 8000 U/mL. This was the first time we could directly see the primer production during IPA. All previous experiments relied on the PCR step to convert primers to gene, and the PCR yield was taken as a proxy of efficient primer amplification. Her results (Figure 37), confirmed the increase in primer production rate with more NBI enzyme activity. A clear delay could also be observed at low NBI activity. This network used no source, but the autocatalyst was triggered by 10 nM of initial v11. 110 These new probes were very convenient to monitor the behavior of the IPA protocol using purified NBI but were not resistant to bacteria lysate (they had 3 phosphorothioate bonds at the 5' extremity to protect them, but none on 3'). It was therefore not possible to use them to try to understand the issues we had later with bacteria lysate (see below). I therefore designed new probes also presented in the annex iii. We found that the best design used two hybridized strands. One of the two strands is carrying a fluorophore and the other has a quencher. When the produced primers bind to one of the strands, the other is displaced by polymerase extention, leading to dequenching and an increase in fluorescence.

Thanks to this new probe it was possible to have a good estimation of the primer production rate according to NBI activity. As shown in Figure 38, we observed that primer production speed is increasing up to 0.3% of NBI enzyme but saturates, or even decreases, for higher enzyme concentrations. According to these results, the IPA reaction would generate about 0.7nM of primers p17r3 (17pb) /min, in contrast to the 6.4nM estimated with the dNTPs measurements. This difference may be due to differences in production rate between v11 and the primers, which could not be resolved by the dNTP-monitoring experiment. We also used this reporter to measure the effect of the initial trigger concentration.

Results on Figure 39 show that increasing trigger concentration is decreasing the time needed to reach a certain concentration of primers (here 100nM, but equivalent results were obtained for 50, 150 and 200nM of probes). The probe, despite it reacts very fast when put in presence of primers, seems to have an effect on the reaction kinetics (see Figure 39). Contrary to the results obtained by Giselle with the hairpin probes, the initial part of the fluorescent curves is not overlapping when different quantities of probes are added in the reactions. It is therefore not possible to estimate the speed of primer production by fitting the slopes. Considering that the probe is affecting the reaction, results obtained with the lowest amount of probe should be used to estimate the speed of primer production. Here, with 80 min to reach 50nM, we find a similar value than with the previous experiment of ~0.6 nM/min. Because of the negative effect of the probe, this value is an underestimate.

We also looked at the effect of the addition of pseudotemplate or the usage of the source template on the primer production curves. Figure 40 shows that using 2nM of source template rather than 10 nM of the trigger strand v11 to start the reaction allows a faster production of primers. In these conditions, the pT has little effect with a source while it is delaying the Recording of primers production speed in vitro according to NBI concentration using a double stranded molecular probe. 50nM of the molecular probe dsp17r3_F/dsp13r3_Q was added to an IPA reaction in the IPAV0 buffer in vitro with various quantity of NBI enzyme, aT 40nM, ppT 20nM, T 20nM. Fluorescence increase, followed in a real-time PCR machine, indicates binding of the produced reverse primers p17r3 on the probes. Primer production speed increases up to 0.3% of NBI and saturates above that concentration.

112 production when a trigger is used. We also observe a slight delay between the production of the two different primers, with p25f2 being produced slightly faster than p17r3.

In conclusion, we can estimate the primers production rate by the Isothermal Primers Amplification to be around 0.6nM/min in the IPA V0 buffer with 20nM of aT, 10nM of each ppT and 10nM of T. This can allow us to reach about 40nM of primers within one hour. This amount of primers should be sufficient to perform a PCR reaction (Figure 88).

We showed that we can link NBI activity to a differential primer production. This link staurate for high NBI concentration. We will see in the next part how this IPA step connects with the PCR reaction.

Figure 39. Measurements of primers production by dsp17r3 probes in IPAV0 buffer according to v11 trigger concentration. Top: Primer production curves for various initial v11 concentration, aT 20nM, ppT 10nM; inset: Time to reach 100 nM of reverse primer p17 according to v11 concentration. Bottom: Production of primers followed using different concentration of probes. The probe has a strong delaying effect on primer production. Red points are the time when the different concentrations of primers are reached.

In vitro implementation of the IPA-PCR

Optimizations to link IPA and PCR steps

Buffer optimization

The nickase NBI used in the IPA, and which we want to evolve, requires a buffer with relatively large monovalent salt and Mg 2+ concentrations. The buffer NEB 3.1 recommended by NEB contains 10 mM of MgCl2. On the contrary, PCR reactions are usually run in buffer with ~2 mM of Mg 2+ and with low salt concentration. The standard Taq buffer from NEB contains only 1.5 mM of MgCl2 and the thermopol buffer that can also be used for classical PCR with the Taq polymerase, 2 mM of MgSO4. Higher magnesium concentration can increase the polymerase error rate or completely prevent the reaction to happen. Since the IPA and the PCR reaction will eventually be run in the same droplet, they need to be run in the same compatible buffer. During the experiments, several sets of primers have been used, to amplify either the full length of the NBI gene or the first 780 bases. This short fragment assay was first used for convenience, to have shorter reaction time during optimization.

When I started the project, an IPA buffer containing 5 mM of Mg 2+ and 20 mM of extra NaCl salts compared to NEB thermopol buffer had been arranged. When performing the IPA-PCR with this buffer in a real-time PCR machine with various concentration of the NBI enzyme (see Figure 44) the PCR seemed to perform well, with a correlation between NBI activity and the PCR efficiency. But, on the gels, a lot of byproducts appeared. We therefore tried to reduce the quantity of salt and/or Mg 2+ for the IPA step. As we can see in Figure 42, with 4 mM of Mg 2+ instead of 5 mM originally, the IPA can still run, albeit with a lower rate. We decided to decrease the magnesium concentration and to use a new IPA (nIPA) buffer with only 4mM of magnesium. As it appeared that even with the recommended PCR buffer (from New England Biolabs) the PCR was not performing well with some primer pairs, we also optimized the primer's length (see below).

PCR was then run in different buffer with variation of NaCl and Mg 2+ concentrations.

Results on Figure 41 show that the PCR can be run with higher magnesium concentration but the size of the amplicon and/or the primers sequence can be of critical importance. Therefore, it was decided to keep the nIPA buffer.

Primers optimization

The primers were initially chosen short because it is easier to work with short sequence when working with EXPAR or NEAR. It was reported that oligonucleotide products of 10 to 22nt give the best results (30). This effect is certainly linked to the necessity of strand displacement activity for longer strands. An experiment to try to estimate the effect on the product length is presented in the annex ix. The NBI gene sequence being A/T rich, it resulted in quite low melting temperature primers. For the amplification of the 780 first bp of NBI, we ordered some new primers of either 25 or 40 pb with higher and more conventional melting temperature around 59°C for the 25pb and 64°C for the 40pb. To amplify the full-length gene only the reverse primer needed to be changed but its melting temperature was already 59°C and increased too much for longer primer. PCR was then performed using the 9 different combinations of primers for the short length PCR and 3 combinations for the full length first in the classic thermopol buffer and then in various buffer with higher salt or magnesium concentration. As a result, the 25bp primers seemed to perform better that the initial ones in thermopol buffer. Moreover, they are still giving correct PCR results in the new version of the IPA buffer with 4 mM of Mg2+ and 20 mM of NaCl (Figure 43).

Further optimization

With the change of primers and magnesium concentration, it was still possible to perform IPA-PCR and to visualize different PCR efficiency according to the enzyme activity on the RT-machines. But the products on the gel electrophoresis were not clean enough. The intercalating dye used in the real time PCR revealed to be one of the factors leading to the bad quality of the PCR product. Removing it or reducing it to 0.2X final allowed to perform PCR in better conditions. Bst Large Fragment, which we used initially, was changed for Bst 2.0 WS and gave better results. Interactions between the templates and PCR was also investigated as possible reason for the PCR quality issue. The triggers produced, or the templates themselves could interact with the PCR reaction and create these by-products. On another side, several attempts showed that a minimum template concentration is needed for the IPA step to amplify enough primers within 1 hour. With less than 40nM of total templates there was no PCR. With an IPA incubation of 50 min and 20nM of cv11r2p (aT), 10 nM of v11 (T) and 10 nM of each ppT IPA-PCR could be observed. Finally, with more stable primers, the use of the Bst 2.0 WS and the modification of the buffer it was possible to improve greatly the quality of the obtained PCR product (Figure 45).

Study of the selection function

The results presented above demonstrate that we can indeed link the NBI enzyme activity to the yield of replication of its own gene in vitro. To have a better idea of the selection shape, I decided to use the method presented by Genot et al. 130 , which was developed at that time in the Fujii lab, to perform a scanning of the NBI concentration in droplets. The scheme of the droplet-scanning experiment is detailed in Figure 46. Two solutions containing the molecular program, the PCR reagents and both an intercalating agent and a specific PCRreporting Taqman probe (zenphex) were prepared (see Material & Methods). One of them was supplemented with 0.5% of commercial NBI and 1µM of Alexa 647, a red fluorescent dextran. This fluorophore is not interacting with any part of the system and is used as a barcode to follow the concentration of NBI. A microfluidic device is used to produce droplets where the two solutions are mixed. Flows for both solutions are controlled by a pressure pump. Small continuous variations of pressure in each channel allows to change the contribution of each solution to the droplets. By doing so, we can scan continuously for various NBI concentrations, a proxy for various NBI activity, and this concentration can be obtained a posteriori using the fluorescent barcode. The droplets are then incubated in the PCR machine for the IPA-PCR and subsequently observed under the microscope. Results presented on Figure 48, show the barcode fluorescence, the fluorescence level for the PCR-specific reporting probe, indicating PCR yield, as well as the merging of the two channels. Reorganizing droplets according to the barcode level gives an idea of the shape of the selection function in droplets. In this case, we observed a quite linear relation between activity and replication. The merged bigger droplets were not removed from the analysis.

Giselle MacCallum, a master intern I supervised, tried to reproduce this experiment during her internship. The idea was to use this method to study the different possible selection functions we can build with the molecular programs. Unfortunately, it did not work as expected in the new parisian lab. Surfactant, as well as fluorophore cross-talks issues prevented her to reproduce quickly the experiment. Finally, she obtained the results presented in Figure 47 in the same condition as in my experiment. Here, the IPA-PCR seem to plateau around 0.25% of NBI. An explanation could be a change in the IPA activity as different stocks were used in the experiments. Yet, this experiment still demonstrates a monotonous relation between PCR yield and NBI activity. With this in mind, and knowing that small changes in conditions could strongly affect this curve, we moved to the implementation of the IPA-PCR reaction with NBI activity actually coming from a bacterial lysate.

Controlled lysis of the bacteria

In the CSR protocol, lysis of the bacteria is simply done by increasing the temperature to 94°C when starting the PCR amplification and incubating for 5 min instead of the classical 30s [START_REF] Ghadessy | Directed evolution of polymerase function by compartmentalized self-replication[END_REF] . In the case of the PEN CSR, although NBI is a thermophilic enzyme, it cannot support such elevated temperatures, and thus an alternative way of lysing the bacteria had to be explored. According to NEB, the commercialized NBI can work at 55°C for one hour but experimental results in the lab suggest that NBI half-life (t1/2) is only 5 min at this temperature.

In their paper about picoliter cell lysate assays 166 , Kintses et al. perform chemical bacterial lysis, without elevated temperature, in microfluidic generated droplets. They use two aqueous 121 flows, one containing the cells and the other one the substrates and lysing agents. They perform their incubation and droplets sorting on the microfluidic chip. The droplets stay on chip for about 12 min between their production and sorting and lysis seems to occur in the first few minutes, just after droplet formation. Such small delay for cell lysis seemed well adapted to our selection process, so we tried to use the same lysing agents. Kintses et al. use 0.4% of rlysozyme (rlys) and 17.5% of BugBuster (BB) at room temperature in their lysing solution. As this solution is mixed equally with the aqueous solution containing the cells, these concentrations are twice more concentrated than in their final droplets. Bugbuster being a detergent, it can interact with droplet formation and destabilize the emulsion. According to the authors, who are using fluorinated oil and EA surfactant (like us), higher concentration of bugbuster indeed hinders the droplet formation.

Direct observation of bacteria lysis

We first tried to visualize the lysing effect by realizing LIVE/DEAD bacteria assays under the microscope. This system is based on the usage of two fluorescent stains with different properties regarding their entry in the bacterial cells. The green syto 9 dye can penetrate alive or dead cells and stain their DNA while the red propidium iodide can only penetrate dead cells.

Unfortunately, no particular change in bacteria color was observed when putting the stained bacteria in lysing solutions containing BB and rlys. We then tried to observe lysis of bacteria expressing GFP. These bacteria appear as green points under the microscope, which should disappear when the bacteria are lysed. Imaging the bacteria under fluorescent microscope it was concluded that lysis seems to take around one hour, with some bacteria still apparently intact at that time. These results were not in agreement with the report from Kintses et al.. To reproduce as closely as possible the reported protocols and better quantify the lysis phenomenon, we encapsulated the GFP bacteria in droplets. We then followed the lysis over time by performing a time-lapse with the fluorescent microscope at 45°C (running temperature of the IPA). Bacteria encapsulation with BB confirmed that we could use 17.5% of BB without perturbating the droplet generation. But, since the ratio was not always equal when setting the flow in the microfluid chip, BB was problematic during the adjustment time. This problem and bulk experiments presented later led us to decrease the amount of BugBuster but also rlysozyme used in the lysing solution. We also tested the effect of a complete suppression of the BugBuster detergent. The bacteria were resuspended in the master mix at 4°C, and then a temperature jump was applied to the final IPA temperature. We then counted the number of intact bacteria, in one microscopy image, as a function of time. The results are shown in Figure 50. Fitting the decay of bacteria counts with exponentials allowed to distinguish several types of behavior.

With 10% of BugBuster we obtained 3 populations:

• Non lysing : 8% (+/-1%)

• Slow lysis:15% (+/-2%); lysis rate =0.017 (+/-.003) per min; t1/2 = 40 min

• Fast lysis: 77% (+/-2%); lysis rate = 0.28 (+/-.03) per min; t1/2 = 2 min 30 Without BugBuster we obtained 2 populations:

• Non lysing: 30% (+/-7%)

• Slow lysis: 70% (+/-7%); lysis rate = 0.04 (+/-.007) per min; t1/2 = 17 min 20

These results seem to show that the bacteria are not completely lysed by addition of the lysing agents, and that the bacterial population contains distincts phenotype with respect to lysis. Using BugBuster should help us increasing the lysis efficiency by decreasing the proportion of non-lysed cells and accelerating the lysis. But having different population with various lysing speed is problematic for our experiments and would introduce selection bias.

Also, these observations are not in agreement with Kintses et al. data. Bulk experiments presented next are in a favor of a partial lysis that would be sufficient for NBI enzyme release out of bacteria within minutes.

Measurement of NBI activity release during lysis

To assess efficiency of the NBI enzyme release by bacteria, we performed lysate assays with the protected loopact probe (sequences given in Material & Methods). Interpretation of the obtained results is not always easy. Since the height of the final plateau can vary from a condition to another it is sometimes difficult to estimate the real degradation speed of the probe when this plateau is not reached during the timescale of the experiment. Another protected probe, similar to loopact, but with a mutation in NBI recognition site, unspecific loopact, was used to distinguish specific activity from eventual unspecific activity released from the bacteria content. Figure 51 shows that there is very little unspecific activity released from the bacteria lysate, as the unspecific probe is almost not degraded and Taq expressing bacteria do not cleave the specific probe. It also shows the efficiency of the rlys and BB lysing agents at the concentration used in the paper of Kintses et al. as they greatly increase the amount of released NBI activity. Unfortunately, when performing an IPA-PCR in vitro with the commercial NBI with the lysing agents, we noticed that the reaction could occur, with almost no effect in presence of either BB at 17.5% or rlys at 0.2% but not when they were used simultaneously (Figure 52).

We therefore tried to investigate the effects of the two lysing agents and their combination. The results presented in Figure 53 suggest that the negative effect of combined rlys and BB is only observed in the absence of bacteria. It is possible that in the absence of bacterial membrane to lyse the BB and/or rlys can interfere with the enzyme or the reporting system, but this does not happen (or at a smaller and unmeasurable scale) when bacteria are present. To study this point, we set up a series of experiments, in which bacteria (at 50 or 100 bact/nL) were incubated at 45°C with the protected loopact probe. The panel D of Figure 53 confirms that BB can have an effect on the commercial NBI enzyme or the probe in absence of bacteria. At 17.5% of BB almost no activity is detected. Even if this effect is not observed in presence of bacteria, it is still possible that BB harms the IPA reaction. Still, Figure 53 A seems to confirm the microscope observation that the use of BB increases the lysis, compared to the use of rlys alone. On panel C, various concentrations of rlys were tested in order to see if increasing rlys concentration could replace the use of BB. Counterintuitively, after some concentration threshold, less and less activity is observed. No more activity is observed after 0.8% of rlys. It here also seems that rlys can interact with the enzyme, its activity or the probe signal. We also tested bacterial NBI release, with various concentrations of BB in presence of 0.2% of rlys (panel B). There is also a decrease in activity for 17.5% of BB compared to reaction with only 10 or 5% but the decrease is not as important as in the case of the commercial NBI as it is still more efficient as with no BB.

As BB and rlys can interact with the reaction, we tested a limited heat shock as an option to lyse the bacteria. We observed that activity is already released from bacteria at 45°C without any lysing agents (Figure 54 A). Addition of a 50°C 1 min heat shock pulse does not seem to We also tested heat shocks at higher temperatures (panel B) but none yielded an increase in the loopact digestion slope. These experiments indicate that elevated temperature between 45°C and 65°C have similar effect on NBI release. It seems that the porosity of the bacterial membrane is not increased when the temperature is increased within this range.

Still, the experiment shown in Figure 53 C suggests that not all NBI activity is released by incubation at 45C without lysing agent. Further investigation then tested the combination of rlys with heat shocks, on the assumption that fragilization of the membrane by rlys could improve the heat shock efficiency. In Figure 55 A and B, degradation of the protected loopact probe has been assayed with various concentration of rlys at 45 or 50°C. Both temperatures give the same profile with an optimal rlys concentration. The measured activity is higher for 127 reaction performed at 50°C than at 45°C. In the light of the previous result on the heat shock effect, it seems that this increase in speed is more due to the enzyme working faster at this temperature that really to an effect of the temperature on enzyme liberation. Furthermore, we observe that there is no delay in the reaction and no increase of the reaction slope over the beginning of the reaction. Thus, it means that no extra activity is released overtime. rlys is a mesophilic enzyme and it is usually recommended at room temperature (as in Kintses et al. experiments). Working at 45°C could prevent its action. Pre-incubation of the bacteria at room temperature in the presence of rlys was tried (panel C), but these experiments show that incubation up to 25 min does not increase much the activity release. Here also an additional heat shock at 49.4°C had no effect, compared to direct incubation at 45°C.

As bacteria do not seem to lyse completely in a few minutes and heat shock are not increasing the activity released, we wondered if the measured activity was not coming from leaky release of NBI activity in the resuspension buffer. This could be problematic, as it would mean that in droplet experiments, a part of the activity in each droplet would be resulting from an average library activity. In a first experiment, presented on Figure 56, several washes of the bacteria were done, and the supernatant activity was tested for NBI activity with or without rlys. rlys would allow us to know if the activity is coming from residual bacteria in the washing buffer of just for free enzymes.

In the complete protocol, after enzyme expression in bacteria, the culture growth is stopped on ice and small aliquots of 1.5mL are prepared and centrifuged at 5000g for 5min.

The culture medium is then removed, and bacteria pellets are either stored at -80°C or used directly for experiments. The obtained pellet is resuspended in 100µL of resuspension buffer (Tris-HCL pH 7.5 50mM, NaCl 100mM). A washing step consist of resuspension, centrifuge at 5000g 5min and removing of the supernatant. In Figure 56 we look at NBI leakage by assaying the supernatants obtained after the i th washing step. Panel A shows that there is indeed activity in the washing buffer that is not due to bacteria present in the supernatant. However, this activity is really small compared to the one released by the bacteria. Furthermore, the buffer is diluted at least 5 to 10 times more in the actual reaction than it is in the assay. On panel B, the primer production has been assayed with the dsp17r3 report probe. It shows that activity contained in the washing buffer is producing only ~10% of 50 nM of primers in more than 300 min while the experiment with bacteria reach this concentration in less than 75 min. Again, as here the resuspension buffer is a least 5 times more concentrated as what it is in the classic reaction, this residual NBI activity does not seem to significantly contribute a lot to the final activity assayed in bacteria.

According to the previous results, the biggest decrease in buffer activity is observed between wash 2 and wash 3. 2 washes are therefore sufficient to remove most of the free enzymes in the buffer. Most of the leak could come from freezing/unfreezing the bacteria. Still, we always observe some activity in the subsequent washes. It means that even if it is a slight release, enzymes are continuously released in the buffer. Droplet generation is taking some time, during which the concentration of free enzymes could increase and become problematic. So, a last assay was realized with supernatant obtained from bacteria staying at RT in the resuspension buffer for 2 hours or with supernatant retrieved from the premix solution used to generate the droplets after the time of droplets generation. Results presented on Figure 57 show that there is a slight increase in the buffer activity when waiting for droplet generation or at RT for 2 hours, but this activity is still very low compared to bacteria activity. With rlys, more activity is detected in the buffer after droplet generation than without, this could mean that bacteria were not completely pelleted, and some were present in the reaction buffer. This would argue for an even smaller release of activity in the reaction buffer.

The results of this bacteria lysis study show that lysis does not need to be complete to release some of the NBI enzyme in the buffer. rlys and BB can both accelerate or increase the activity release, but their concentration need to be controlled to not interact with the following reaction. Other experiments showed that BB can indeed accelerate lysis in presence of rlys but has little effect on bacteria lysis alone. As BB and rlys interaction can be armful for the reaction, using only rlys should be preferred. Increasing the temperature to 45°C can also be sufficient to release some enzyme activity in the buffer. Higher temperature heat shocks do not increase the total quantity of released enzymes and could destroy the NBI enzyme. We will see in the following parts that further adjustment of lysing conditions can help for sensitive templates.

Enzymes sensitivity to the bacteria lysate

Finding a Taq polymerase

Although we had confirmed the efficient release of NBI activity, our first experiments of IPA-PCR, using bacteria at various concentrations, failed. In parallel, experiments to replicate the CSR process with Taq polymerase (presented in part IV) revealed that the PCR step could not be run at high concentrations of bacteria. Indeed, gene self-replication by bacteria expressing Taq polymerase failed, even with externally added commercial hot start Taq (HS Taq) polymerase.

To explore this point, we performed the PCR step only, starting form minipreped pNBI plasmid, with 250nM of primers and from 50 to 200 bact/nL in the nIPA buffer. The gel presented on Figure 58 shows that, when using the HS Taq, the PCR product is less clean as soon as bacteria are present in the mixture, and that the yield is decreasing with the bacteria concentration. It is known that some enzymes can resist more to biological contaminant like the one that can be found in whole bloods 167 . We therefore decided to test several commercially available recombinant Taq polymerases, first in their commercial buffer. The initial target size for droplet was 1pL (it was changed later). This correspond to an equivalent concentration in bulk of 1000 bact/nL. Similar to what we observed for HS Taq (NEB), the KOD Fx Neo (Toyobo), the Phusion HS (Thermofisher), the Q5 High Fidelity (NEB) and Vent (exo-) (NEB) could not amplify DNA at high bacteria concentrations. Among them, the HS taq was still the best performing, with detectable product up to 200bact/nL. However, some enzymes performed much better: Titanium Taq (Clontech) could amplify a template in up to 800 bact/nL, and finally, Dream Taq (Thermofisher) and One Taq HS (NEB) could still be used at 1000 bact/nL. Maximum resistance values are given in Table 8 and gels of the PCR products are presented in Figure 59.

These experiments were run in the commercial buffer corresponding to each polymerase. The buffer for IPA-PCR was optimized to support both IPA and PCR. So, the different enzymes performing well at high bacteria concentrations were tested in the nIPA or IPA Buffer. The PCR was run with 200nM of primers and this time worked in both nIPA and IPA buffer for all enzymes, strangely, even for the HS Taq up to 1000 bact/nL. After these results, OneTaq polymerase was selected to pursue the experiments. Both DreamTaq and OneTaq seemed to have increased lysate resistance, but more information was available on the OneTaq "enzyme", especially its standard buffer composition, that is nor provided in the case of DreamTaq. This information can help us better tune the buffer and optimize the reaction if needed. OneTaq polymerase is an optimized blend of Taq and Deep VentR™ DNA polymerases. In its Hot Start version, as for the classical Taq, an inhibitor reversibly binds the active site of the enzymes below 45°C. We tried to perform the IPA PCR in the OneTaq buffer supplemented with 8 mM of KCl and 2.2 mM of Mg 2+ to mimic the nIPA buffer. The reaction worked with the OneTaq polymerase, but it was decided to go on with the nIPA buffer because the obtained product was not as clean as with the nIPA buffer, and besides detergents and very small variation in salt concentration, the OneTaq buffer is not very different than the Thermopol buffer DF. OneTaq polymerase also appeared to be more resistant to high magnesium concentration (Figure 60). The Magnesium concentration had been carefully optimized to accommodate both the IPA (high magnesium) and PCR (low magnesium) requirements with a classic Taq, but the IPA is more efficient with more Mg 2+ . So, a new version of the IPA buffer, named IPAV0, was made with a final Mg 2+ concentration of 6 mM.

Looking for a new polymerase for the molecular program (IPA step)

Despite this change in PCR polymerase, it was still not possible to perform the IPA-PCR reaction with 200 bact/nL. We then tried to investigate if the enzymes used in the IPA could work in the bacteria lysate. As we use a simplified network we only use Bst 2.0 WarmStart and the nickase NBI, the latter being provided by the bacteria. Lysate assays of NBI activity with the protected loopact probe already proved that the enzyme is active up to 1000 bact/nL. But Bst 2.0 WS could be not resistant enough to the lysate. Its concentration was increased from the originally used 0.1% to 1%, with no effect (enzyme concentrations are given as final dilution from the commercial stock). Then, similarly to the PCR polymerase, several polymerases were tested.

First, in addition to Bst 2.0, Bst 3.0 and Vent (exo-), both from NEB, were tested. Bst 3.0 was used at 0.1% like Bst 2.0 and was also tried at 3%, Vent (exo-) was tried at 2%, which is the range used by other lab members in their molecular programs. Only Vent polymerase gave a PCR product and with lower yield than Bst 2.0. As these enzymes have different buffer requirement, some buffer adjustment was done to place the enzyme in better conditions (Figure 61). The buffer of the Bst 3.0 contains 150 mM of KCl, 3 times more than the buffer of Bst 2.0 and 5 times more than the IPAV0 buffer. Running a PCR in the IPAV0 buffer with increasing concentration of KCl, it appeared that PCR could still occur with 90mM of KCl but not with 130mM. NBI activity was also tested in increasing concentration of KCl. As shown on Figure 61 D, its activity increases with KCl concentration. No unspecific cutting was observed with increasing salt concentration. The speed of Bst 3.0 was also tested via a turnover reaction (see material & methods). Results are presented in Figure 61 E and show that Bst 3.0 is slower than the 2.0 version in the original conditions but, can reach the same speed when KCl concentration is increased. After this buffer optimization the in vitro-IPA PCR was again tested with various enzyme concentration (Figure 61 F). No improvement was visible for Vent (exo-) with higher or lower concentration of enzyme. But a product could be seen for Bst 3.0 at low concentration (0.1%). The IPA-PCR reaction was then tested in presence of bacteria at 50, 100 or 200 bact/nL and none of them gave a PCR product.

Simultaneously to these tests, molecular program sensitivity to the lysate was also assayed. This development is presented in the next part. With addition of protections on the molecular program templates, it was then possible to run the IPA-PCR in bacteria with low bacteria concentration (50 bact/nL). As it was still not possible to reach higher concentration, further experiments were done with new polymerases to replace the Bst 2.0. John Milligan from the Ellington lab evolved a hybrid Bst enzyme in his paper on a CSR experiment using RCA for replication 168 . I met him at the SEED conference in 2016 and we later asked him to try his polymerase for our reaction. As it was evolved in bacteria lysate we thought it could improve our IPA-PCR in lysate. On Figure 62, John's Bst was tested in a turnover reaction, to estimate the quantity to use in the IPA-PCR reactions. This confirmed that it could work in the IPA V0 buffer and perform a reaction encoded in a molecular program.

However, its use in IPA-PCR with bacteria did not improve the reaction (Figure 62).

Finally, two other polymerases GspM 3.0 and Tin (exo-), both from Optigene, were tried. After concentration optimization (Figure 63 A) they were tested in the presence of increasing concentration of bacteria expressing either the active or inactive NBI enzyme. As observed in previous experiments, Bst 2.0 worked at 50 bact/nL and a slight band appeared even for the inactive NBI mutant. Investigation of this phenomenon is presented in part 3.4. For the GspM 3.0 the phenomenon was inversed, and the reaction performed better for the inactive mutants. For Tin (exo-) a product could be observed up to 200 bact/nL but here also the inactive mutant was strongly amplified. It was decided to not continue with these enzymes, but it could be interesting to test them again, especially Tin (exo-), in the new optimized conditions in which no amplification of the inactive mutant is detected.

Molecular program templates sensitivity to the bacteria lysate

Following the IPA-PCR with reporting probes

Different methods were tested to follow the IPA process in bacteria lysate. The purpose was to determine which parameters could explain the difficulties we had and to observe directly the effects of components modification on the IPA stage. Estimating the primer production and degradation rates with a simple intercalating dye is difficult in presence of bacteria lysate, because genomic DNA interacts with the dye and saturates the signal. As the products (17-35mers of single stranded DNA) are small it is also complicated to visualize them on a gel, especially at low concentration. Therefore, we developed reporting systems with dedicated molecular probes.

In the first tests, the IPA network was replaced by a simpler network, consistaing of only the autocatalyst template (aT) performing a "turnover" reaction. In turnover conditions, the reaction contains a small initial concentration of dNTP, the exonuclease is included, and the reaction is started by a small amount of trigger strand (or some time left to self-start). After the initial exponential phase, the reaction reaches a steady state, where degradadtion by the exonuclease compensate production by the molecular program. Once all dNTPs are consumed, concentration goes back to 0. The characteristic rectangular shape of the time trace is transmitted to the reporting system, and is, in principle, easy to distinguish from experimental artifacts or drifts. Such experiment can be used to check if the probe is not degraded by the lysate and that the fluorescence change that we observe is due to the molecular program reaction. For example, if the probe signal does not go back to its original state, it may indicate that it was degraded by the lysate.

The first reporting system was based on nucleobase quenching 125 , as traditionally used in PEN systems. In this approach (see annex iii) the template itself is modified with a fluorophore on the 5' extremity. When the trigger binds to the template the fluorescence intensity is modified, in most cases decreasing. Figure 64 A shows that this reporting system, so the template itself, seems to be destroyed by the lysate rapidly with 250 bact/nL. Note that the templates used in these experiments were protected by addition of biotin-streptavidin on the 3' extremity, and phosphorothioate bonds on a few bases (3) at both extremities. Both modifications serve as a protection against exonucleases.

On Figure 64 B another reporting system based on a random-coil strand (rcF6cBQ-

Cy5_1

), which open under fixation on the target strand was used. Here, we used much lower concentrations of bacteria expressing another enzyme (Taq), with addition, or not, of 2% of commercial NBI. The probes and the aT are also protected. We see that the probe is cleaved or modified by the bacteria lysate from already as low bacteria concentration as 3 bact/nL. The beginning of the reaction resembles the strand production and could be confused with it. We can also see that for 3 bact/nL we can still see the shape of a turnover reaction with the commercial NBI but not without it. This suggests, that the probe gets indeed partially degraded, but the molecular program still functions to some extent, at least up to 200 min in this case.

Optimization of templates concentration directly in the IPA-PCR reaction

Since it was difficult to follow the IPA with reporting probes we directly tried the IPA-PCR reaction to assess the lysate effect on the molecular program. Using the nIPA buffer and the OneTaq polymerase, we first tried to use template strands protected as in the experiment above. Biotin was added to both extremities of the IPA MP templates as well as 3 phosphorothiate bonds on the 3 first and last bases of each templates. In the following explanations, these templates will be named biot templates, as opposed to unbiot templates. On the Figure 65, concentration of the templates was increased, and various bacteria concentration were used to try to perform an IPA-PCR in bacteria lysate. The ratio between aT and ppT as well as T was kept constant at 2:1 and will be the same for all following experiments. With 100nM of biot templates no PCR product was observed with any bacteria concentration, but a smear could be observed at the lowest bacteria concentration (50 bact/nL), contrary to the other tube and resembling the background smear obtained for the positive control.

Encouraged, we therefore tried to optimize the conditions for low bacteria concentration. On Figure 66, Gel 1 and 2 show successful IPA-PCR reaction in 50 bact/nL for several concentration of templates. unbiot templates do not succeed in the reaction is the same conditions. The protection of the molecular program templates is therefore mandatory to work in bacteria lysate. Also, the template concentration that was used in in vitro experiments (aT 10nM) was not enough to perform IPA-PCR in lysate. On gel 2 we can also see that with 40nM of biot aT, the reaction happens at 50 bact/nL, at 100 to a lesser extent, but not at higher bacteria concentration. The same experiment was repeated with 100nM of biot aT as a replicate and did not yield PCR products at any bacteria concentration. These experiments suggest that high concentration of templates is also deleterious for the reaction and that an optimal concentration exist. This optimal concentration might also be linked to the lysate concentration. Experiments represented on Figure 67, show that a minimum of 40nM of aT is necessary for IPA-PCR to take place in lysate. Higher concentration also allows to perform IPA PCR but with an increasing amount of side products. On the right gel we can also see that even in vitro, depending on the buffer and incubation time, the concentration of templates can be critical for the success of the reaction. On the gel 3 of Figure 66, various time of IPA incubation were tried. Increasing the incubation time yields an increase in PCR product but no improvements for higher bacteria concentration. 40nM of aT was selected as the final concentration for the molecular program. As for the aT template, optimization on the quantity of trigger strand showed that a minimum of 15 nM of T was necessary to have an efficient IPA-PCR in bacteria lysate. As bacteria are resuspended in a resuspension buffer, when higer concentration of bacteria are introduce in the solution, the concentration of added resuspension buffer is increasing. To see if the important part of resuspension buffer could explain the amplification issues at high bacteria concentration, some experiments were run with bacteria resuspended either directly in the IPA-PCR buffer or as usual in the classic resuspension buffer. No difference was observed between both conditions.

Are protected templates degraded by bacteria lysate?

In complementary experiments it was tried to use the gamS protein to prevent templates degradation by the lysate. gamS was expressed in Top 10 bacteria and purified. The gamS protein is binding the RecBCD complex (exonuclease V) and prevents it from cutting DNA 169- 171 . Experiment presented on Figure 66 gel 1 used gamS and 20 nM of aT unbiot templates to perform the reaction in various bacteria concentrations. A light band can be observed for 50 bact/nL but nothing for higher bacteria concentration. Biot templates proved to be more efficient to prevent the eventual degradation by the lysate. In later experiments gamS was tried in combination with biot templates but did not show improvements.

While the previous results show a strong deleterious effect of bacterial lysate, they do not prove unequivocally that the templates are degraded by the lysate. In the first experiments using probe reporting, it seemed that the probe was degraded but not the protected templates at low bacteria concentration. Increasing the template concentration and using gamS did not help to improve the reaction in higher bacteria concentrations. The problem could therefore be linked to another phenomenon that template degradation. To gain a better idea of the fate of templates we tried to incubate 1µM of templates with bacteria lysate for various amount of time and perform a gel electrophoresis in 40% acrylamide to detect the templates and their remaining quantities. The resulting gels are presented on Figure 68.

According to these gels, it seems that unbiot templates are indeed degraded by the lysate, but it is not the case for the biot templates even with 500 bact/nL. The streptavidin should modify the migration of the strand. Still we can see on the top right gel that there seem to have some template at the correct position, suggesting that streptavidin was substoechiometric.

Streptavidin concentration was therefore adjusted. The 1mg/mL stock was used at 0.3% in IPA-PCR reaction giving 10 11 molecules in 10µL reaction so, 4x10 [START_REF] Brannigan | Protein engineering 20 years on[END_REF] available sites. 10 nM of template is 6x10 10 molecules in 10µL, so for 80nM of templates in total, for aT=40nM, ~5 10 11 molecules and 10 12 necessary would be needed for a complete protection. Only taking into account the molecules numbers and not the double tagging, the final concentration of streptavidin was doubled in the following experiments but should be increased more to cover all the templates.

Buffer optimization for a faster reaction

All the previous experiments were run in the nIPA buffer. Because of the change in the polymerase and its ability to work in higher magnesium concentration, the buffer was optimized again. As shown previously, higher magnesium concentration can allow a faster activity of the nickase. We thought that improving the conditions for the nickase and having a faster molecular program could allow the molecular program to function better in the lysate. But the PCR using the selection primers amplifying the full length NBI gene were performing badly in the IPA V0 buffer (Figure 69 B). However, when performing the reaction with bacteria, the IPAV0 buffer (with more magnesium) revealed to be more efficient. The IPA PCR could work up to 200 bact/nL for amplification of short length fragments. In others experiments, clear bands were also obtained for the full-length gene in the IPAV0 buffer. This buffer was therefore chosen for the PEN CSR experiment.

Further investigation on lysate toxicity with resisting probes

After several trials on reporting probes for the IPA presented in annex iii, a probe able to resist bacteria lysate up to some reasonably high concentration was obtained. This irreversible probe mimics the templates and is protected with biotin and phosphorothioate bonds. Interestingly, only one of the probes, corresponding to the reverse primer p17r3 could be used in bacteria. Despite the same protections, the probe for the forward primer p25f2 was destroyed by the lysate. This dspreportp17r3 probe, consists of two complementary single We also tested the effect of streptavidin and rlys on the speed of primers production in vitro (Figure 72). It showed that streptavidin is delaying the primer production, which could be explained by steric clashes with the polymerase. rlys effect is dependent on the buffer and as seen previously can be toxic for the reaction. It is just delaying the reaction in IPAV0 while it was destroying the reporting system and maybe the template in nIPA. This is again an incentive to prefer the IPAV0 buffer.

Inefficient additives

Several other additives were tried to increase the IPA reaction resistance to bacteria lysate. Addition of RNAse A like in the initial CSR protocol did not change the results. Addition of extra junk DNA to saturate the eventual exonucleases was tried by adding λ phage DNA but was not successful. Extra thermophilic Single Strand Binding (ETSSB) was tried at 0.5 or 1% in hope to protect better the unbiot templates. Trehalose (0.5M) was also envisioned to improve the reaction, with no success. We also tested an increase of dNTPs concentration to test the hypothesis of a complete depletion of them by the IPA at high bacteria concentration. After the change of bacterial strain described in part II, the gamS enzyme was tried with no strong or clear effect. Other extra junk DNA was tried by adding Χ sequence (Χ sequence is the sequence for which the Exonuclease V has a particularly high affinity.). This results in a lot of primer dimers but no IPA-PCR product. In order to test the hypothesis that the Bst polymerase may be sequestered by the bacterial genome, we tried to digest it with the mesophilic EcoRI or the thermophilic TspRI restriction enzymes. None of these solutions were satisfactory.

Toxic components are contained in the pellet of bacteria lysate

When testing all these additives, an experiment was conducted with only the supernatant corresponding to bacteria concentration of 50 to 200 bact/nL (see Figure 73). Cells were lysed in resuspension buffer with 0.2% rlys for 20 min before centrifugation. The results were interesting, as it showed that with only the bacteria supernatant the IPA-PCR was working well in all concentrations up to 200 bact/nL. Unfortunately, we cannot centrifuge each bacterium before putting it in the droplets. But this indicates that the toxic components of the reaction are in the membrane or the other components that can be removed by centrifugation.

Some templates are more sensitives to bacteria lysate than others

After using the same ppT templates for a long time, changes in the plasmid sequence due to library cloning forced us to change the selection primers. They were originally designed to only amplify the gene of NBI but when changed, we decided to amplify a slightly longer fragment. This would allow us to perform a second "cleaning" PCR or "nested" PCR after the selection, with a pair of primers inside the originally amplified strand. We should therefore amplify only the right product and not the primer dimers or side products generated by the suboptimal PCR in lysate. Changes in the templates revealed that some templates can be more sensitive to bacteria lysate toxicity than others. On Figure 74, several primers were tested for the PCR of the NBI gene. We picked two, and generated ppT templates, but they did not yield any PCR product after an IPA-PCR at 100 bac/nL. We checked if these primers needed longer time to be accumulated during the IPA stage.

To do so, several combinations of the new and old ppT were used in in vitro IPA-PCR with Another explanation could come from the different size of the produced primers. In the case of the old templates, best PCR amplification conditions were obtained with a forward primer of 25 bp and a reverse primer of 17 bp. A differential primer production dynamic could result in unbalanced concentration as the IPA progress over time. Some experiment with asymmetric primers concentrations done by Rémi Sieskind showed that unbalanced primers concentration can prevent PCR to happen if one of the primer concentrations is less than 50% of the others.

The obtained results did not explain the trouble happening with bacteria as the new templates seemed even more stable in IPA-PCR reaction. In another attempt of IPA-PCR with 100 bact/nL using the new templates (Figure 76), a light band appeared for 50 bact/nL and a lighter one for 100 bact/nL. This suggested that the new templates might be more sensitive to bacteria lysate. In another experiment presented on

Finding working conditions for sensitives templates and testing heat shocks

Already tested additives, such as ETSSB or gamS were tried to try to protect this new template strand but did not improve the situation. Random primers were also tested to try to saturate the lysate's exonucleases. Increasing the template concentration or addition of primers corresponding to the sensitive templates did not yield to any progress. Finally, heat shocks of either one or two minutes were tried in hope to inactivate the bacteria lysate toxic components.

The first trial was done using the old reverse template to assess which temperature could be supported by NBI. Results on Figure 79

Efficient implementation in bacteria

The trigger v11 promotes amplification of inactive genes

After successful implementation of the IPA-PCR in 100 bact/nL with the new IPAV0 buffer, mock experiments were run in droplets to select the active WT gene among a large excess of inactive mutants. These experiments are presented in part IV (p.244). In the first runs of the mock experiments, no or very little enrichment for the active gene could be obtained after a selection cycle. In one control experiment we noticed that surprisingly some PCR product could be obtained from self-replication of the inactive mutant in bacteria lysate (Figure 81). We first thought that this was due to the small residual activity of the inactive mutant, which we could observe with the purified inactive enzyme in vitro. But, similar IPA-PCR experiments with bacteria expressing another enzyme (inactive KlenTaq) in presence of the plasmid of NBI also yield to some gene amplification, this time with absolutely no activity. Decreasing the trigger concentration to 10 or 5nM instead of 20nM helped to get rid of this effect but resulted in lower quantity of obtained product for the active gene. One hypothesis for the amplification of inactive mutant is that the v11 trigger can bind the primer-producing template and be extended by the polymerase even in the absence of nickase activity. Once the trigger in elongated it could serve as a tailed primer in the PCR reaction. Since, as we will see in the part 3.5, the PCR can perform at very low primer concentration, 20nM of extended trigger could be enough to initiate the PCR. Explanation on why this is only happening in lysate, and not in purified conditions, still needs to be found.

Replacing the trigger by an NBI-dependent source template to initiate the reaction

To prevent self-replication without activity, we decided to use a source template (sceT)

as initiating template instead of the trigger v11. The sceT ssv11 is a hairpin loop who possesses NBI recognition site and generates v11 triggers during extension-nicking cycles with a polymerase and the NBI enzyme. Using the sceT, NBI activity is necessary to produce v11, and no v11 is present at the beginning of the reaction, therefore this approach excludes the leaky generation of tailed primers. On Figure 82, the ability of v11 or ssv11 to trigger primer production in vitro were compared to find out the concentration of ssv11 that should be used in Experiments run in bacteria lysate with v11 or ssv11 on primer production rate showed that it is easier to adjust the kinetics by using ssv11-based molecular program (Figure 83).

Increasing the quantity of ssv11 is impacting the production speed of primers while v11 based

program seem to all have similar primer production speed. It is possible that the linear contribution of ssv11 to the global generation of v11, and hence primers, is significant for the highest concentration tested.

We also thought that using the sceT, which is protected against exonucleases, would possibly increase the program resistance to bacteria lysate. However, IPA PCR experiments in lysate, with increasing incubation times, show no strong difference with the v11-triggered case:

both reactions fail if incubated longer than 45 minutes (Figure 84). This is another observation suggesting that template degradation is not the issue for short IPA incubation times with protected templates.

Overall, these results confirmed the capacity of the sceT-based circuit to select only active genes (Figure 85). In addition, other experiments with increasing bacteria concentration showed that using the sceT could improve a bit the molecular program capacity in bacteria lysate: V11-based molecular programs could run in up to 120 bact/nL whereas ssv11 circuits performed up to 160 bact/nL.

IPA-PCR yield according to NBI activity

We then performed similar experiments, this time performing the whole IPA-PCR in 100 bact/nL of inactive bacteria lysate with increasing NBI quantity. We had to adjust the gel electrophoresis protocol to be able to correctly estimate the DNA quantity (we tested 5x dilution before loading and staining with SybrGreen after the run, instead of in the well). We also used a gel analyzer, the TapeStation from the LBC laboratory. Here samples were diluted 55 times to not saturate the signal. According to both gel electrophoresis and gel analyzer, the IPA-PCR saturates quite fast with NBI activity. The results on Figure 87 show that a 15 min IPA incubation provides an interesting dynamic range in the context of looking for more active mutants. In these conditions, there would be a clear discrimination between the wildtype and better mutant enzymes, with saturation at around 5 times the wildtype activity. At 30 min IPA, the wildtype is already almost at the plateau, and this regime allows mostly negative selection of deleterious mutations.

PCR saturating with primer concentration

Saturation of the IPA-PCR process is not only due to the saturation of the IPA itself but also of the PCR reaction. Several experiments showed that the PCR yield was the same for any primer concentration from ~50 to 500nM. Changing the magnesium concentration could eventually extend a bit the low concentration region, but no major changes were observed.

Modifying the annealing temperature did not change the results either. On Figure 89, qPCR was done on PCR products to estimate their quantity without any measurement bias. These measurements revealed that the PCR is indeed saturating before 100nM of primers. Therefore, to maintain the dynamic range, low concentration of primer produced by the molecular program should be targeted. On Figure 88 we can see that between 0 and 50nM of primers various PCR yield are obtained. This partially explains why we observed a better discrimination in IPA-PCR for an IPA incubation of 15 min.

Robustness of the experiments

The previous experiments gave us an idea of the shape of our selection function in bacteria. However, several issues can arise from the method used here. The first one is that the so-called 'inactive' bacteria are expressing an inactive version of the enzyme. This inactive version cannot cut the DNA but can eventually bind to the recognition site and partially inhibit the added active enzyme. Secondly, in these experiments, the increase in activity is produced by increase in the concentration of the enzyme. In future selections, we do not want to select for a better expression (corresponding to an increased concentration of enzyme in the droplet)

but for an improvement of its catalytic properties. The saturation that we observe at high NBI concentrations may not be representative of what would happen with an increase in kcat at constant concentration. Another way to better study the shape of the selection function would be to pick several mutants with various activity levels and use them to draw the selection function. This option was tried during the course of the project but among the picked clones who seemed to have various activities, few of them appeared to have mutations that could explain this variability. Variability in expression level from batch to batch could explain the observed differences. Some details about these clones are given in Part II.

The primer reporting probes could eventually be used in the selection process to monitor primer production in droplet and decide when to launch the PCR step. Unfortunately, when adding the probes in the IPA-PCR process it was not possible to obtain any PCR product at the end, even with probes concentration as low as 10nM, a concentration hard to detect under the microscope and giving very small signal in real-time PCR machine.

Conclusion

To have an efficient positive selection, short IPA incubation times should be preferred.

The amount of expressed NBI might also need to be adapted and decreased to be in conditions where the primers production by the molecular program is not saturated. Solution to control the level of expression are presented in Part II. For negative selection, using longer IPA incubation can allow to have a ON/OFF system where all the active enzymes survive and inactive ones are discarded.

II.

Library construction

Library construction methods

The sequence space is the set of all protein sequences that could possibly be generated starting from the wild-type sequence. Directed evolution methods are especially well suited for exploring the sequence space of a given protein without taking care of any information. Their with continuous axis is theoretical and highly misleading but allows to visualize the problem arising in DE experiments (Figure 90). The exploration of this fitness landscape strongly relies on the design of the library. The introduced mutations enable to move the enzyme through the landscape and should, to be efficient, avoid the hollows of inactive mutants and direct the enzyme towards the highest fitness peak, if the goal is to improve an enzyme. That's why smart libraries should not only reduce the size of the studied sequence space but also find the best trajectories in the fitness landscape. Experimental results have allowed to draw some conclusions about the distribution of fitness effect of mutation, that means the relative frequencies of neutral, deleterious and beneficial mutations. Advantageous mutations are rare, and the strongly beneficial ones are exponentially distributed and therefore difficult to access among the other possible mutations 173 . For this reason, focused mutagenesis libraries exploit the available structural, functional and phylogenetic data to preselect positions or regions that should be preferentially mutated to observe more beneficial mutations. Because of the redundancy and structure of the genetic code, the distribution across the 20 amino acids at the protein level is not uniform and randomization of a codon does not allow to have non-biased probability to find one or the other amino acid 174 .

Smart libraries have therefore to meet many criteria. Their purpose is to provide highly evolvable sequences, that can span evenly the sequence space with no bias comprising few or no inactive sequences and therefore a lot of at least functional but preferentially improved ones.

Libraries able to generate the most variability at the level of amino acids sequence with lower variation of the genetic codes will be preferred as they allow to explore a larger sequence space with reduced library size. Avoidance of high proportion of the functional wild-type gene, used as a template to introduce the mutations, is also major criterium as it decreases the throughput of the experiments 175 . Relatively easy and short protocols will also be preferred as evolution experiments usually require a lot of cycles that can be already quite long according to the used method. Methods allowing to explore the synergic effect of beneficial mutations or eventually epistasis phenomenon by mixing mutation are also necessary.

The different type of base mutations

The three types of mutations that can be introduced in genetic sequences are substitutions, insertions and deletions (Figure 91). Substitutions are divided in two kinds of mutations, the transversions and the transitions. A substitution is called a transversion when there is a change in the nature of the base, meaning that a pyrimidine (T or C) is changed in a purine (A or G) or conversely, giving 8 possibilities. The 4 possible transitions are the substitutions that do not change the nature of the base (A<->G or T<->C). According to the used mutagenesis process, one or the other will be favored but among the different types of transition or transversion some can be preferred over the others. Ideally, methods that does not have any bias in one or the other substitution would be preferable to explore evenly all the fitness landscape. Insertions and deletions, which respectively introduce or remove one or several bases in the sequence, are generally not wanted. This is because they are more likely to generate inactive clones by shifting the frameshift of the sequence. So, the best method should reduce the frequency of apparition of these mutations unless they can provide insertion and deletion of in frame codons.

Library metrics

Metrics have been developed and should be taken in consideration when designing a library. One must discriminate between the library size referred as the number of screened or selected variants and the library's effective size. The second one is the number of distinct protein variants in the library that is greatly variable according to the used methods and can greatly influence the results. As the latter is more difficult to evaluate, library size usually refers to the former. Some metrics, such as the full coverage, expected coverage and the probability of having the best variant in the library can be defined and estimated 176 . The full coverage is the probability that the library contains all variants in the studied sequence space and, the expected coverage is the fraction of sequence space that is expected to be represented in the library. Good library generation methods should try to maximize these metrics.

According to the evolution purpose, the protein size and available information possibilities and methods, choices are not the same. In the next paragraphs different library creation methods are presented. These methods can be of great interest to deactivate genes at random for a genome-wide experiment, but they suffer from important bias in the spectrum of reachable mutations. Yet, they are cheap and relatively easy to implement [START_REF] Packer | Methods for the directed evolution of proteins[END_REF] .

Increasing error rate in DNA replication in vivo

• Mutator strains Naturally occurring or engineered bacterial strains called mutator strains with defects in the proofreading efficiency of their replication polymerases and/or also in the mut xiii DNA repair system 183 can be used to introduce random mutation in gene sequences [START_REF] Packer | Methods for the directed evolution of proteins[END_REF] . In a wild-type E. coli bacteria DNA replication by the polymerase III happens with an error rate of 10 -10 mutations per replicated base. To compare, a plasmid transformed in the widely used mutator strain XL1-RED (Stratagene) will have an average error rates of 10 -6 per base per generation.

In this strain, mutations of the three enzymes mutT, mutS and mutD are combined giving an average rate of 0.5 mutations per kb on the overall genome after 30 generations of growth.

xiii MutS, MutH and MutL proteins detect mismatch in DNA sequence and direct the repair machinery to it.

166

Plasmid propagation in such strains revealed a rich spectrum of base modification like single base insertions, deletions and substitutions along the entire plasmid. Observed mutations are mainly transitions for 85%, transversion for 10% and frameshifts for 5% 184 .

Like the chemico-physical techniques mutator strains don't require complex procedures and represent a very cheap option to generate libraries. They do not require specialized equipment nor experience in recombinant DNA cloning techniques. A large library can be achieved in 2 or 3 days. But they also have a lot of drawbacks. As mutations accumulate all over the genome, the organisms can have highly variable phenotypes and a limited genetic stability. Mutation load can generate slower growth rates for highly mutated cells causing asynchronous growth over the population and an overgrowth of the cells with reduced mutator phenotypes. Also, as one can see with the XL1-RED strain, several days are needed to generate more than 1 or 2 mutations per gene so faster methods must be preferred for high-frequency mutagenesis. And last but not least, the many widely spread mutations all over the host genome can interfere with the selection process. For instance, changes related to the target protein expression regulatory elements could select for a more expressed protein instead of an improved one.

• Mutations located on the target plasmid Some methods have been developed to solve the issues related to the accumulation of mutation on the genome of the carrying organism. They involve an external mutagenic polymerase inserted on a plasmid.

In one implementation the mutational load is reduced by introducing temporarily the mutator plasmid transforming transiently the strain in a mutator strain. The plasmid carries a mutated version of the proofreading subunit of the DNA polymerase III that can replace the natural one increasing the mutation rate by 20 to 4000-fold during this time 185 . Then the temperature-sensitive origin of replication allows the rapid removal of the mutator plasmid restoring a stable strain.

Others managed to restrict the mutations to the target plasmid only, by using a plasmid specific polymerase, the Pol I polymerase specialized in ColE1 plasmid replication. Using an error-prone low-fidelity Pol I and a strain expressing a temperature-sensitive allele of the natural Pol I they ensure that the mutated Pol I is predominant at 37°C and limit mutagenesis to the ColE1 plasmid 186 .

Changing of host organism for the eukaryotic S. cerevisiae, CC Liu and his colleagues took advantage of the nucleus compartmentation and used a cytoplasmic plasmid together with a specialized error prone polymerase to constrain the mutation load in the cytoplasm leaving the host genome intact 187 .

These techniques allowing to limit the mutational load over the organism are more sophisticated and require more cloning work. But they did not fix critical issues of in vivo random mutagenesis methods. The mutation rate is still quite low and there is absolutely no control in the library design. One cannot choose the regions that should be mutated, adjust the mutation rate or easily tune the mutational spectrum.

Increasing error rate in DNA replication in vitro

• Error-prone PCR Various methods exist to generate random mutagenesis in vitro. The most widely used is the error-prone PCR (epPCR). It was first described in1989 by Goeddel and co-workers and rely on the modification of PCR buffer condition altering or enhancing the natural error rate of some DNA polymerases 188 . Such methods can reach an increased mutation rate of 10 -4 to 10 -3 per replicated base against 10 -5 for a wild-type Taq polymerase 189 or 10 -7 for the high fidelity Q5 polymerase (NEB). The Taq polymerase is often used due to its naturally high error rate but some specially designed enzymes, like the mutazyme (Agilent), with higher mutation rates and various bias in the induced mutational spectrum can be preferred. Buffer modifications mostly play on an increased magnesium (Mg 2+ ) concentration that will stabilize noncomplementary pairs, the addition of unnatural polymerase cofactors like the manganese (Mn 2+ ) that will increase the enzyme error rate, the use of unbalanced concentration of dNTPs and finally, the addition of mutagenic dNTPs (e.g. 8-oxo-dGTP or dITP) that can pair with wrong or any other base. Other buffer modifications like the ion concentration or the use of additive can also help. Mutation frequency as high as 20 nucleotides per kb have been achieved simply by varying the nucleotide ratio and the amount of manganese 190 .

Control of the error rates can be achieved by varying the number of PCR cycles and/or the DNA starting concentration. As mutations accumulate at each cycle the more cycles are run the more mutations you can obtain at the end. Starting with few copies of the template give rise to more mutation as, for each new cycle, the chance to use an already copied and mutated version as template increases.

Other amplification methods can be used with the same modifications towards higher error rates in replication. One interest of using the error-prone Rolling Circle Amplification (epRCA) (see F.b) for instance, beside the absence of thermocycling and specific primers design, is that the product can directly be transformed in the host, letting the homologous recombination of the gene happen in vivo 191 .

epPCR methods are relatively easy to implement and necessitate just some basic cloning techniques to introduce the obtained mutated genes in a vector. Like above presented random mutagenesis methods, epPCR generates libraries that are biased toward some specific base changes from a base to another. This is highly dependent on the polymerase type and buffer conditions and can be adjusted using multiple enzymes with different bias, like in the mutazyme II system (Agilent), or unbalanced dNTPs concentrations. But, exploring evenly all the possible base change is impossible. This natural bias, added to the fact that point base mutations are introduced, create a bias in the resulting amino acids spectrum that can be studied. Indeed, it is very unlikely that random mutations happen on consecutive nucleotides, so some amino acid changes will never be reached using these modifying the sequence. In this method, contrary to the RID, there is absolutely no control over the randomly inserted bases.

Similarly, in the sequence saturation mutagenesis (SeSaM) 196 the gene is split in a pool of ssDNA fragments of random size distribution, this time using modified α-phosphothioate nucleotides. Mixing these nucleotides with normal ones allows to have random apparition of phosphothiate bonds in the sequence, which can be specifically cleaved in a subsequent reaction. The use of biotinylated forward primers provides a way to keep only one of the two strands. Like in the RAISE technique, a terminal transferase is then used but this time to add one or several copies of the universal base deoxyinosine, that can pair with any other base.

Therefore, when these fragments are used as primers in a PCR, random nucleotides will be put at deoxyinosine positions leading to up to 30% of consecutive base substitutions. This technique still has some bias due to the use of the deoxyinosine base that is favoriting transversion, so it can be interesting to use in compement of the epPCR that is transition biased. In the transposonbased approach 197,198 an engineered nonspecific version of the Mu transposon inserts itself at random positions in the sequence. Removal of the transposon by a restriction endonuclease creates one break per gene with a loss of 3 bp that can be kept by ligation or change using DNA cassettes thus generating deletion, substitution or insertion. Combining such random codons insertion techniques and error-prone replication techniques can allow to navigate more evenly on the amino acids fitness landscape but these methods cannot give a full understanding of a finite landscape as the number of sequences needed to explore all the sequence landscape of a protein with random mutations is too big.

Focused mutagenesis and recombination methods are presented in annex 1i.1 and i.2, p.316. These methods give much more control on the position of mutations insertions.

Therefore, smart libraries can be constructed by using structural or phylogenetic information.

Assisted design

To assist directed evolution practitioners a lot of computational tools have been developed for the design of the libraries. On one side these softwares can help in choosing the best mutagenesis methods, in the design of primers or oligonucleotides you need or in optimizing your library diversity. On another side they can determine the region that has to be mutated or reduce drastically the size of the library by simulating some identified hot-spots mutation in silico using structural information.

Softwares that help on the practical side of the library design ( The second type of software have specific applications according to the goal of the experiment and available data. They are sometimes the same that are used in rational design.

Some are based on structural information and designed to detect amino acids or regions involved in protein-ligand interaction, some are analyzing the binding pocket itself or the access tunnel that allow the ligand to access the binding or catalytic site. For the identification of targets for increasing thermostability both structural and phylogenetic information can be used.

Amino acids with high mobility in molecular dynamics experiment are better candidate to target as more rigid structures are usually more stable. Phylogenetic information can also help finding stabilizing mutation as consensus amino acids have been shown to generally increase protein stability [START_REF] Bershtein | Intense Neutral Drifts Yield Robust and Evolvable Consensus Proteins[END_REF] . They also provide more evolvable starting point for the evolution. On a same manner, conservation profile of amino acids throughout a protein phylogenetic family is precious. Indeed, mutating these conserved amino acids in the sequence is generally not recommended unless they are in the catalytic site and the goal of the experiment is to change the catalytic function or activity. They often are involved in the global structure stability and there is high probability that modifying them lead to structure disruption. Avoiding mutations on these amino acids improve the proportion of functional mutants in the library. Besides, limiting amino acids substitutions to the one already found in natural evolution, although it is obviously restricting the scope of possibilities, improve the fraction of functional mutants in the library.

Disruption of the structure is also an important problem when constructing family chimeras or recombining mutants. Some softwares can help finding the crossovers size and number that should be targeted to avoid misfolding by determining subdomains boundaries. In silico simulation of mutagenesis and calculation of resulting free energy or global protein rigidity can help eliminating some specific amino acids substitutions to reduce the pool of aa targeted in saturation mutagenesis method for instance. Using phylogenetic information can also help predicting mutations effects. In the Reconstructing Evolutionary Adaptive Paths (REAP) approach theoretical ancestral proteins are used as the foundation for library variants.

Ancestral sequences and signatures of functional divergence allows to statistically evaluate which amino acid mutations in which sites have higher probability to produce functional proteins with varied phenotypes.

Finally, some programs and model can use information obtain from previous evolution cycles to propose the next mutations to assess. When designing a library using all these available computational tools the limit between assisted directed-evolution and rational design become very tiny as the library size can become very small. Using the Framework for Rapid Enzyme Stabilization by Computational libraries (FRESCO) 199 Wijma et al. shrank the library to only 64 mutants by selecting mutations improving predicted free-energy of protein folding, testing sulfide bonds apparition along the backbone and finally performing in silico screening of the selected mutations. Such small library size does not require a full directed evolution scheme. 1.3 Should we still use random mutagenesis?

As explained earlier, it is now acknowledged that designing a smart library is critical for the DE experiments. And random is not smart. So, is there still a place for these random mutagenesis techniques? Random introduction of mutations will always be an absolute necessity for a protein with no known structural or phylogenetic information as there is no way to create a rational library without knowledge. It can be used all over the directed evolution process or serve to identify regions of the protein that should be targeted for engineering. If structural data are available, it can be used to test hypotheses developed from the existing structure. In some cases, knowledge of the structure might not allow to determine specifically which amino acids are involved in the targeted behavior. However, it may still be possible to determine a target region. In such a situation, focusing random mutations in that area can be very useful and reduce the studied sequence space to explore.

Structures may say quite little about function. Cristal structure are frozen in a specific configuration, which may be different from the functional one. Also, deducing the determinants of protein function at the amino acid level using structural information can be very challenging.

It is sometimes not possible to determine where mutations should be introduced according to the goal of the DE experiments. Typically, projects targeting an increase in stability, thermoresistance, folding capacities, allosteric changes, increased stability in a particular buffer or research of epistasis will generally involve amino acids all over the sequence of the proteins 200 .

Even if as we have seen phylogenetic information can help to increase stability and thermosresistance, using random mutagenesis stay the only possible way to explore the sequence space for this purpose once phylogenetic information have been used or if only the structure is available. In the case of experiments targeting catalytic activity, it has been discovered that mutations distant from the active site could have significant impact [START_REF] Romero | Dissecting enzyme function with microfluidicbased deep mutational scanning[END_REF]201 that are even difficult to explain going back to the structure. So, random mutagenesis can be used to probe mutations that are distant from obvious site location or not present in naturally occurring orthologues.

Rationally designed libraries are thought to be the key for more efficient evolution experiments, but they shrink the fields of possibilities and reflect only current understanding of enzyme function which is limited. They put on blinders on researchers that will stick in the sequence space of known or envisioned possibilities based on preconceived notions. They do not let space to the unpredictable and surprising options that life could have to offer. So even if some sequences appear as surely dumb to try, and the process is longer, random mutagenesis stay the best way to unveil the secrets of structure-function relationship and discover the inconceivable. Beside some researchers use completely random library enriched in coding sequences only extracted from soil to try to discover new enzymes activities that are not known to exist 202 .

Outside of these rational considerations results speak for themselves and it has to be noted that random mutagenesis has already yield to rapid gains in fitness for some promiscuous functions and a lot of valuable improvements in a lot of study. On a more practical side, random mutagenesis methods are easy to implement, do not require a lot of molecular biology steps and even if oligosynthesis prices are going down they stay cheaper than most other techniques. That is probably why they are still widely used by many laboratories.

Smart libraries were first developed because of the small screening capacities of DE methods. Arrival of high throughput methods, like the one we are developing changes this problematic. Generally, few mutations can be sufficient to find an improve protein, therefore, if the DE methods allow to test enough possibilities in this 2-mutations far landscape, the design of the library is not that important. High throughput allows to cross valleys of the fitness landscape and to detect epistasis effects which would be hard to detect in a smart library.

One major problem of the random mutagenesis technique is that you can reach only a small portion of amino acids exchanges. But this inconvenient can transform in an advantage as residues introduced by single base substitutions are slightly more likely to be tolerated 174,203 .

As we can see in the Figure 92 single mutations generated at the level of the GAA codons lead to amino acids that are either in the same category of amino acids so that are more likely to not completely disturb the structure. Indeed, the probability of staying with a negatively charged amino acids (Glu, Asp) is 0.3 as well as the one to reach non-polar aliphatic amino acids (Ala, Val, Gly). To reach a positively charged (Lys), a polar uncharged amino acid (Gln) or a stop codon is only of about 0.1 and there is absolutely no chance to reach ring or aromatic amino acids.

Random mutation techniques are not perfectly random, and bias are introduced differently according to the chosen techniques so even if they are not based on information, they can beneficiate from some "rational" design. In order to increase the proportion of reachable amino acids, Cambray and Mazel designed an algorithm to construct a synonymous sequence of their target gene systematically including the codon with the most different evolutionary perspectives 204 . Both wild-type and the recoded gene were used and greatly improved the diversity of generated mutants. The mutagenesis assistant program presented earlier can typically help to find the best random mutagenesis technique to use according to your protein target. Of course, random mutagenesis techniques can always be used in combination of focused mutagenesis techniques into a single diversification step or in alternative cycle of evolution to increase the explored sequence space.

Analysis of the obtained libraries

Sequencing is obviously the best way to analyze a library diversity, bias and so on. But, it is not always possible to use it. Due to the large size of the library, next generation sequencing seems to be the more appropriate, but NGS sequencing costs are still quite high and most of them are restricted to short fragments size of 300 bp (or 600 pb for paired-end illumina sequencing). The MinIon nanopore sequencers can perform long reads but with high error rates not really compatible with this particular application (minimum 5% of error vs 0.1% for Illumina NGS methods). But for simple assessment of the mutation rate, NGS sequencing of a part of the gene can be sufficient. Sanger sequencing a few picked clones can also give an idea of the library diversity and mutation rate. Other indirect strategies can be used. Assaying the activities of some picked clones in 96 well plates and compare it to the wild-type can provide an estimate of the distribution of activity of a library. According to some researchers an error rate resulting in 30-40% of inactive mutants (defined as <10% of the parent enzyme) is suitable

for evolutionary experiments 205 . The number of inactive mutants is highly correlated to the mutation rate but is also dependent on the protein sequence, so this rule should be adapted to the studied protein. Estimation of the wild-type remaining sequences can also be made from the targeted error rate in random mutagenesis according to a mathematical model 205,206 . To assess recombination methods average number of crossover and their relative locations, labeled probes hybridization on nylon membrane containing spotted colonies of the library can be run 205 .

Statistical analysis of the signals provides an estimate of the crossover length and of the proportion of actually recombined gene.

Limitation on the library size

The sequence space that can be studied for every protein is incredibly large and researchers working in the field of DE have developed several strategies to increase the number of parallel tests one can run at each evolution cycle. But, the number of mutants that can be screened at every cycle remains limited regarding to the size of sequence space. For in vitro DE methods, the bottleneck in the library size comes more generally from the number of compartments or display elements that can be used or generated to run the screening or selection tests in one evolution cycle than from a limitation in the diversity generation methods. A library in a mRNA ribosome display technique can reach 10 13 mutants. But, in the case of evolution techniques relying on an organism to express the protein, this bottleneck is generated at the transformation step. Furthermore, this display technique is particularly adapted to binding assays and not easily adaptable to catalytic ones.

The transformation efficiency varies a lot depending on the host. Up to 10 10 CFU/µg of DNA can be reached for E. coli, 10 7 per 10 8 cells for S. Cerevisiae, 10 7 for mammalian cells or 10 5 for microalgae 207,208 . These values are all given for the best conditions of transformation.

For E. Coli an efficiency of 10 10 will be obtain in the best case for a supercoiled and rather small plasmid (2.7 kb). As soon as the plasmid size increase and the plasmids are relaxed, or nicked, efficiency will greatly decrease. Efficiency of ligation product transformation is known to be at least 1 to 3 orders of magnitude less efficient than the one of a supercoiled plasmid 205 . As a result, usually large library sizes in supercompetent E. Coli cells are around 10 7 mutants and can reach 10 8 for the biggest ones. Moreover, several individual transformations are often combined to reach such values.

The way libraries are designed and cloned can greatly influence the library size and composition. In the classic restriction/ligation cloning method precaution must be taken to minimize the amount of self-circularizing plasmid, so-called background ligation products, and carry over of the initial plasmid templates that have been used for PCR. As miniprepped templates gets transformed way easier than ligated product they can overtake the population, leading to almost no mutant in the final libraries. The background transformants are decreasing the diversity of the library and are also wasting selection efforts. In the special case of loss-offunction screenings they will necessarily be confused with interesting clones and such products must be totally eradicated. In the case of classical DE experiment generally up to 1% of selfligating product are tolerated 205 .

To avoid the above-mentioned problems, most methods use the DpnI restriction enzyme that can digest methylated DNA obtained from bacteria or artificially methylated by the dammethyl transferase. This allows to remove the template plasmids. Carefully chosen restriction enzymes with non-palindromic overhangs enable easy directional cloning and limit the creation of vector dimers or recircularization. But generally, even such precautions are not enough, and the cloning protocol must be optimized, through a time-consuming process with additional steps decreasing DNA yield. Several new methods have emerged not exclusively based on restriction cloning but on megaprimers annealing [209][210][211] . In the MEGAWHOP library generation technique 212 , the mutated gene is used itself as a megaprimer to PCR the full plasmid. This way, after a digest by the DpnI enzyme, no template nor empty vector or multiple insert vector is present in the library. The double nick generated by this method are repaired inside the bacteria.

Despite the advantage of leading to library with 99,9% of plasmid with a single insert, it seems that the efficiency of the method is relatively low. Depending on the source one can find only 500-1000 colonies for "standard" competent cells of an efficiency of 10 7 /µg, 2000 transformants in cells with an efficiency of 10 8 /µg and up to 10 5 transformants in the best case 176,205,212 . In a similar fashion the AXM mutagenesis 213 protocol also uses the mutated gene as a megaprimer but claim to reach 10 8 clones in a single E. Coli electroporation. Here, only one strand of the gene is used as megaprimer thanks to the use of one phosphorthioated primer that allow the conservation of one of the two strands after digestion of the PCR product by an exonuclease. Contrary to the MEGAWHOP method the megaprimer is then used on a singlestranded and uracilated circular phagemid DNA. After transformation, the uracilated strand is degraded in the bacteria therefore the megaprimer mutation are preferentially kept in the host.

After E. Coli, the yeast S. Cerevisiae is the second most used host organism in DE 207 .

Not only yeasts are preferable for expression of eukaryotic proteins or heterologous membrane proteins, but they possess a gap repair system that can allow to transform open plasmid and vector with homologous sequences at both ends. Homologous recombination in the yeast will complete the plasmid ligation. Researchers thought about using the yeast high frequency of homologous recombination to create highly shuffled libraries without the need of cloning. In Another advantage of the yeast is the potential of expressing genotoxic enzymes that cannot be stably transformed in bacteria, thanks to the nucleus compartmentation. But the global efficiency remain quite low as such experiment typically gives 10 000 clones.

Up to date, best techniques to reach the largest library size in vivo remain the continuous evolution techniques. In continuous evolution all the steps carried out in directed evolution must be supported in some self-sustainable manners that do not require the intervention of researchers. Gene expression, selection or screening and introduction of new mutations should be autonomously done. By definition these techniques do not require a transformation step, apart from the initial one. They can allow to propagate gene through many rounds of evolution with constant introduction of mutation. The natural error-rate frequency of host organisms is generally not very high as their goal is to maintain their integrity. Providing that a mean is found to link the activity of the evolved protein to the propagation of the host organism, this is the main limitation of the technique. Using mutator strains or the derivatives presented in the random mutagenesis techniques like plasmid specific error-prone polymerase can allow to increase this natural mutation rate and reach huge (although difficult to evaluate) library size at the end of the experiment. Having more than 2 mutations per generation is essential to cross fitness valley that are not crossable otherwise.

GM3 culture system 215,216 has been developed to allow to change culture chambers and improve continuous evolution of bacteria getting rid of the bacteria forming biofilm in the culture chamber. Methods using orthogonal extranuclear yeast replication system 187 or the synthetically optimized retrotransposon Ty1 217 allow to introduce mutation on a target gene in yeast allowing for continuous evolution. In another approach, using the yeast sexual mating to introduce recombinant mutational cassette between evolution cycle Cornish's group 218,219 managed to apply selection to 10 13 mutants. Such library screening capacity is quite impressive as it would require at least 10 5 to 10 6 cycles of in vitro evolution to reach similar values. But, Figure 95. What is the best library generation methods according to your goals, means, available information and evolution stage ? Despite the light tone of this decision tree, going through all the possibilities allows to appreciate the different reasons to choose a method over another, the pros and cons of using one or another method, the variety of library generation methods and the important place of the random mutagenesis despite the recognized need of constructing smarter and smaller libraries. Assistant programs presented in the section 1.4 can of course be combined with all the proposed mutagenesis techniques.

2. Expression of the toxic Nt.bstNBI in E. Coli

The Nt.bstNBI enzymes is toxic for the host bacteria as it can cut its DNA at several positions. On the genome of the takara BL21 strain for instance, already 3462 NBI recognition sites can be found. Expressing the enzyme in a prokaryotic host might sound at first difficult but the enzyme is commercially available at New England Biolabs, that manage to produce it at a large scale.

Co-expression with a corresponding methylase

Expression strategy

New England Biolabs wrote a patent explaining how they manage to express the NBI enzyme using bacteria 220 . As NBI is closely related to two restriction modification systems MlyI and Ple I, that possess the same recognition site, NEB team decided to use the Ple1 methyl transferase from Pseudomonas lemoignei to protect the DNA of the host cell (despite the existence of the corresponding Nt.bstNBI_M methyltransferase).

We decided to use the same strategy for the expression of the NBI library. The PleI methylase methylates the GASTC (S= G or C) site on the adenine base. To first try the system on the wild-type version of the enzyme, Kazuaki Amikura from the Kiga lab and the master intern Ruochi HSU from the Fujii lab, who were initiating the project before I arrived, ordered the genes of the nickase and the methylase to a gene synthesis company (IDT). Some modifications were made to the sequence so that it does not possess any NBI recognition site.

One was present near the position 221 in the amino acid sequence. So, the codon TCT coding for a serine has been changed to AGC, also coding for a serine. Another important change was made at the level of the T7 promoter used for the NBI gene, changing slightly its sequence from TAATACGACTCACTATAG to TAATACAACTCACTATAGGG. As described in the patent, the NBI endonuclease was cloned in a low-copy plasmid, here kanamycin resistant pSB3K3 (10-12 copies /cells) under the modified T7 promoter, a patent-derived rbs and supplemented with a histidine Tag. The PleI methylase was cloned in the high copy ampicillin resistant pUCIDT (500-700 copies/cells) under the control of the lacI gene and promoter, to allow a leaky expression of the methylase.

First expression in the KRX strain

The first transformations of the plasmids were done in the KRX strain (Promega). Cotransformation of the two plasmids in chemocompetent cells failed, suggesting that successive transformations were necessary. We thought this might come from the need to first protect the cell DNA by the methylase, before introducing the toxic gene, as recommended in the NEB patent. But surprisingly, transformation of the pNBI gene alone in the KRX strain led to some transformants. It is actually known that E. coli can tolerate low-level expression of some Nicking enzymes in the absence of the cognate methyltransferase 140 . Nevertheless, after transformation of the KRX bacteria by the pleI plasmid, the cells were made chemically competent and transformed again with the pNBI plasmid leading this time to colonies with both plasmids.

KRX colonies bearing either only the pNBI plasmid or both pleI and pNBI plamsids were used to express the enzyme. As shown in the Table 11, the growth was really slow especially for the not genome protected bacteria (without ple1) who needed 7 h to reach an OD600 tub of 0.6.

After an induction time of 3h30, bacteria were collected to purify the protein using an his-tag purification column. The bacteria carrying only the pNBI plasmid did not grow after induction, their OD600 even decreasing during that time.

The Figure 96 shows the SDS-PAGE results of the different fractions during the protein purification. We can see that the protein was correctly expressed and purified when pleI is present but only a very faint band is observed in the wash buffer when pleI is not present in the bacteria. We tried to introduce the pRare2 plasmid to increase expression level of NBI. pRare2 is coding for 7 rare codons tRNA genes. Its usage, in addition to increase NBI expression level, could allow to avoid expression bias due to codon usage of the different mutants. Unfortunately, it required the use of chloramphenicol that impaired strongly the growth of the bacteria, which was already very slow, so we gave up. When we equipped the new lab in Paris, we decided to buy an electroporator, as it is known to lead to higher transformation yields. Even a simple 10-fold increase in transformants can be of critical importance to explore better the fitness landscape. We were especially concerned by the fact that the feasible droplet throughput was 100 times higher (~10 7 ) than the transformation throughput obtained with chemocompetent cells.

Surprisingly, it was not necessary to transform the electrocompetent cells with the plasmids one after the other. Co-transformation lead to high transformation yields in OverExpress C43 bacteria. The OverExpress bacteria are known to be a suitable host for toxic proteins expression. They exist both as chemocompetent and electrocompetent cells, contrary to the KRX cells, which are only available commercially as chemocompetent. Four different versions exist, two different strain (C43 and C41) with or without the pLysS plasmid, producing the T7 lysozyme, an inhibitor of the T7 RNA polymerase, reducing basal expression of the target protein. They were all tried for NBI expression and only the C43 exhibited activity after induction, with a reasonable growth rate. As pLysS is a chloramphenicol resistant plasmid, growth of bacteria carrying the plasmid was also greatly reduced.

After our first tests with newly transformed bacteria, the IPA-PCR reactions done either with OverExpress C43 bacteria or KRX comprising the ple1 methylase and the expressed NBI enzyme revealed the insertion of a big > 1500 bp sequence at the beginning of the NBI gene (see Figure 98). Sequencing of a few clones and BLAST research showed that this sequence was issued from the transposon IS5 221 . Although this insert seemed to be linked to the toxicity of the enzyme, it was surprisingly not decreasing the enzyme activity as bacteria carrying the gene had normal activity level according to loopact assays. Digging further in the literature, it was found that this insert can have a regulatory effect on translation. Depending on its orientation, it can inhibit or enhance the expression [222][223][224][225] . This insert being a self-splicing intron it should not change the sequence of the enzyme. Its insertion site is known to be CWAR where W = A or T and R = A or G and this indeed corresponded to its insertion point, just after a CTAG site in the NBI sequence. However, we observed that the bacteria still had quite high level of expression of NBI. The only noticeable difference was a faster growth.

Investigation going back to the plasmid extracted from the KRX cells showed that actually this insert was already present in a fraction of the plasmids. Indeed, transforming the miniprepped plasmid back in KRX bacteria led to two kinds of colonies, some bearing the insert, and some with the initial sequence of the gene. We therefore thought that the toxicity was coming from the fact that the enzyme was transformed without the protective ple1 methylase. It was surprising to find that the inserted mutant still had NBI activity in vitro. As KRX strain was supposed to have a tight regulation of the expression control it seemed possible to use it to clone and amplify the plasmid, because toxic expression would be efficiently prevented. But, it appeared that it was not the case.

To try to overcome this issue, several strains supposed to not express proteins under a T7 promoters were tried. The classical NEB5α and, NEB10β, but also NEB F'iq, which is supposed to allow the cloning of toxic proteins, were transformed with the pNBI plasmid. In all these strains the insert appeared much faster than in the KRX strain, or very few clones were obtained after transformation. It was possible to keep the intact plasmid in KRX strain during an overnight on plate just after plasmid transformation and even for a liquid overnight. But, using these strains not expressing the T7 polymerase led to apparition of the insert within a few hours after the transformation. Currently, no solution has been found to clone and recover the pNBI plasmid with confidence about its sequence integrity. Interestingly, when transforming the plasmid obtained from KRX strain back in KRX, two kinds of colonies appear. Some small colonies that usually contains the right gene, and some bigger colonies that contains the insert.

Growing the small colonies overnight generally allows to maintain and recover the vector with the correct gene after a miniprep.

To try to overcome this insert problem, I thought that NEB researcher might have a trick for the plasmid construction. Going back to the NEB patent I noticed one major point that was not taken into account back in Japan. The strain used for expression in the patent was a particular NEB strain (ER2566) that was deficient in the After gel electrophoresis, the PCR product is higher than the expected size of 0.6 kb. This experiment shows the apparition of the IS5 intron in the bacteria strain and testify for its absence of effect on the expression level and activity of the enzyme since bacteria lysate can still perform the IPA-PCR reaction.

did not help for plasmid cloning but could improve NBI expression.

We contacted NEB to know if it was possible to obtain this ER2566 strain and they pointed us toward one of their commercial T7 express strain (#C2566), which is lacking this restriction system. This new strain was not available as electrocompetent cells, so we had to make it competent in the lab. The chemocompetent cells were first transformed with pleI so that 1) the host DNA would be protected before introduction of the nickase and 2) cells to be made competent would carry a resistance to an antibiotic to avoid contaminations. Even if the co-electroporation seemed to work in the previous electrocompetent cells, we thought that possibly the absence of genome protection in the host cells could counter-select highly active mutants. Furthermore, incomplete protection of the bacteria genome could be the reason for the apparition of the insert. Efforts to make efficient electrocompetent cells are depicted in the annexe vi. In the T7 express strain, induction of the T7 polymerase is done by IPTG, thus when using this strain IPTG will induce both the expression of NBI and the ple1 methylase. After successful transformation of the NBI gene in the newly made competent cells, NBI could be expressed properly. The T7 express pNBI/ple1 bacteria grew normally until induction, and even if the optical density of the culture was barely increasing during induction, the bacteria lysate assay using the protected loopact showed higher nicking activity than the former KRX bacteria.

Adaptation of culture conditions

It is important for our experiments to control the average expression level of the enzyme, as too much activity can slow down or saturate the IPA. It is also preferable to be placed in conditions were the concentration of the enzyme is limiting and it is really its activity that will control the self-selection. We performed tests, with the KRX strain, to search the best way to control the expression level of the enzyme. Culture time after induction, starting OD600 and inducer quantity were screened and results are presented on the Figure 99. The results show that the main criterium to control the NBI quantity/activity in the bacteria is the induction time.

Compared to the starting induction OD600 or the inducer quantity, it offers a widest range of activity and thus a finer control. For T7 cells, NBI expression level was better controlled by inducer concentration (see Figure 102).

It is known that expression level can vary from cell to cell [226][227][228] . We therefore performed experiments to assess cell-to-cell expression variability in the new T7 express strain. Activity assay revealed that NemGFP-NBI (for which the bacteria that had regular shape) was inactive, contrary to that NBI-T7-GFP and NBI-CemGFP. This suggested that the long shape was related to NBI activity and toxicity. Observation of the non-fluorescent bacteria expressing either NBI or its inactive counterpart confirmed that the filamentous phenotype was due to NBI expression (see Figure 100). Such changes in shape can be observed in bacteria growing in medium lacking nutrients or undergoing various forms of stress 229 . It has been suggested the long shape may help survival under stressed conditions, or it could be due to perturbation in the replication and dividing system. In our case, it indicates that something is going wrong with the bacteria expressing the enzyme, despite the presence of the pleI gene and our careful selection of the strain.

The non-canonical shape of the bacteria is a problem for many reasons. Even if the bacteria are not dividing and have the same genotype/phenotype, the final concentration of bacteria lysate in droplet is increased which is highly problematic due to the weak resilience of the IPA-PCR process to high concentration of bacteria lysate. The shape could also prevent bacteria encapsulation as they could not fit in small droplets. The filamentous bacteria also promote the formation of films in the microfluidic device filters (see part III). Finally, this shape reveals stressed bacteria that may have an unusual metabolism and express a variety of undesired activities.

First, we tried to grow the bacteria in a medium supplemented with 0.1% of Triton.

Triton X-100 is a detergent that should destabilize the membrane and prevent aggregation of the bacteria [230][231][232] . Results obtained on the different bacteria are presented in Figure 101. Triton was damaging for the culture and did not prevent apparition of bacterial filaments.

Then, we tried to change the concentration of the inducer (IPTG). Results, presented in Figure 102 show that above 50µM of IPTG, NBI induction is deleterious as bacteria OD600

decrease during the induction phase of the growth. Lysate assays using the protected loopact probe and primer production probes revealed that under 50 µM of induction, NBI activity was strongly reduced. We finally decided to go on with 50 µM of IPTG, as it allowed to not put too much burden on the growing bacteria but keep a relatively high expression of the enzyme. Despite the change of IPTG concentration, observation of bacteria under the microscope showed that they were still having an abnormal shape. To improve the culture conditions, we tried to use an autoinduction medium (AIM). The autoinduction media are supplemented in glucose and lactose. When bacteria have used all the glucose, they start using lactose as energy source which, like IPTG, will induce the Lac promotor and drive the protein expression. We also tried to decrease the culture temperature. As NBI is thermophilic, growing bacteria at lower temperature could decrease importantly its toxicity during the growth. The AIM strategy did not yield enough NBI activity, as no or very few primers were produced from bacteria grown in AIM. Furthermore, they still had an elongated shape. However, decreasing temperature to 30°C, in the classical Luria Broth (LB) medium, allowed to express NBI with high-yield and the bacteria had a more normal shape (see Figure 103).

Variability of expression

Bacteria containing the same plasmid can have various profiles of expression in the same culture conditions. This could be a bias in our experiments. We tried to estimate the importance of this bias. Bacteria expressing the NBI enzyme were encapsulated in droplets in order to have 1 or 0 bacteria per droplet. They were then lysed in presence of the loopact reporter. Observation of the lysis under the microscope and following of the increase in fluorescence level in each droplet allowed to assess activity in individual droplet, thus for individual bacterium (Figure 104). We observe that approx. 15% of the droplet contain a bacterium, so that from the Poisson law, we expect approx. 8% of the bacteria-containing droplets to have more than one bacterium. We thus fit the histogram with a sum of gaussian, fixing the area of the second one at 8% of the first one. From this we obtain a mean slope of 0.78 au, and a standard deviation of 1.05 au. Giselle also tried to measure the bias with the fluorescent bacteria. Results presented on Figure 105 show that the expression level could vary from ± 30%.

Nt.bstNBI library construction

To address increasingly challenging directed evolution campaign, it is critically important to test the largest possible number of mutants during each evolution cycle. Therefore, in addition to be carefully designed according to the experiment purpose, the size of the library should be on the order of the throughput of the experimental droplet platform. In our case, as we were looking for versatile libraries that could be submitted to various selection pressures, we selected error-prone PCR (epPCR) to introduce diversity in the gene of NBI. In addition, since we are targeting a very high throughput for our selection platform, attempts were made to obtain high transformation efficiency of the library into E. coli cells.

Diversity generation

• Why error prone PCR ?

The main goal of my PhD project was to develop a method of directed evolution based on programmable DNA circuits and, using the nicking enzyme NBI as a model for the study.

Therefore, random mutagenesis and especially epPCR has been chosen to introduce diversity in the gene as it is relatively easy to implement. Given that developing the different modules already took a lot of time, we also wanted a mutagenesis method that did not take too much time. Of course, the goal was to improve the enzyme, or at least to show that it can be improved with our method. Random mutagenesis methods have provided interesting results in a number of studies, even with relatively small number of selection cycles [START_REF] Chen | Tuning the activity of an enzyme for unusual environments: sequential random mutagenesis of subtilisin E for catalysis in dimethylformamide[END_REF][START_REF] Ellefson | Directed evolution of genetic parts and circuits by compartmentalized partnered replication[END_REF] . In addition, doing a few random diversification cycles at the beginning of an evolution campaign can provide better starting point libraries [START_REF] Bershtein | Intense Neutral Drifts Yield Robust and Evolvable Consensus Proteins[END_REF] , even if more specific library creation methods are used later.

Two targets were to increase thermoresistance and the catalytic rate of the enzyme.

Phylogenetic information could have been spiked into the sequence for the first purpose, or catalytic site targeted for the second one. But, as explained earlier, random mutations can sometimes give access to unpredictable beneficial mutations sometimes far from the active site or at positions completely different than the consensus ones. On a fundamental aspect comparing the obtained selected mutations with prediction from library design software would be very interesting.

Finally, we also wanted to use the results of these experiments to study epistatic effects in enzymes. As our enzyme is too large for a systematic construction of all possible double mutants, epPCR presented an appropriate and flexible alternative.

• Choice of the epPCR technique Two epPCR techniques were tried; using MnCl2 to increase mutation rate in a classical PCR or using the Mutazyme II kit (Agilent). The first technique was set to work with various MnCl2 concentrations but led to very few colonies after transformation. We then decided to opt for the second option. The few transformants we got in these attempts were not sequenced, so there is no information on the library diversity and the mutation rate using this technique.

The Mutazyme II kit is an epPCR enzyme blend formulated to generate libraries with minimal mutational bias. Two epPCR polymerases with different mutational spectrum are mixed; Mutazyme I from a former proposed kit and another novel Taq polymerase mutant with increased misinsertions and misextensions frequencies. Using these two polymerases allow to have equivalent mutations rates between A's and T's vs G's and C's. Mutations rates of 1 to 16 mutations per kb can be achieved. Mutation rate can be controlled either by the number of PCR cycles or by varying the initial amount of target. As mutations are introduced by the wrong replication of the polymerase at each cycle, starting with a small concentration of target ensure to have more duplication of the template strands and therefore, a higher mutation rate than starting with high number of templates. Likewise, the number of cycle will decide the mutation rate by setting the number of duplication. The kit is designed to give high yield of product and the epPCR protocol is straightforward. Several libraries with various amount of initial template were generated and are characterized in part II_4.

Library cloning strategy

The choice of the vector antibiotic resistance is important to maximize the library size.

The vector pSB3K3 is kanamycin resistant, which compared to other antibiotic like chloramphenicol, it is less damaging to the bacteria and does not delay the growth or reduce the number of surviving cells after transformation. It is even more complicated to produce large number of vectors containing the wild-type NBI gene than the empty vector. Because of the toxicity issues presented earlier, bacteria without appropriate methylase protection are prone to introduce an insert in the gene. So, for epPCR starting templates, we used PCR products amplified with Q5 high fidelity polymerase and methylated by the DAM methyltransferase. The methylation here provides a way to remove the template DNA, which would increase the proportion of wild type sequence in our library.

Different ligases and ligation protocols were also used. The electroligase from NEB is supposed to facilitate efficient electroporation, so it was tried in addition to the T4 DNA ligase.

Efficiency of ligation is one of the main concerns in library preparation for directed evolution experiments. Ratio of insert and vector should be optimized to get the most ligated product. But most importantly, self-ligation of empty vector should be avoided or at least strongly minimized. In routine cloning, one can pick several clones to find the correct one, so that empty plasmid is not a big issue. For libraries however, having a large number of empty clones may result in a substantial decrease of the library size. The first experiments led to satisfactory transformation yields but with a lot of empty vectors, sometimes more than half of the library. Several strategies were tried to limit their formation like dephosphorylation of the vector, biotinylated primers for purification of the digested DNA with streptavidin coated beads and so on. None of these techniques gave a significant improvement. Control ligation reactions without the insert led to high transformation yield and colony PCR on the transformed vector + insert ligation colonies confirmed the large proportion of empty vectors.

A second cloning strategy using Gibson Assembly (GA) was therefore designed. As it is necessary to introduce errors only in the NBI gene sequence, the presence of a his-tag in our vector is limiting the possibility for cloning as the repetitive his-tag region allow multiple binding possibilities for primers. Despite these limitations, we designed primers and used Hi-fi Assembly kit (NEB) for the library generation. Rocio Espada did several tests and got comforting results with a 100-fold difference between the number of transformant for vector + insert GA versus vector only GA. Unfortunately, when I tried to use this approach to clone the previously selected library, it resulted in a lot of empty vector or primer dimers insertion. The obtained library had no measurable activity. A more careful protocol with gel purification of the correct band amplified from the selected libraries allowed to recover some activity but colony PCR showed that here again half of the colonies had no insert, despite the 100-fold difference between the vector only or vector + insert GA transformation yield. Results of the different experiments and tested improvements are presented in Table 12.

Table 12. Different cloning experiments and their resulting library sizes

The two protocols we tried, restriction cloning or Gibson assembly, are not completely satisfactory. The first protocol successfully allowed us to generate the library used in the selection experiment of section IV.3. However, in subsequent attempts in the same conditions, it yielded very few clones with the insert. The second one yielded 50% of empty vector for the recloning of the library but lower yield of 10% were achieved by Rocio Espada in mock library generation experiments consisting of cloning the inactive version of the gene. It is possible that cloning of the product of selection is more difficult than a routine cloning.

Increasing cloning efficiency

Although the library contained a lot of empty vectors, we also tried to increase the bacteria transformation efficiency itself. Electrocompetent cells transformation generally leads to more transformants that heat shock transformation of chemocompetent cells. The NEB-5-α strain has a transformation efficiency of 1-3x10 9 cfu/µg of pUC19 plasmid for its chemocompetent version versus more than 10 10 cfu/µg of pUC19 plasmid in its electrocompetent versions. More generally a broad survey of transformation methods reports that the transformation efficiency of chemocompetent cells can vary from 10 5 to 2x10 9 on average and is between 0.5-5x10 10 on average for electrocompetent cells 233 . Electrocompetent cells were therefore used for the library transformation. The NEB T7 express are not available as electrocompetent cells, so I made my own T7 express ple1 competent cells containing the methylase plasmid. Optimization of protocol to create the electrocompetent cells is explained in annex vi. Having cells pre-transformed with the ple1 methylase also in principle provides cells with a protected genome at the time of transformation with the NBI plasmids.

After several transformation trials with various conditions, it appeared that the only way to increase further our transformation yield was to increase the amount of DNA used for electroporation. We thus concentrated the product of ligation using Zymoresearch columns eluted in a very small volume (up to 6µL) of elution buffer. Doing several transformations and combining them can also be an option. We discovered that in some directed evolution protocols [START_REF] Ellefson | Directed evolution of genetic parts and circuits by compartmentalized partnered replication[END_REF]234,235 , some researchers actually use electroporation cuvettes intended for yeasts with a 2mm gap instead of the 1mm gap cuvette used for bacteria. Doing so, they can transform 200µL of bacteria instead of the 25 we can usually transform in classical cuvette. Transforming the bacteria in 200µL cuvettes indeed yield in increase of the transformation yield without the need of repetitively transform tens of bacterial pellets. Achieved library sizes are currently between 10 6 and 10 7 transformants. The total quantity of vector and insert we can put in a ligation or Gibson assembly is limited. Increasing the volume of these reactions to be able to concentrate and transform more DNA could allow us to increase even more the library size. After removal of the blocking group and the fluorophore of the added base, a new cycle can start. This approach is called sequencing by synthesis. The highest-output Illumina platform, HiSeq X, generates more than a terabyte of data per run, with read length of 150 bases and high accuracy, allowing human genome scale sequencing at costs under 1 000$. In order to compensate for the short-read length, paired-end sequencing (sequencing starts from both ends) doubles the reads length. More than 12 billion of single reads can be obtained in one run.

• Iontorrent: The Iontorrent systems is also using polymerization of clusters of DNA strands but clonal amplification is made on separated beads. Here, the detection of H + release during the base addition is used to determine the sequence thanks to a solid-state sensor. The bases are flushed and removed one by one in the reaction chamber so that only one type of nucleotide can be added at a time. When the right base is added H+ release can be detected, whereas no signal is sense when wrong bases are added. In the presence of homopolymers, the H+ release signal increase proportionally to the number of added bases, but homopolymers are more difficult to resolve than on Illumina platforms.

The two previous methods use a preparation step in which clonal clusters of strands are generated, from which they the extract population-averaged sequencing signals. Limitations of the reads' length is due to the limited number of oligonucleotides flows that can be used in one run but also to the divergence in signal that arose after several flows. PacBio and Oxford Nanopore technologies can provide longer reads of thousands of bases because they are based on single molecule sequencing, and this avoid the dephasing between members of a cluster.

But, since the signal is not an average of many simultaneous reactions, the error rate increases.

• PacBio: PacBio system is also performing a sequencing by synthesis of the new strand but here only one DNA strand polymerization is optically monitored inside a zero-mode waveguide xiv . It can provide reads of thousands of bases using detection of fluorescently modified nucleotides in a very small volume.

The Single-Molecule Real-Time (SMRT) instrument is the most widely proven long-reads technology and produces ~888 000 reads per 16 cell instruments with an average read length of 10 000 nt that can sometimes reach 40 000 nt.

Unfortunately, this technique results in high error rates around 15% per single reads. It can be compensated by using circular consensus sequencing allowing several reads of the same sequence, or by sequencing multiple times the same template.

• Oxford nanopore (MinION): The Oxford Nanopore technologies is not using xiv A zero-mode waveguide is an optical waveguide that guides light energy into a volume that is small in all dimensions compared to the wavelength of the light.

strand synthesis but measuring the translocation of the DNA strand through a nanopore protein sensor 237 . The pore is inserted into a membrane and the current modifications due to the passage of DNA in the pore are measured. Profiles of 6-mers passing through the pore are known and used to determine the sequence.

The first proposed platform was The MinION, a small chip bearing ~2000 pores packaged in a device of few centimeters, which can simply be plug by USB on a computer. It is the lowest-cost DNA sequencer with an instrument price of similar quantity of each sample. Figure 109 shows that the ratio is more or less the same for our experiments. But for each fragments of the gene the repartition is very different (see Figure 109), especially for the last and longer fragment that is always underrepresented. Here, several parameters must be considered, the unbalanced quantities of each part but also the difficulty to fully sequence one particular fragment. In future experiments, each amplified part of the template should be quantified and mixed in equal proportion before barcoding. Primers should be redesigned so that the last fragment is smaller, and the primer regions face each other (instead of overlapping), in order to fully read all the sequence and mutations.

Characterization of the library activity

Another way to characterize the library is to assess its activity. Comparing the activity of the libraries with the activity of the wild-type (Figure 110) using a lysate assay with the fluorescent probe protected loopact we found that Lib 50, Lib 100 and Lib 200 had respectively 12.7%, 15.5%, and 19.1% of the activity of the wild-type. This result for the bacterial library can be transferred to the genetic library if one assumes that the cloning was done at the same perfect efficiency (i.e. without empty vectors) for all the libraries.

93 clones from the Lib200 were grown, induced and their activity tested in lysate with the protected loopact probe. In Figure 111 activity have been normalized with respect to final OD600 pr and baselined with a known inactive mutant. The results show that ~56% of the library 112). Among them only one did not contain an insert, 3 appeared to have the same sequence as the wild-type and the others contained different mutations.

A particular mutation, W116stop, appears in many clones as a partial replacement on the Sanger sequencing chromatograph. The clones carrying this mutation, even if it is the only one, display an intermediate activity. It is possible that this mutation emmerge in the population during the induction to deactivate the toxic gene.

The idea was to use these 13 clones to construct the selection curve of the assay, that is the curve linking directly the enzymatic activity of a given clone to its PCR amplification yield, but the experiment did not yield a successful amplification. However, we could measure the primer production rate for each clone, and found a good agreement with its nicking activity as measured by loopact. Note however that some clone seemed to perform better on one test that the other. Still, we can globally conclude that our molecular circuit correctly transfer the information concerning the enzymatic activity of variants, at least at the primer production stage.

Prediction software

It can be interesting to use a library composition prediction software to have an idea of the library composition. The Table 17 gives the PEDEL_AA 239 prediction for a library of 10 7

variants with a PCR efficiency set to 0.6 and 30 PCR cycles with mutazyme II parameters for nucleotides mutations matrix and indels occurrence. We can see that increasing the mean number of mutations per sequence is not impacting too much the number of probably inactive variants (with indel and stop codons) but it increases the number of single unique sequence, decrease the proportion of wild-type sequence and increase the mean number of amino acids substitutions. This suggest that using high error rate could be beneficial for our selection experiments. These observations agree with the modeling of D. Allan Drummond et al. saying that high error rate random mutagenesis libraries are enriched in functional and improved unique mutants while low error-rates library contains lots of functional but similar sequences 240 .

We can also look at these resuls regarding the "x-factor" 241 . It is the probability that a random amino acid replacement will lead to a protein's functional inactivation. Guo et al. determined it as 34% in 3 different similar mutagenesis conditions (2, 4 and 6 aa mutations per gene). This would mean that among or ~70% of sequences with no indel or stop codons, between 5 and 20% would be wild-type and 34% would be inactivated, only ~15 to 30% would therefore be functional and carrying mutations.

Using MAP2.0 3D 242 , various information including mutations information regarding the position in the structure can be obtained but there is no information about the parameters used for the simulation. The average number of mutations per gene or the number of PCR cycles are not given and nowhere to be found in their platform. With this software we find an average amino acid substitution per residue of 7.45, 7.39% of stop codons, 6.71% of Gly/Pro residue modification known to destabilize the structure, 21.90% of preserved amino acids and a codon diversity coefficient of 3. our case, the disperse phase is an aqueous phase and the continuous phase is oil. Emulsions are therefore made of small droplets. These droplets are only metastable stable and tends to merge to form bigger and more stable droplets, ultimately reaching the thermodynamic equilibrium of two separated phases. This phenomenon is exemplified by the vinaigrette, an emulsion that forms two separate phases when left immobile for a long time, reaching a minimal energy state.

However, droplets integrity on the time scale of the experiment is required for the use of droplets as microreactors. To stabilize the emulsion, a surfactant must be added. This compound prevents droplets coalescence by providing an energy barrier and decreasing the surface tension. Surfactants are amphiphilic molecules with a hydrophilic and a hydrophobic part that locate at the interface between the two phases. Surfactant choice is crucial in the formation of the emulsion and its stability. Droplets of various size can be produced. To avoid bias in the selection, all compartments should have the same size. This is because the selection method that we plan to develop are sensitive to the local catalytic activity, which in turn depends on the specific activity of the enzymatic variant AND its concentration. The polydispersity of an emulsion evaluates the variability in the droplets size. Methods producing small droplets with low polydispersity should be preferred.

Variability of droplets generation methods

Various methods can be used to generate droplets (Figure 114):

• Sonication Sonication can be used to initiate cavitational xv events in liquid/liquid interfaces. This results in droplets generation with a mean particle size of 0.2-2.0 μm with most droplets between 0.1 and 10µm 243 . This acoustic emulsification process is dependent on the ultrasonic power and the chemical composition of the system. Such emulsions are for example used in separation techniques to remove some components of a solution 244 .

• Rotor stator

The rotor/stator is composed of a rotor of two or more blades included in a stator with a stator comprising vertical or biased slots around the wall of a homogenizer cell. The movements of the rotor draw liquid in and out of the assembly by vacuum generation. Homogenization power and residence time of the emulsion droplets in the shearing field are the primary parameters controlling droplet size. Viscosity of the liquids, surfactant type, volume size, and

xv Cavitation is the formation of vapour cavities in a liquid, small liquid-free zones, that are the consequence of forces acting upon the liquid. It usually occurs when a liquid is subjected to rapid changes of pressure that cause the formation of cavities in the liquid where the pressure is relatively low.

• Membrane

Membrane emulsification is used since the late 1980s and were first produced with a glass membrane called Shirasu Porous Glass (SPG). Nowadays, this membrane is still widely used but other types like ceramic, metallic or polymeric membranes or various type of porous material are also used. Membrane emulsification is producing narrow droplet size distributions and does not necessitate lots of energy. Several emulsification modes exist. In the direct mode, droplets are generated at the interface between the membrane and the continuous phase by forcing the dispersed phase through the membrane. In the premix mode, the two phases are first roughly emulsified before going through the membrane. The direct method necessitates to be in a relatively low membrane flux to maintain the size-stable droplet production resulting in low throughput and long production times. And, to be efficient, detachment of the droplets from the membrane needs to be enhanced by shear stress, agitation by stirring or rotation or vibration movements of the membrane. But, the obtained droplets have low polydispersity and can have size ranging from 0.2 to 100µm according to the used membrane. In the premix mode higher rate and smaller droplet size can be achieved and there is no need of movements introduction do detach the droplets from the membrane. But droplets have a higher polydispersity. In both cases, hydrophobic or hydrophilic nature of the membrane should be adapted to the disperse phase nature, as well as the charge of the membrane 247 .

• Self-assembly Garenne and al. proposed to use fatty acid self-assembly to encapsulate proteins in vesicules 248 . The used fatty acids forming coacervates xvi . These coacervates create locally a crowding effect that tend to generate protein accumulation. When a pH variation is applied, the fatty acids spontaneously sequester proteins, creating small vesicles. Enzyme-rich vesicles were produced and reaction measurements inside these vesicles was confirmed. This system could be adapted for applications that are not sensitive to pH or working in the pH range used in the paper. Other surfactant self-assembly methods are also used in the food industry or for therapeutics applications but generally to produce nanoscale emulsions 249 .

• Emulsion by shaking

Several methods of directed evolution use relatively simple shaking systems to generate their emulsion [START_REF] Ghadessy | Directed evolution of polymerase function by compartmentalized self-replication[END_REF][START_REF] Ellefson | Directed evolution of genetic parts and circuits by compartmentalized partnered replication[END_REF] . Simply vortexing the mixture of oil with surfactant and aqueous phase allows to generate droplets 250 . Adding of the water phase in the spinning oil phase while stirring continuously [START_REF] Ghadessy | Directed evolution of polymerase function by compartmentalized self-replication[END_REF][START_REF] Ellefson | Directed evolution of genetic parts and circuits by compartmentalized partnered replication[END_REF] or using cell lyser like the TissueLyser LT (Qiagen) can also be an option.

Cell lysers use a bead in a tube with a diameter slightly smaller than the tube width. The tube is then placed in the cell lyser apparatus. By generating vibration, the machine creates shear xvi Organic-rich droplets formed via liquid-liquid phase separation. stress at the border of the tube that allows to generate droplets. Obtained droplets are generally very polydisperse. This is generally viewed as an issue for selection experiment but can be highly time-saving.

• Microfluidics

All the above presented methods lead to broad droplet-size distributions 251 . Up to now, microfluidics is the best strategy to obtain monodisperse droplets. Capitalizing on the strong expertise of the hosting Japanese lab, and later the proximity with a dedicated technological platform (IPGG), we naturally decided to turn towards this method. More details about this technique are given below.

Droplet generation by microfluidics

Microfluidics can be considered both as a science or as a technology. It is based on the manipulation of small volumes of liquid, from microliters to attoliters, using micrometer-sized channels 252 . At least one dimension of the channels must be in the micrometers range for a device to be consider as microfluidic. Because they greatly reduce the Reynolds number to much less than 100 and often less than 1, the small dimensions of the channels allow to work Starting in the 1990s, the first microfluidic systems were based on silicon technology.

With the emergence of soft lithography using polymers to replicate structures from microscopic scale mold, the cost of device production decreased and the field expanded. The most used polymer is Polydimethylsiloxane (PDMS). This polymer has unusual rheological properties, is optically clear, inert, non-toxic, and non-flammable. It can be used for many applications, including biological ones. The fabrication process of a microfluidic chips is depicted in Figure 115. Soft-lithography is the molding of a polymer mixed with its curing agent on a photoresist master. Variety of complex devices can be made including multiple layer devices or valves 253 .

Microfluidics is a very interdisciplinary field involving chemistry, biology or physics.

Lots of applications are targeted. On a technological point of view, microfluidics is used to handle fluids in machines. Using microfluidics, it is possible to use lower amounts of reagents and manipulate very small particles like cells. This can allow to spare important amount of money when using expensive reagents. Reduction of distance by a 10 3 factor results in a volume reduction of 10 9 . Small volumes allow very fast analysis and efficient detection even when only little amount of a sample is available. The size of the microreactors make them perfect for manipulation of genes, cells or small organisms. Integration of microfluidics systems on a single chip led to the creation of miniaturized laboratory platforms named lab-on-chip (LOC), micro total analysis systems (µTAS) and point-of-care diagnostic devices 254 . Such devices allow single cell analysis, physics fundamental studies or observation of reaction dynamics.

Not only microfluidics chips possess channels controlling the flow direction and particles interaction, it can be combined with electronics or optical systems like lasers to perform several operations on the studied system. The small involved liquid volumes, the all-in-on possibilities and the small device sizes make it possible to develop compact and portable systems that can be used in biotechnology, pharmacology, medical diagnostics, environmental monitoring.

Clinicians have a growing interest in microfluidics for clinical research or diagnostic applications 255 . In 2013, for instance, an integrated chip was built to perform sample pretreatment and detection of HIV-1 by nucleic acid amplification 256 .

A sub-branch of microfluidic is the droplet-based microfluidic or digital microfluidics 257 . This branch of microfluidics uses small droplets compartments to encapsulate reagents, particles like beads or biological elements like cells. These produced microreactors can therefore be used for single cell analysis 258 but also to perform digitalized reactions like [START_REF] Taly | Droplets as microreactors for high-throughput biology[END_REF]166,[262][263][264][265][266] .

Several techniques, represented on Figure 116, exist for droplet generation:

• Flow focusing

In flow focusing devices, the dispersed phase is squeezed inbetween two incoming perpendicular flows of the continuous phase. The two phases are forced through a narrow region of the channel called the nozzle. The accelerated jet destabilizes generating droplets either at the level of the nozzle or a bit downstream 267,268 . Symmetric shearing forces generated by the continuous phase enables stable and controllable generation of droplets. The droplet size can be controlled by the flow rate ratios of fluids, the interfacial tension, the viscosity ratio, the size of the nozzle, or the geometry of the channels 268 . However, there is no general law to predict precisely the size -or production frequency -of the droplets, and some empirical adjustment is generally required 267 . 1. The resin is exposed to a U.V. light under the mask so that channel parts react. 2. The remaining part are then removed with chemical agents (developer). This results in a mold with protruding channels parts. 3. Liquid PDMS mixed with its curing agent in then poured on the mold and will take the shape of the channels. 4-5. After baking to make it solid, the obtained carved PDMS is bound to a surface, often a glass surface to create the channels. The mold can be reused many times, but PDMS chips are typically used only once. Tubing are then connected to the inlet and outlet of the chip thanks to holes in the PDMS.

• •

Step emulsification

The step emulsification process is inspired by membrane emulsification techniques and require the construction of a layered mold. The disperse phase pass from an inner chamber to the surrounding outer continuous phase chamber via a small channel. The droplet generation process is similar to the one of the T-junction with droplet burgeoning and detachment. The abrupt transition between the inner chamber and the outer reservoir has a shape of a step were the stream of disperse phase breaks into droplets. Theses device are sometime constructed with parallel transfer channels to increase the throughput 269 . The droplet size has been shown to be tightly controlled by the microchannel cross section dimensions. Combining this step emulsification and flow-flocusing or co-flow, it has been shown that the droplet size is not controlled by the microchannel cross-section but by the smallest spatial dimension of the inlet microchannel 270 . This allows to produce smaller droplets with controlled size at high rates.

Here, the size is less dependent on the applied flows.

In all these different geometries for droplet production, several modes can be observed.

In the dripping regime, droplets are formed near the meeting points of the two phases. In the jetting mode, droplets form from an extended stream of the dispersed phase, downstream in the channel. In the squeezing mode, growing droplets obstructs the channel blocking the flow of the continuous phase. This generates a dramatic increase in the dynamic pressure upstream of the forming droplet in the continuous phase. The induce pressure increase force the droplet to detach 267 . The three regimes are depicted in Figure 117.

To increase the rate of droplets production some layered designs have been produced.

These designs target industrial applications with hundreds of radially arranged nozzle 271 .

In addition to the use of hydrodynamic forces, active droplet generation can be achieved using electrical fields, magnets, centrifugation, mechanical or optical control. Active droplets generation methods offer higher flexibility in droplet size control and production rate but also shorter system time-response 272 .

The geometry of the channels is crucial to determine droplet production rate and size, but the oil and surfactants that are used are also of foremost importance. Solutions are connected to the chip inlets using various kind of tubing. The nature of the tubing, its length and its diameter can influence the reactions. According to these parameters the time the sample spent in the tubing can be variable and interactions with the samples can happen. Among all possibilities we chose to use polytetrafluoroethylene (PTFE) tubing. PTFE tubings are transparent, chemically inert, non-toxic, feature chemical resistance and a surface that facilitates flow. It is easy to handle, quite flexible and relatively high pressure resistant.

Several tools can be used to drive the flows in the microfluidic chips. The most used ones are mechanical syringe pumps and pressurized pumps. When using syringe pumps, solutions are introduced in the syringe and the syringe needle is connected to the chosen tubing system. This method creates a dead volume equal to the tubing volume capacity and the volume staying in the needle head. With pressure pumps, the tube is put under pressure and the tubing is directly connected into the solution. There is less volume in this case. Syringe pumps allow to set a flow-rate whereas pressure pump apply a preset pressure but there is no control on the flow rate. If two solutions have different viscosities, applying the same pressure will not result in the same flow rate. Also, microfluidic device impediments, appearing because of mold defects for instance, can change the flow rates induced by a pressure pump. Yet, an advantage of the pressure pump is that theoretically any reservoir with desired volume can be connected to the pressure pumps while the volume handled by the syringe pumps volume is limited by the syringe dimension (which need to remain reasonable in order to have a good control of the flow rate). Finally, pressure pumps offer much better time-responsiveness. Changes in pressure result in almost immediate adaptation of the flow, whereas change in flow rate have a slower response. Both methods are used equally but they do not result exactly in the same outcome 276 .

Microfluidic-based systems of droplets generation are currently the best way to produce emulsions with monodisperse droplet population. However, since droplets are generally produced one by one the production rate can be considered as quite low compared to some other techniques. There is also a limitation in the size of the droplets that can be generated, as fluidic resistance increases with decreasing channel dimensions, reducing the flow rate. Moreover, simple microfabrication techniques may not be precise enough anymore 246 .

Very recently, a method called templated-emulsification was proposed to overcome microfluidics limitation and accelerating monodisperse droplets generation. The principle is to use porous gel beads mixed with the solution to encapsulate and generate emulsion just by shaking of the tube with an oil phase. The gel beads act as template and fix the size of the droplets. The idea seems interesting for experiments needing several successive encapsulation steps. In this paper, the gel beads were first produced using a microfluidic device. One can imagine that if monodisperse gel bead are commercially available such emulsification method could be of great interest 277 .

Development of a microfluidic device

Device for droplets with controlled diameter

The device used for bacteria encapsulation should fit several requirements:

• Make droplets of controlled and consistent size to ensure the best possible selection performance.

• Have two inlets, in order to mix bacteria and rlysozyme at the last moment so that bacteria are not lysed before entering the droplets. This is important to maintain a tight phenotype/genotype linkage.

• Generate droplets at a high rate for high-throughput selection experiments.

Step-emulsification devices appeared as the best way to control the size of the droplets with precision. In this droplet generation method, the size of the droplet is controlled by the channel cross-section or, if the step is combined with a co-flow strategy, by the smallest dimension of the inlet channel. Another advantage of step devices is to decrease the global resistance of the device. The hydrodynamic resistance of a micro channel scales with its length and the inverse square of its section. Restraining the total length of channels with low height allow to relieve some part of the resistance. In turn, a smaller resistance in the chip give the possibility to use higher flow-rate in the channels and accelerate the emulsification process.

Along the project, target droplets size evolved. 10µm diameter droplets were first targeted based on the publication on the CSR. Finally, faced with the impossibility to run an IPA-PCR with high bacteria concentration, the target droplet diameter was increased to 27~28µm. During the internship preceding my PhD work, I used the mold of a device designed by Jongmin Kim from the Fuji lab to try to produce 10µm diameter droplets. It allows me to train on bacteria encapsulation and to have a first idea of the design problem I could encounter.

The device presented is presented in Figure 118. Droplet size was variable, not stable at all. The best stability was obtained for droplet of 20µm in diameter not for the expected 10µm. One other important problem was the filters, which were not efficient enough. The device clogged quite rapidly, if not immediately after starting using it. And finally, the maximum supported pressure was very low, and the general flow rate not satisfying for high throughput experiments.

The following devices design was inspired from the publication of Shim et al. 278 , as suggested by Kim sensei. In 2013, they proposed a device able to generate droplets smaller than 10fL at a rate of up to 1.3x10 6 per second. No clear explications were given to the reason of their success. They concluded "that the locally narrow flow-focusing nozzle design and the low viscosity of the fluorinated oil are the key features that enables [them] to generate femtoliter droplets controllably at frequencies above a million hertz." 278 So, the geometry of the nozzle was reproduced, in the hope to observe the same droplet production yield. Figure 120 shows the comparison between the device and the published design.

The first device consisted of a step 10µm high for the nozzle and filters and a height of 30µm for the remaining part of the device. It was not perfect but did produce small droplets of 10µm diameter with relatively high yield. This device was less sensitive to small variation in pression than Kim's device. In the Table 18 you can see the different pressure used in the different devices.

Improvements

This first device filled the requirements but had a lot of room for improvement of the practical aspects (Figure 121).

• Filters modifications

As the nozzle is very small and sensitive to clogging, improved filters were needed to avoid that small dusts could reach it (Figure 119). First filters were not adequate. As bacteria are 1-3µm sized, very small filters with 10µm spacing were designed in hope to prevent the entrance of anything else than bacteria in the device. They were placed on the step level to leverage this decrease in size. But the quick reduction of the channel dimension often resulted in inlet channel in dusts bottleneck blocking completely or decreasing drastically the incoming flow. They were changed a first time by filters with gradual increase in size stringency, staring with 20µm spacings, through a portion with 10µm spacing, and ending with 6µm spacings (limiting size for the mask resolution). But, this was not sufficient, as the change between the 50µm thick inlet and the step of the filter (10µm in thickness) was still the limiting region, prone to clogging. In the last version, this gradual filter was linked directly to the large part of the inlet and its exit was also enlarged. These last changes improved considerably the device.

In the final filter design the last row of plots with 6µm spacing was also removed. In previous device the mold resolution was not high enough for them to be correctly printed in the PDMS.

Figure 122 gives examples of the different troubles appearing with the different filters. The last filter is the most satisfying design but the of the filter area is still a problematic region were the flow slows down and bacteria tend to accumulate, build films and impede the flow (see Figure 130).

• Shrinking the device

The size of the device was progressively reduced for many reasons. First, it allows to decrease the global length and resistance of the design. Then more devices could be made from one mold and eventually they ended up being all fitting in a single glass slide. Having 8 devices on the same glass slide allow to drastically decrease time for chip preparation and therefore increase the device production yield. The compactness of the device also allows easier navigation under the microscope between the different parts.

• Introducing fluidic resistances upstream of the nozzle.

Serpentine channels increasing the path length and therefore its resistance, while not taking too much space, were added between the nozzle and the inlets. With such design the resistance is larger in the channels before the nozzle, compared to the resistance between the nozzle and the outlet. This avoids backflows when the inlet pressures are temporarily unbalanced.

• Output mixing turns

As in Alex and Anthony's device presented in Figure 125 and used to map the bifurcation diagram of some PEN molecular programs, a waving output channel was used in the device. This shape allows for droplet content mixing after their generation and stabilization of the interface 279 . Compaction of the channel and reduction of the curve should allow for a better mixing.

• Inlets plots

Plots were added in the inlets and outlets to avoid them to collapse. Due to the small aspect ratio of the inlet ports ceilings sometimes ended up partially bond on the glass slide. This lead in some cases to completely unusable inlets. It was also tried to fight against Poisson law by ordering the bacteria via circular channels inserted after the inlet 258,[280][281][282] . Due to the small size of bacteria, a long channel needs to be used. The resulting design was very large and would have drastically reduce the production frequency of chips.

The step design for the nozzle proved to be very useful way to control the droplet size.

Several heights, nozzle widths were tried, as well as using the same geometry without a step.

The resulting droplets size are presented in Table 18. Anthony's and Alex device 130 (Figure 125) was also tried and together with other results confirmed that the step strategy is the best way to control and predict the droplet size. The final device is presented in Figure 123, anda tridimensional view is given in Figure 124. The channels are all 50µm wide when not in the nozzle or filter parts. Filters and nozzle are drawn of the same mask to allow their printing in the same step of 20µm height. The width of the nozzle being 20µm this result in a nozzle section of 20µm by 20µm. The remaining of the device has a height of 40µm. The device can produce droplets with diameters from 23 to 29µm by adjusting pressure ratio of oil and water channel.

By comparison, the same design without step and with a global height of 20µm produces droplets of 50µm diameter (Table 18). This final device, or some adaptations, are now widely used by the other team members to generate droplets of the size they need. The device was compacted so that 8 devices fit on 50x60 mm rectangle that can be directly bounded to a microscopy glass slide of 72x56mm. Plots were added to the inlet and outlet to avoid bonding of the PDMS ceiling on the glass. Filters were connected to the inlets with a wide channel to avoid having dusts clogging and to decrease the global resistance of the section. In the main part, channels are 50µm wide.

Table 18. Variations of parameters in tried microfluidic device. The droplets seem to be quite monodispersed. Comparing these results with the one of a similar device without step, one can directly notice the importance of the step. The same device with a nozzle channel 20µm wide and a global height of 20µm lead to droplet of 24.4µm± 0.6 of radius. The monodispersity is quite good also but it demonstrates the interest of the step. In the 2-step device the size of the droplet is corresponding to the nozzle section more or less, while here the droplet size seems less predictable. Changing the flow rates could allow to reach smaller size but with the 2-step device fewer variations are observed when the pressure is changed. Finally, one can compare the obtained droplets with a device adapted from the Millipede device of Amstad and al. 269 by Rémi Sieskind, another PhD student of the lab.

This device allows very high-throughput production of droplets using 500 parallel nozzles with a rate of 2.5 mL/min. The produced droplets are within the same size range with a mean radius of 11.2µm ± 2.5. But, the size dispersity is wider. Two population of droplet size could even be noticed. The monodispersity of this device is increasing when used with viscous solutions such as or water-glycerol mixtures. Authors of the paper work in 20% glycerol and Rémi is using polymeric gel precursor to produce beads but in my case, I do not want to add extra thickening agents. So even if this device is increasing drastically the droplet throughput (~10 times) it would not be ideal in my application. Furthermore, its current implementation does not allow to separate bacteria and lysing agents before encapsulation. Advantage could be taken of the high speed of the droplet generation time to have an encapsulation faster than the lysis of bacteria but rlysozyme action is quite fast and bacteria are lysed in few minutes according to some other-directed evolution experiments 166 . 19. The usual slope in the PCR machine is of 6°C/s. Looking at the miscroscopy images, it seems that decreasing it to 3°C/s or 1°C/s could maybe decrease the merging. But, when looking at the percentage of droplet with conserved size after the PCR, it seems that there is not so much difference. 1°C/s and 3°C/s slopes are giving very similar data. And it seems that merged droplets in the case of the 6°C/s slopes are bigger but not more numerous. The slope was then kept to 6°C/s in further experiments. Ellie could count 25% of non-amplifying droplets and the effect seemed to disappear when using only 2% of RAN. This suggests that the excess amount of surfactant had captured or inactivated a fraction of the plasmids or proteins. This was the first time DNA capture by oil/water interface was suspected and it encouraged us to keep using 2% of surfactant in the oil.

It was surprising, as fluorinated oil and fluorosurfactants are generally thought to be compatible with single molecule detection 273 , but coherent with other observations in the lab.

Later on, when performing selection experiment with bacteria in droplets, recovery of the DNA from IPA-PCR amplification became problematic. No PCR product was visible on gels after PCR amplification. It was first thought that the reaction was not functioning in droplets, but the large amount of genomic DNA usually retrieved in bulk (and clearly visible in the loading wells of the gel) was also absent. Several hypotheses were tested, and the results of the many resulting experiments are presented in Figure 129.

Performing the PCR reaction in mineral oil and Span 80 surfactant as proposed in the original CSR paper lead to a correct recovery of DNA. This suggested us that the problem was coming from the fluorinated oil and its associated surfactant. Droplets were directly put in an electrophoresis well with loading dye and the genomic DNA of the same droplets were run next to it. We noticed a difference in intensity between the genomic DNA band between after and before extraction and started thinking that the extraction process could be the result of DNA capture by the oil/water interfaces. Several buffers were also tested to see if they had an influence on the capture. Decreasing the surfactant quantity from 2 to 0.5% was tested but this resulted in unacceptable level of merging. Besides, no significant difference was observed between the different percentage of surfactants. The difference between EA and RAN surfactant was also tested and did not yield to remarkable changes. Different techniques to break the emulsion were tried. Some not requiring the use of PFO (1H,1H,2H,2H-perfluorooctanol, see

Material & Methods), including a freeze-thaw process or electrostatic breaking of emulsion with electrostatic gun (data not shown) did not yield to any difference with the classical PFO technique. Adding PFO alone or diluting the water phase with MOPS while adding the PFO was also tried with no significant difference. Interestingly, when performing all these tests, DNA was recovered in almost all conditions contrary to when the same protocols were applied on the IPA-PCR results. We then tried to use the droplet stabilizer (Raindance) also lent by Valérie Taly's lab to improve DNA recovery but the results were mitigated with sometimes few DNA recovered sometimes none. Finally, we learned, again from Clément Nizak and Dany Chauvin, that adding 1ng/µL of yeast RNA and 0.4% of pluronic f-127 to our water phase could solve our issues. Indeed, these two additives allowed us to retrieve a bright DNA band on the gel, similar to the one from the bulk experiments.

It is thus thought that nucleic acids can associate irreversibly with the droplet interface during thermal incubation, and that the unspecific RNA and/or the pluronic surfactant have the ability to passivate this interface, by creating a protective coating on the surface. Possibly the effect of Pluronic f-127 is more on the protein side, to promote good reactions in droplets but it was not tested alone. Note that our buffer already contains by default a relatively large amount of passivating protein (Bovine Serum Albumine) that should also coat the interface and prevent protein adsorption, but no experiment was done to test its effect.

The search for a solution to recover the DNA from the droplets was very time consuming and render the results analysis complicated. It was difficult to know if the reaction of IPA-PCR in droplet did not work or the DNA was just capture in majority by the water/oil interface.

Using qPCR on the low amount of recovered DNA helped us to know if something happened or not. But, like surfactant formulations, researcher's recipes to avoid adsorption phenomenon in droplets are difficult to find.

Non-specific adsorption phenomena are well known, and some information can be found about it. Surfactants can interact with proteins 283 and cationic surfactants, in particular, are known to interact with DNA 284,285 (note that the fluorinated surfactants that we used are most probably all neutral) 286 . Proposed solutions to control the protein adsorption are mainly modification of the surfactant head groups which may requires the synthesize of a custom surfactant. And anticipating the interaction is complicated when you do not know the surfactant structure. Information about the kinetics of adsorption 273 tells us that above a critical droplet size (between 20 and 70µm) it is controlled by diffusive processes, while below this size binding kinetics take the relay. So, since we are near the critical zone maybe the adsorption processes are changing between slightly different size of droplets. This may explain the diversity of observations and the low reproducibility of some of our experiments.

Surprising little mention of this type of problem can be found in other directed evolution or digital amplification reaction protocols from the scientific literature. In the directed evolution community, most techniques do not use PCR and can therefore use reduced quantity of surfactants. This could explain why they do not face the issue 166 . Protocols that do use PCR such as CSR or CPR are performed in mineral oil, conditions were we indeed observed less problems [START_REF] Ghadessy | Directed evolution of polymerase function by compartmentalized self-replication[END_REF][START_REF] Ellefson | Directed evolution of genetic parts and circuits by compartmentalized partnered replication[END_REF] . But regarding the application of digital PCR, this adsorption problem should be of principal interest and could yield to underestimate of the DNA quantity.

Bacteria films in filters

Filters of the microfluidic device have been optimized to prevent clogging of the channel by dusts. But, another problematic phenomenon appears at the level of the filters. During the time of the droplet generation, bacteria form biofilms. The films formation probably starts in the filters as the speed of the liquid decreases in this part of the device (due to the large overall section necessary to avoid clogging). These biofilms then grow and extend downstream to the channels. The films also sometimes detach and perturbate the flow, but the biggest issue is that they tend to increase the resistance in the fluidic channel. Since I am principally using pressure pumps it is problematic because it means that the flow rate will decrease in the bacterial channel.

Indeed, the bacteria are originaly separated from rlysozyme in two master mixes connected to 2 inlets. To reconstitute the correct reaction, their contribution in each droplet should be balanced. Biased contribution, with lower amount of the bacterial stream would results in a lower lambda value and increasing concentration of rlysozyme. While the former effect may not be critical (it just decreases the throughput), the latter may be more problematic as rlysozyme concentration must be carefully controlled and higher concentrations are detrimental

(see I.3).
We tested the use of syringe pumps instead of the pressure pumps. We first had to readjust the flow parameters to mimic the pressure-controlled conditions, with water flow rates set at approximately 1µL/min and the oil flow rates around 10µL/min. For some reasons, although we tried to reproduce the flow conditions of the pressure pumps experiments, we observed that more oil was required to produce a given amount of emulsion. Also, preparing the solution to insert them in syringe was taking way more time than plugging the tube in the pressure pump. During this time, the sample were not kept at cold temperature. The syringe pumps also have also a quite large dead volume. I am working with small volumes of 100-200µL, and almost 50µL was lost in the tubing. (This problem can be eventually be fixed by adding extra oil phase at the rear of the sample.) Finally, as I learned the hard way working with needles and tubeing can be dangerous even when carrying gloves. So, I decided to go on with the pressure pumps which I thought were more convenient. Fluigent pressure pumps can also be used with flow-rate measuring instruments. We tried them in the lab, but they were causing too much troubles in the tubing sealing and the low rate recording was not reliable at low flows.

To assess in realtime the relative contributions of the two flows, a fluorescent dye was added in the rlysozyme solution. This way, we could directly visualize under the microscope the separation between the two laminar flows in the channel just upstream of the nozzle. This revealed that, to ensure an equal contribution of the two water channels, the flow need to be adjusted continuously during droplets formation. Note that the presence of the flow separation exactly in the middle of the channel corresponds to an equal contribution of the two inlets only if the solutions have the same viscosity. Indeed, some experiments in the lab showed that two fluids with different viscosity were not meeting in the middle of the channel when run at the same flow rate with syringe pumps. Therefore, if the bacteria concentrated enough to strongly change the liquid viscosity, the method we used may not be very precise. Finally, we noticed that a line between the two flows could be observed under the microscope even in the absence of the fluorophore. The bacteria are also clearly visible when strong illumination is used, and we therefore abandoned the use of Alexa 647. Finally, after starting using the pluronic f-127 and yeast RNA in the two solutions it seemed that the flows were better equilibrated (in the sense that same pressure could be kept during all the collection time with no change in the two flows contributions). Also, the residual volume after droplet generation was more similar in both inlets. This effect still needs to be confirmed as it varied from experiments to experiments.

The preparation of the bacteria so that they do not form films, or stick less, could also be investigated.

IV. Selection Process

1. Re-implementation of the Compartmentalized Self-Replication process and the efficiency of DE protocols

During the development of the method based on molecular programs, replicating the CSR experiment was explored as a way to master the several aspects of the compartmentalized selection in droplet. The idea was also to benchmark the efficiency of our method against the one presented by the team of Holliger. This step ended up being not as straightforward as expected but revealed us some general problems with this type of approach. In the end, it helped us to fix some issues we had with the IPA PCR, such as finding a lysate-resistant Taq polymerase. Additionally, this sub-project prompted us to reexamine the way the efficiency of DE protocols was generally evaluated, and to propose an improved metric in this purpose.

Taq Polymerase expression in bacteria and first CSR attempt

Expression of the Taq polymerase

To first try to repeat CSR protocol the pakTaq plasmid containing the Taq polymerase gene was ordered from Addgene and cloned in a pET14b vector under a T7 promoter with a histidine tag at the N-terminus. Successful expression and protein purification were achieved.

To perform mock selection experiments, an inactive version of the gene was also created by site directed mutagenesis. In their study on the catalytic site motif of the DNA polymerase I, Shinkai and al. discovered that it was highly mutable, except on the first position of the motif 287 . In most of the cases, random mutations introduced in the catalytic site was not completely depriving the polymerase of its activity, unless the mutation appeared on the first amino acid of the motif. Catalytic site motifs of the DNA pol I and the Taq polymerase being similar, the aspartic acid 631 (GAC in the sequence) of the DYSQIELR active site motif was changed to a glycine (GGC) by introducing a point mutation at the 2 nd base (from A to G) by site-directed mutagenesis. Like the wild-type gene, plasmid containing the mutated version of the Taq polymerase gene were transformed in KRX bacteria cells. After 4 hours of induction

Taq polymerase was purified using his-tag column purification. The activities of the two purified versions of Taq were tested after dialysis by running PCR reactions. Figure 132 shows the purified proteins and the corresponding activity assays. Unlike the wild type version of the gene, the purified inactive mutant indeed did not succeed in PCR reactions, despite the presence of the plasmid gene in the bacteria and a correct expression.

Self-replication tests in bulk

Subsequently, the CSR protocol (presented in Figure 133) was run in bulk, using bacteria carrying the active version of taq and the PCR reaction was followed in a real time PCR machine. We tried with concentration of 50 or 200 bact/nL but no amplification was ever visible even when adding external Hot Start Taq, extra plasmid or RNAses (as in the original paper) in the reaction mix. Later we realized that the droplet size of 15µm diameter announced in the CSR paper accounts for a bacteria concentration in droplets of ~500 bact/nL.

We next tried to optimize Taq expression. The CSR paper did not give enough information about the Taq expression conditions, so we had no idea of the induction time they used, and we were also not using the same expressing cells (KRX instead of TG1 strain). 3 inducer concentrations (0.05%, 0.1%, 0.2% L-rhamnose) and 2 different induction OD600nm (0,5 or 0,7) were used to screen expression condition on 2 different starting colonies, with the same induction time of around 4h30. Indeed, in this first stage of expression tests, we noticed that some colonies seemed to express more enzymes than others and that a second purified band, around 35kDa, was not always present with the same intensity. All the Taq enzymes expressed in the screened conditions were quickly purified using heat precipitation in presence of 10% of PEG 8000 and their quantity was analyzed on an SDS PAGE gel. Despite the changes in expression conditions, Figure 134 show that there was no strong difference in the expression yield achieved at the end. The bacteria expressing the Taq genes with these different screening conditions were also test in a self-replication assay. None of them produced PCR amplification, when used at 100 or 200 bact/nL. In very diluted (6 bact/nL) conditions, the Taq expressed by the bacteria was not able to perform self-replication, but we noticed that addition of external commercial Taq gave some PCR amplification of the bacterial plasmid. Complementary experiments ran for NBI gene amplification (see 3.2, p. 131) showed that indeed the natural Taq polymerase was not able to perform PCR with high bacteria concentration. Gels show that for more than 300 bact/nL there is no PCR product and that the PCR yield and accuracy is decreasing from 200 bact/nL. This does not fully explain why the self-replication reaction was not working, as we tried with smaller concentration and never got any amplification. It is still possible that, with longer induction time, higher amount of Taq polymerase per bacterium can be obtained and would allow Taq self-replication. But we decided not to go in that direction for the Taq tests and, as we did for the NBI evolution scheme, we opted for the use of a lysate-resistant Taq variant for our CSR trial experiments.

Given that the culture conditions, the vector and, the bacteria strain that we used for the expression of Taq are not the same than in the Holliger CSR paper, we cannot directly compare our results with the reported ones. It still seems unlikely that self-replication could happen in the very small droplets (15µm in diameter) that appear to represent the majority of their emulsion. However, CSR, as well as the CPR experiments is run in emulsion generated by shaking and not with microfluidic, so the droplets are not monodisperse and we can imagine that some bigger droplets allowed the self-replication to happen. One can also imagine that mutants able to perform PCR reaction in high concentration of lysate arose from the beginning of the experiment. In the case of the CPR, it is not sure what is the concentration of bacterial lysate, because the authors give no direct information about bacteria concentration nor droplet size, but one can note that the induction time is of 7h, instead of the 4 to 5 h we tried in our experiments. Other groups reported work to accelerate the speed of polymerization of the Taq polymerase using the CSR but there, little information was provided about the exact experimental conditions [START_REF] Ghadessy | Directed evolution of polymerase function by compartmentalized self-replication[END_REF] .

1.1 Klentaq, a Taq polymerase less sensitive to bacteria lysate

Using a lysate resisting version of Taq

To overcome the lysate toxicity issues, a more robust version of the Taq polymerase was tested. The Klenow fragment of the polymerase is a truncated version of the enzyme with a N-terminal deletion of 278 amino acids 288 . It is known to have an improved fidelity and thermostability 289 but also to be less inhibited by classical PCR inhibitors like whole blood. In its natural form it was found to be 5 to 10 times more resistant to whole blood than the full Taq polymerase 167 . Iaroslava Golovkova, an intern I co-supervised ordered a codon optimized version of the Klentaq gene and cloned it in the same pET14b vector under a T7 promoter, this time without any histidine tag. The same inactivating mutation as for Taq was introduced via site directed mutagenesis by changing the aspartic acid 332 (GAT here) in a glycine (GGT) in the DYSQIELR motif to generate an inactive mutant. Expression was realized in different strains. The success of expression, and later self-replication, appeared to be highly dependent on the bacterial strain used. It was not possible to obtain KlenTaq expression with standard conditions in BL21 bacteria, but the expression and self-replication worked in the KRX strain.

I used the two constructs to transform electrocompetent TG1 bacteria, the same strain as in the CSR paper but did not manage to get successful self-replication.

Finally, KRX bacteria were used and selection efficiency test was performed to allow us to calibrate our selection process. Figure 135 shows that wild-type Klentaq expressed in KRX bacteria was able to perform auto-PCR amplification at high bacteria concentrations, up to 1000 bact/nL. However, the yield of the PCR reaction was greater for lower concentrations of bacteria. Furthermore, in order to compare with the IPA-PCR experiments, we needed to have similar droplet size. The droplet size was then chosen to be of ~24µm diameter (~7.2 pL).

Figure 135 also confirms that the inactive mutant of KlenTaq is not able to self-replicate, despite the presence of the gene in the bacteria.

Selection in droplets

Bacteria were encapsulated in the reaction buffer with primers flanking the beginning and the end of the gene sequence in ~7 pL (~24µm Ø) droplets, using the last version of the microfluidic device and fluorinated oil with 2% of EA surfactant. At the end of the droplets generation, emulsions were placed in a PCR machine to undergo temperature cycles. After this, the emulsion was broken using PFO. A simple selection run with a λ of 0.9 and an initial starting fraction of active Klentaq of 10% led to a final fraction of 90% of active KlenTaq gene in the population in one round. 1.2 Evaluating the efficiency of directed evolution protocols and selection/screening processes in droplets

Mock experiments

One way to check the efficiency of the selection is to perform mock experiments with "libraries" of know composition. Various way of designing these experiments can be used. In most of the case a fake library is generated where an active wild-type (WT) gene is mixed with a large excess of an inactive or low activity variant of this same gene. But other options are available, as long as one can discriminate between two very different levels of activity. For example, the active WT gene can be tested against a more active previously selected mutant, another enzyme or a protein that as no activity related to the experiment or just an empty vector.

One can also just count the active and inactive clones from a library before and after selection.

Measurement of inactive and active variant quantity

According to the composition of the mock library, different methods can be used to evaluate the quantity of each genes or types of clones before and after selection. Often, the inactive mutant is created via a deletion in the gene sequence. Therefore, it may be possible to detect the different versions of the gene on a PCR gel because of their distinct sizes. PCR or comparative PCR gel analysis are the most used methods to quantify active and inactive genes.

Other modifications in the inactive gene, like the insertion of a restriction site can also allow to perform a digestion by restriction endonuclease before the PCR to discriminate between the two genes. When it is not possible to easily discriminate between the different genes, white/blue screening of colonies or colorimetric activity screening on agar plate can be used provided that the gene is linked to a colorimetric output. In some in vivo screening experiments, the inactive cells can be stained and one count the remaining fraction after the selection. Finally, some clones can be picked and have their activity assayed on plates.

Importance of the mean occupancy λ in high-throughput compartmentalized directed evolution

The methods presented above can be used to measure the ratio or fraction of active over inactive genes before and after the selection process.

An important feature of high-throughput in vitro protocols is that they use a random distribution of the mutant library in the artificial micro-compartments (usually droplets, but also liposomes, micro-patterned arrays, etc). Because of this random compartmentalization step, a main concern is the co-encapsulation of mutants in the same droplet. For emulsions, regardless of the droplet-generation technique (shaking, filter extrusion, microfluidic, ...) the process generates compartments comprising different number of objects in each droplet. Even in the case of monodisperse emulsions, assuming for example that the mean droplets occupancy is 1, the proportion of compartments containing 0, 1 and more-than-one genotypes will be roughly one third each. Knowing the mean number of genotypes per compartment 𝜆, the distribution of occupancy n can be computed according to the Poisson law (see annex iv, p.332):

𝑃(𝑛, 𝜆) = 𝜆 𝑛 𝑒 -𝜆 𝑛! Intuitively, we can expect that the value of 𝜆 will have a strong impact on the success of the selection. Asymptotically, an infinite value for 𝜆 correspond to the bulk case, and therefore no selection at all.

To get more insight on this issue, we compiled data from efficiency tests collected in 25 publications on selections performed in droplets using various protocols. Table 22 lists experiments corresponding to selection methods were emulsion were generated by bulk shaking. Table 21 and Table 20 correspond to screening experiments realized respectively in microfluidic generated emulsions or by bulk method (shaking or extrusion through filter).

Values with a * were estimated according to the data given in the paper but not explicitly given by the authors.

Several things are interesting to notice. For example, we observe that among these 25 papers, the 7 based on selection strategies did not implement microfluidic emulsions and also used relatively high 𝜆 values, close to 1 or even higher. In the screening methods, both microfluidic or not, the value of 𝜆 is often much smaller, typically below 0.4. Also, most reports concerning screening methods using microfluidic systems present methodological developments on mock libraries, but do not actually evolve the enzyme. It is therefore hard to see directly from this data if working with lower 𝜆 and polishing the way droplets are produced, sorted and screened has a strong impact on DE itself. Due to the diversity of the enzymes targeted in the different papers it is anyway difficult to make a correlation between the obtained enrichment, the apparent selection efficiency of a method and the enzyme improvement achieved during the actual evolution experiment.

What are the effects of λ and p in experiments?

In an attempt to characterize the quality of a given high-throughput directed evolution protocols, many papers report a so-called enrichment factor ϵ by simply taking the ratio of the fraction of active variant before and after one selection round. In some cases, the enrichment factor is instead defined using the ratio of active variant before and after, instead of its frequencies. This can make methods comparison difficult, especially when the enrichment is not defined. In their paper about the FADS [START_REF] Baret | Fluorescence-activated droplet sorting (FADS): efficient microfluidic cell sorting based on enzymatic activity[END_REF] Jean-Christophe Baret and al. define the enrichment as the final fraction of active mutant over the initial one but decide to use the initial and final ratio of the active vs inactive mutant instead. According to them "it enables a quantitative comparison of enrichment over a much wider range of initial and final conditions" than the defined one. Indeed, if the ratio of positive mutants increases a 10-fold after selection the ratio enrichment will also increase a 10-fold contrary to the fraction enrichment that will depend on the initial fraction of active to inactive genes.

In their work, the theoretical enrichment using the Poisson law parameters is defined as:

ϵ = 𝜂 𝑚 = 𝑁 +,𝑚 𝑁 -,𝑚 = 1 1 -𝑒 -𝜀 0 𝜆/(1+𝜀 0 )
Where ηm is the theoretical enrichment, N+,m is the number of active variants at the m round, N-,m the number of inactive variants at the m round, ε0 the initial ratio of active over inactive clones and λ the initial mean number of cells per droplets. This raise a problem of having potential infinite enrichment factors. They choose to set a limit and record this special case as enrichment >1000. We chose to not use this definition but rather the ratio of the fraction. This ϵ parameter should be used to estimate the effect of the various biases regularly found in selection processes.

However, the usage of ϵ as a measure of efficiency of a directed evolution method comes with a number of issues:

• First, ϵ depends strongly both on the initial fraction of functional mutants p and on the value of 𝜆. As can be seen in Figure 138, measurements of enrichment factors made with different initial fractions generally yield values of ϵ that vary over many orders of magnitude. Similarly, the dependence on 𝜆 is not considered. Note also that sets of experiments done at higher 𝜆 appear to lie typically below protocols that use smaller 𝜆 values.

• Second, the upper theoretical limit of ϵ is infinity when p or 𝜆 become small. This reflects the fact that in principle, a flawless selection or screening assay should get rid of the poor phenotype in a single round. This problem was already noticed by Baret et al. 76 who decided to set an upper limit at ϵ = 1000. It remains difficult to estimate what should be a correct, or acceptable, value of ϵ. Moreover, in practice, small values of 𝜆 tend to yield quite poor ϵ. This can be due to statistical issues, difficulties in the recovery of the genetic material, or to parental contamination, among other possibilities. In any case, measurements at very small 𝜆 are not really useful in assessing a protocol meant to provide the highest possible throughput, as the throughput is actually proportional to 𝜆. Note that the theoretical lower limit of ϵ is 0 at high p or high 𝜆. Every value below 1 indicates a process that counter-selects for the good genotype.

?

* not explicitely given by the authors, estimated from available data in the paper **estimated from gels given in the paper or sup mat by eyes § enrich is an alternative mode of screening, the other most use mode in this paper is called the purify mode †ratio : ratio has been chosen to evaluate the method efficiency instead of fraction † 2 not defined : the formula of the enrichment is not given ¤ gating : Upper % percentile in UV fluorescence gate * not explicitely given by the authors, estimated from available data in the paper **estimated from gels given in the paper or sup mat by gel analysis with ImageJ †ratio : ratio has been chosen to evaluate the method efficiency instead of fraction † 2 not defined : the formula of the enrichment is not given N.B. : ratio enrichment is written when despite using fraction the enrichment is calculated over the ratio

Screening experiments with emulsion generated by microfluidic Altogether, it is clear that ϵ is not a very good way to assess efficiency and to compare different protocols in terms of their ability to enrich libraries in desired mutants. In line with the existing literature, we have still computed enrichment factors to analyze our experimental results. However, together with Anton Zadorin, we also searched for a better metric. We introduce here an improved performance index called Selection Quality Index (SQI), which is based on the two control parameters p and 𝜆 , and the observed value p'. SQI is expressed as a fraction of the theoretical (achievable) value for the selection of a functional variant from a pool of lethal variants and normalizes for the effect of p and 𝜆 . An SQI of 1 denotes a perfect protocol, whereas an SQI of 0 indicates a complete failure, i.e. the absence of any enrichment effect (negative values indicate counter-selection). Accordingly, this index -in combination with the throughput of the protocol-can be used to assess the true potential of a given experimental approach to concentrate rare catalytic variants. It can also be used to compare different protocols, or different variants of the same protocol.

How to select a value of 𝝀?

All random-compartmentalized selection processes are highly dependent on the 𝜆 value.

It is the fact that the co-encapsulation effect controlled by 𝜆 interplays with other experimental contingencies to affect globally the efficiency of selection, that makes the analysis difficult.

Anton Zadorin, researcher in my lab during my PhD, performed a theoretical analysis of the effect of random compartmentalization on the performance of selection processes 303 . The mathematical analysis assumes additivity of activity for compartments containing multiple genotypes. It was done in the limit of infinite number of droplets and objects but the results seem to be applicable to smaller realistic and finite situations. Some of the relevant conclusions are given below:

• The best genotypes are still enriched, regardless of the value of 𝜆, but at rates typically decreasing with 𝜆.

• The presence of multiple genotypes per compartments is less deteriorating that intuitively thought. Additionally, one should distinguish screening-like process, were the fitness (the number of copies in the next generation) of a good phenotype is not affected by co-encapsulation with a bad one, and selection-like processes, where it is (it is divided by two). We refer to these situations as nonsharing and sharing respectively. Counterintuitively, the latter is more robust to co-encapsulation.

• The efficiency decrease is strongly related to the shape of the replication function, i.e. the function that describes the link between activity and fitness.

For linear functions, the decrease in efficiency is approximately inversely proportional to 𝜆 at high 𝜆 values. However, in the case of exponential functions, the negative effect disappears and the process is altogether insensitive to the value of 𝜆 (of course this is in the limit of infinite population and infinite growth potential in each droplet).

• Using libraries size exceeding the number of droplets (λ > 1) can be more favorable than reducing the initial population diversity. For example, in the case of polynomial selection functions, increasing 𝜆 will never reduce the ability to select more than it increases in throughput.

• To obtain the highest final mean activity in a selection process the best strategy is always to split the droplets "budget" in as many rounds as possible. This advocates for protocols were the "cost" of a round is low, such as the approach that we develop here.

• Knowledge of the population phenotypic structure and the shape of the replication function is enough to define the best 𝜆 value for a given experimental setup.

A new quality metric for selection protocols

To use this mathematical approach in order to properly assess the quality of various directed evolution protocols, we need to first consider the factors that affect the change in frequency of a mixture of active and inactive mutants. These factors can be separated in two groups.

The first group contains controllable or known parameters of the experimental design: 𝜆, the sharing status, and the replication/selection function. These effects are detailed below:

• As mentioned above, when 𝜆 increases, a fraction of the compartments may contain more than one phenotype, leading to the occurrence of "hitch-hikers". These are inactive mutants, which should not have survived the screening or selection process, but happened to share a compartment with a better phenotype. In principle, knowledge of the compartmentalization process allows us to describe the distribution of occupancies.

For example, in the case of random compartmentalization which we consider here, this distribution follows a Poisson law.

• A second effect, also linked to random co-encapsulation of variants, is fitness sharing.

253

This occurs when the presence of the poor phenotype is actually detrimental to the good one. This is typically the case in selection experiments: an equal fraction of the replication potential associated with one compartment will be used to amplify each local variant, whether or not they actually contributed to the replication activity. This sharing effect typically does not happen in screening protocols, which works by discarding the low activity compartments, rather than replicating the genotypes in the good ones. In other words, a variant present in a supra-threshold compartment contributes one genotype to the next generation irrespective of the presence of co-encapsulated variants in his compartment.

• Third, the replication/selection function, which describes how many copies each phenotype is able to transmit over one round, has a strong impact on the selection dynamic. In screening for example, the value of the function is one if the phenotypic activity is above a given threshold, and 0 otherwise.

The second group consists of experimental peculiarities that are not directly controllable. Together, they explain the observed deviation from a perfect assay. These factors can be based on a variety of causes, some known or guessable, other altogether unknown. For example, in screening test, the sorting machinery may not be 100% efficient and have a significant error rate (which typically increases when sorting is attempted at higher frequencies) (#3). Noise in the expression stage, where compartment containing the same genotype may end up with various level of observed activity (typical when bacterial expression systems is used.

i.e. lysate assays) can also play a role. Additionally, leaks, poor genotype-to-phenotype linkage (e.g. in mRNA display strategies), hidden selection biases (e.g. linked to toxicity effect), nonhomogeneity or polydispersity of the encapsulating compartments, copy number variation, carry-over of the parent genomes in selections, tendency of bacteria to stick to each other and a myriad of other causes may affect the result. One typically does not have enough information to precisely describe or model these effects, and is rather interested in quantifying their collective negative impact on the selection protocol, in order to empirically try to minimize it.

Anton used his previous mathematical derivation of selection under random shuffling to compute theoretical (expected) value of the fraction of active variants in the selected library, p' as a function of the initial fraction p, and the value of 𝜆 (Figure 139). This function is discontinuous at p=0, reflecting the effortless invasion of the population of dysfunctional mutants by a functional one. Therefore, the value of the jump Δ provides a good absolute characterization of the selection efficiency at very small p, as it depends only on the known parameter λ. Since it is not possible to perform experiments at infinitesimal p, we have to use a linear approximation between p->0 and p=1. The corrected jump is therefore equal to Δ + (1-Δ) p. Finally, we can use the difference between expected and observed jump to compute an unbiased metric of efficiency, which we call Selection Quality Index. In the non-sharing situation, which is typical of screening protocols, we have:

𝑆𝑄𝐼 𝑠𝑐𝑟𝑒𝑒𝑛 = (1+𝜆)(𝑝 ′ -𝑝) (1-𝑝)
. In the sharing (selection) case, 𝑆𝑄𝐼 𝑠𝑒𝑙𝑒𝑐𝑡 = 𝜆𝑒 𝜆 (𝑝 ′ -𝑝)

(𝑒 𝜆 -1)(1-𝑝)
. These formulas provide an absolute measurement of protocol quality, that is independent of p, 𝜆 and the replication function f, reflecting only the experimental contingencies belonging to the second group.

Application to reported data.

Applying this approach to the reported data listed above immediately reveals some interesting trends. The corresponding plot is shown in Figure 140.

First, many of the experimental points tend to collapse to some particular values. For example, the points of #6 or #11, which were taken for different p above 1/100, now give a single value for SQI, independent of the initial fraction. The trend that associated higher values of 𝜆 to lower enrichment scores is not visible any more. Additionally, for relatively high initial fractions, we note a cluster of data points that achieve a value of SQI close to one, indicating that the best achievable enrichment has been attained.

Second, for the lower p values, the selection efficiency always decreases with decreasing initial fractions of active mutants. This stands in striking contrast with the trend observed in Figure 138, where enrichment factors seemed to indicate that assay performed much better at lower initial fraction. This was in fact only due the fact that it is easier to invade a population of dysfunctional mutants, rather than a population already containing a significant fraction of functional mutants. Our analysis and metric correct for that mechanistic effect. It thus highlights the fact that other experimental contingencies actually dominate at low p, making experimental protocols less and less ideal as p decreases. This is probably due to a decreasing signal-to-background ratio, where the background noise can result from a variety of effects, such as contamination by parent DNA in selections, sorting errors in screening or the difficulty to recover very small amounts of genetic material in the general case.

Third, protocols can be separated according to their type: only screening using microfluidic compartmentalization appear to yield efficiencies close to 1. In contrast, protocols using screening of bulk emulsions have a lower performance. This seems to indicate that the size-dispersity of the compartment is an important factor contributing to the selection efficiency. Note that some selection processes are able to obtain high index without resorting to monodisperse emulsion, which could be related to the superior robustness of selections versus screenings, with respect to co-encapsulation, mentioned above. Note that reports concerning selections tend indeed to use higher 𝜆 values.

Measurement of active and inactive fractions of the mock KlenTaq library using rhQPCR

As in our case active and inactive KlenTaq genes have just one nucleotide mutation between them it is not possible to distinguish them on a PCR gel. To have access to the initial and final quantity of the two versions of the gene we used a new qPCR method called the rh-qPCR able to differentiate single mutation in a sequence. Detailed presentation of the methods can be found in the annex v, p.334. For each sample 2 qPCR reactions were run with a different rh primer matching the original or the mutated base. This allowed to access separately to the 138. Lines connect data point originating from the same report and done with a single value of λ (indicated next to the line). The plot legend refers to the reference number. Disks are used for protocol using screening and bulk (nonmonodisperse) emulsions; diamonds, for protocols using screening and microfluidic (monodisperse) emulsion; Stars, for protocols using selections in bulk emulsion. The dotted line at SQI =1 represents the theoretical maximum efficiency. The general trend now indicates that lower initial fractions negatively affect the process, in line with experimental knowledge. Protocols get less efficient when one uses lower initial fractions of active mutant. It is also clear that screening protocols with polydisperse emulsions perform worse than the monodisperse case.

SQI

quantity of each version of the gene in the sample.

Selection efficiency of the CSR in our conditions

Inactive and active version of the KlenTaq enzymes were used to assess selection efficiency of the CSR. An initial fraction of 10% of active clones among inactive clones was used to estimate by rh-qPCR the final fraction of active clones after CSR in our conditions with various λ values. No DpnI digestion of the obtained PCR product was done before quantification so initial genes are still present in the final obtained measurements. Experimental details can be found in Material & Methods.

In a first assay, rh-qPCR was used to infer the active Klentaq gene fraction before the selection and either after running the selection experiment in droplets. Measured initial fraction using rhqPCR was 13.5% and the final fraction after CSR in droplet with λ ~0.3 was 89.2%. This correspond to an enrichment factor of 6.6 (or 9 if we use the initial fraction to be the expected 10%). The value of SQI is 1.013 (1.019 using 10% of initial fraction), which means that we have an optimal selection.

In light of the previous discussion on the effect of the Poisson law in the selection, two other sets of experiments were performed, using various lambda. For the second experiment we modified the rh primers, because the first generation of primers gave poor realtime amplification curves. The second experiment may thus be considered more reliable than the first one (see Table 23). Note that in this second set, the initial fraction of 10% of active clone was measured this time to be more around 20% despite the method used to measure bacteria concentration using optical density was the same as in the previous experiments. The results show that the efficiency is decreasing with higher lambda: the enrichment in one round is less pronounced because of the carryover of inactive mutants by active ones. When we compute the SQI values, we observe that it is very close to 1 for 𝜆 below one, and larger than one at higher 𝜆.  and SQI for the CSR report could be estimated around 15-80 and 1.6-1.7, respectively, with 𝜆 = 1.7. To conclude this chapter, these values allow a few remarks: First, at low 𝜆, the efficiency of the CSR method to select PCR polymerases seems very good, as it performs almost optimally, at least with relatively rich libraries. It is surprising that the original protocol, which used very polydisperse emulsions, seems to perform as well as our cleaner microfluidic approach. This observation has to be taken with care however, as the report is not very precise, and we had to estimate some parameters. The best way to get a conclusive answer would be to perform "our" Klentaq CSR using "bulk emulsions".

Second, it is notable that for large 𝜆, both the original report and our re-implemention yield SQI values well above the theoretical limit of 1. Figure 146 comparing the theoretical final fractions also shows this difference between the results and prediction. A possible explanation for this repeated observation would come, somehow counterintuitively from lysate toxicity. A consequence of this toxicity would be that multiply occupied droplets may not efficiently amplify and thus, contribute less to the composition of the selected library. In other words, the reaction is somehow naturally immune to carry-over by co-encapsulation. But contrary to the case of NBI where above 120-160 bact/nL IPA-PCR is not working anymore it is not the case with KlenTaq as shown in Figure 145.

Third, in light of the literature survey presented above, it would be interesting to measure the SQI value of CSR, and more generally in vitro self-selection experiments, with lower initial fractions. This is indeed an important parameter to characterize the behavior of a selection process on real libraries submitted to strong selection pressures, were active variants may be extremely rare. This has not yet been attempted and is left for further studies.

Compartmentalized self-selection process

Similarly to the KlenTaq selection experiments, mock experiments where first used to assess the efficiency of our new selection method. Those experiments allowed us to select the best molecular program parameters.

Inactive mutant of NBI generation and characterization

For mock selection experiments, we first created an inactive mutant of NBI by site directed mutagenesis. In their comparative study of MlyI, PleI and NBI endonucleases, the researchers of New England Biolabs performed amino acids substitutions at some positions, which they thought to belong to the catalytic site, according to sequence alignments. Four substitutions from aspartic or glutamic acid to alanine lead to inactive or almost inactive enzymes 138 , E418A (0% activity), D456A (0%), E469A (1%) and E482A (1%). The position 418 was therefore chosen for our mutagenesis and the GAA codon coding for the aspartic acid was changed into a GCA codon coding for an alanine. Because it was expected to be completely inactive, the mutated plasmid was transformed in the KRX strain with no methylase protection.

The enzyme was expressed with an induction of 3h30. It was then purified using a his-tag column, dialyzed to a storage buffer and its activity was assessed with the loopact activity reporting probe (Figure 148). The growth of the bacteria during expression was normal and they reach an OD600 of ~0.6 within 3 hours. Despite being described as inactive by NEB researchers our version of the enzyme showed a very low, but detectable activity level. This difference may be due to the difference in sensibility of the activity measurement: NEB researchers used gel electrophoresis of T7 phage DNA or pNBI plasmid to detect the activity, whereas here we used a molecular probe allowing a finer, and real time, detection of activity.

This first trials were done in Japan, before the discovery of the incorporating transposon IS5 and the use of T7 express bacterial strain. So, when the selection process was about to be tested, the plasmid carrying the active and inactive clones were transformed in T7 express cells, this time both containing the pleI plasmid so that both inactive and active gene were in the same conditions. After transformation, a colony PCR revealed that the insert IS5 was still present in some active genes but also in some inactive genes. The Figure 147 shows the colony PCR results. The clones numbered in dark on the figure were grown and their activity assessed directly from lysate with 100 bact/nL. They were all chosen to not have the IS5 insert except the supposedly inactive NEB_pNBI-_1, for curiosity. As it is shown in the table in Figure 147 B, among them some did not grow well, notably the inactive genes comprising an insert. 25 andTable 26. In these experiments, I first prepare 2 premixes. They both contain all the necessary reagents, but one only contains the bacteria and the only other the lysing agents. These 2 premixes are connected to the 2 inlets and various pressure is applied. This ensures that bacteria contact the lysing agents just before encapsulation and limits the amount of leaked enzymatic activity present in the bulk solution. Bacteria are then lysed after the droplets formation. The workflow of these experiments is schematized in Figure 151. Starting from the initial conditions, which have been shown to allow nickase-induced gene amplification in bulk, the first trials (1 and 2) starting with 1% of active bacteria did not yield to any selective amplification. We tested percoll as an additive, because it has been reported to increase the density of the buffer, and therefore prevent bacteria sedimentation and than what would the Poisson law predict. As explained in the part III about microfluidics no significant difference was observed between the 3 temperature ramps we tested. And no selection happened in the three conditions. Trial number 4 was also the first time the stepdevice, producingdroplets of about 28µm diameter was used. All the following experiments were performed with this device.

In trials 5, 6 and 7, droplet and non-constant surfactants effects, as well as bacteria capricious behavior toward lysis or experimental flow could be part of the answer. Some stability needed to be found for proper and reliable selection to happen.

In the next experiments, the bacteria were grown using the final optimized conditions at 30°C and with 50µM of IPTG for 2 hours. These changes avoid the production of filamentous bacteria, a correct and homogeneous shape is important because it allows us to control properly the concentration of bacterial lysate droplets. Also, it is possible that bacteria grown in these conditions may have a better membrane integrity and avoid the potentially problematic leakage of enzymes before encapsulation.

In the trial number 8, we wished to test the possible effect of unbalanced mixing of the 2 master mixes at the droplet forming junction. Because of bacteria forming films in the filters, the pressure constantly needs to be adapted in the bacteria containing channel to maintain an equal contribution of the two flows. Also, the relatively dense bacterial suspension may affect the viscosity of the solution. Some experiments done by Rémi Sieskind in the lab made us realized that to have equal contribution of two solutions in the droplets, trying to spread the two solutions equally in the channel is not an appropriate solution when working with solutions of different viscosity. When setting the same flows with syringe pumps for gel beads generation, it appeared that the separation of the two flows was not visually in the middle. We thus did an additional bulk control experiment, were we biased the volume ratio to 1/3 of the solution containing the bacteria and 2/3 of the solution containing the rlysozyme. This biased bulk resulted in a faint band on the gel, suggesting that our protocol was indeed sensitive to the precise mixing ratio (although in principle, only the bacterial and rlysozyme concentrations should be affected). This could thus explain some of the difficulties of our experiments. In the droplets no selection was observed for this trial.

In the 9 th experiments, syringe pumps were used instead of pressure pumps to avoid unbalanced contribution of the two flows. Droplet size and disperrsity was not directly measured, but was estimated to be similar to the previous experiments. It was also the first time that we replaced the droplet stabilizer with a combination of pluronic f-127 and yeast RNA, following the advice of Dany Chauvin from the LBC laboratory (see III.4.2, p.231). Syringe pumps did not improve the selection. Going back to pressure pumps in the 10 th experiment no change was observed. But the use of pluronic f-127 and yeast RNA indeed improved DNA recovery after breakage of the emulsion.

In the 11 th experiment, we tested the possibility of exchange, or leakage between droplets. Droplets containing either the active or the inactive mutants were generated separately.

Then they were mixed together to obtain the usual 10% ratio. No preferential amplification of the active mutant was observed. rhqPCR correctly measured about 100% of active and 0% of inactive clone in the respective tubes. This time, a product was observed on the gel from the inactive gene bulk control experiment and, to a lesser extent, in the droplets containing only the inactive bacteria. In previous experiments bulk control experiments were just bulk replicates of the droplet experiment. They were only constituted of the proportional mix 10/90 of the two solutions used for droplet generation. It was assumed that the inactive mutant could not amplify.

But, the band on the gel was almost as bright for the inactive mutant than from the active mutant (10 times less concentrated though).

Next experiments focused on the molecular programming adaptations presented in part I. It was hypothesized that the trigger initially present in the mix could be extended by the polymerase on the primer-producing templates and could be used as a tailed primer in amplification. While PCR is usually recommended with 200 to 500 nM of each primer, we had shown that, in our particular conditions, PCR could happen with very low amount of primer (e.g. 10 nM; see Figure 88), and that the final yield (after 30 cycles) reached rapidly a plateau for primer concentration above 50 nM. So, the small concentration of potentially extended trigger (~20 nM) could be enough to amplify consequently the inactive gene. In the 12 th trial, concentration of the trigger strand was divided by two. Starting with an active gene fraction of 10%, this resulted in an effective selection, with a final fraction of active gene of 60%. This result was encouraging but selection efficiency was not reaching the one of the CSR. As a reminder, in CSR mock experiments, the active gene reached a final fraction of about 90% in similar conditions. If comparing the SQI values we can find 0.66 for the 12 th trial (considering we started with 10% initial fraction) versus 1 for the CSR.

To further increase selection efficiency, it was tried to decrease Mg2+ concentration of the buffer in the 13th trial. The small activity of the inactive clone, recorded for the purified enzyme, could also be a reason for primers production. As relatively high concentrations of Mg2+ are necessary for otpimal activity of the nickase, this newIPA buffer was used in hope to render the primer production more difficult and favor the active clones. But, this change finally decreased the selection efficiency and a final active gene fraction of ~20% was recovered. This result reinforced the hypothesis that the main issue was the nickase-independent background production of tailed primers by the molecular program, and not the residual activity of the inactivated mutant

Selection efficiency assessment using a modified molecular program

The molecular programming experiments (in test tubes) presented in part I had shown that using a source template instead of the trigger could solve the background primer generation caused by the trigger strands. This source template also accelerated the reaction and a very small amount amount was needed to start the reaction.

Indeed, in droplets, a concentration of 2nM in IPA V0 buffer, with all the previously validated modifications yield to a successful and efficient selection of the active NBI gene.

Depending on the IPA incubation time (15, 30 or 45 min), the initial fraction of active gene of 10% was respectively converted into a final fraction of 91%, 88%, 76%. These final fractions correspond to the following SQI values: 1.09, 1.05, 0.89. These results presented in the Table 24 and in the last line of Table 26 as trial 14 suggest that the selection is more efficient with a short incubation time in these conditions. In Table 24 the column AmpliON represents the amplification of the active gene, that means, the ratio of measured quantity of active gene after selection and before selection. The measured AmpliON values indicates an amplification of at least 10 4 times in droplets PCR for an active gene increasing between 15 and 30 min of incubation. The amplification of 2.2x10 4 obtained for only NEB+4 active bacteria is just slightly smaller than the one in bulk ( -25%). The corresponding AmpliOFF seem to point to a general contamination of the droplets by free enzymes. In the bulk reaction, no amplification is observed for the inactive gene (ratio of 2.6) whereas it is amplified ~100 fold within the droplets for the same amount of time. The decrease in selectivity with IPA incubation time seem to be linked to the increasing amplification of the inactive variants while the active variants amplification stays constant. Free nickases encapsulated in droplets with inactive variants, and increasing slowly the primer concentration in those droplets, could explain the observed phenomenon. Exchange of primers between the PCR in droplets could also be an explanation. Gadhessy et al. observed an exchange phenomenon between droplets during thermocycling [START_REF] Ghadessy | Directed evolution of polymerase function by compartmentalized self-replication[END_REF]273 . Surfactant bilayers could form and be porous to certain small molecules.

Gadhessy et al. observed the phenomenon for dNTPs when trying to evolve the Nucleoside Diphosphate Kinase which produces dNTPs to allow a PCR amplification. But they did not investigate the possibility of primer exchange as primer concentration in their case was the same everywhere. It has been demonstrated that exchange rate is a function of hydrophobicity and size of the molecules in the case of perfluorinated oil 273 and theoretically, our oligos are not hydrophobic and not that small. Many experiments are run in droplets in our laboratory and no oligonucleotides exchange have ever been observed during isothermal incubation. That is why the first hypothesis is preffered. Furthermore, we have seen in I.3.1 that leak of activity can happen by measuring supernatant activity.

In fluorinated oils, the transport of small molecules in emulsions was shown to depend on surfactant concentration [57], an indication that there is no significant energy barrier to partitioning in emulsionsor at least in fluorinated oils 274 .

Selection of a library of mutants

A first experiment was run on three previously generated libraries (Lib 50, Lib 100 and Lib 200; see part II) and led to very few amplifications. After many trials to amplify the recovered product and the original libraries for sequencing we noticed that the primer generated by IPA was actually targeting a part of the plasmid that had been modified during the cloning of the library. Strangely, this primer with at least 6 bp mismatched in 3' still produced some amplification of the correct product according to sequencing data. It is possible that the high concentration of magnesium used in the IPA PCR process decreases primer specificity and results in a higher tendency to amplify PCR products from primers with mismatch. The molecular program primer-producing templates were then changed to target the correct library construct. This change in template sequence unexpectedly resulted in new discovery about template sensitivity to bacteria lysate. Indeed, after trying many different primers and associated templates, it appeared that some sequences are more sensitive to the lysate and are degraded faster. More details are given in part I. Unfortunately, one of this sensitive sequence was necessary for our amplification scheme. After further optimization, decreasing rlysozyme quantity by half (it was put back to its initial quantity of 0.25%) appeared like the best solution.

The IPA-PCR reaction was even observed to work fine with no rlysozyme at all. Soft lysis of the bacteria, or maybe some pore opening at 45°C, seems to release enough activity for IPA, while avoiding a strong release of template-degrading factors from the bacteria.

With these new modifications, the library Lib50, amplified with mutazyme kit II from 50ng of template DNA was submitted to the selection process. This library is the one with the highest mutation rate of 3 mutations per kb. The library underwent either a neutral selection with an IPA of 30 min or a heat shock selection with a 3-minutes incubation at 60°C preceding the 30 min IPA. In this condition we expect the wild-type to be unable to perform IPA-PCR.

The selection was run with a λ of 0.7 and about 60µL of emulsion were collected to ensure a 20X coverage of the library.

qPCR assessment of selection

After selection, amplification was not visible in the gel electrophoresis, neither for the bulk equivalent nor for the product of the droplet experiment. Since the library has a modified behavior compared to the wild type this lack of amplification in bulk did not seem problematic.

But investigation needed to be made to know if some selection occurred and some genes were amplified. As it is not possible to run a rhqPCR to measure the selection efficiency on the library, a simple qPCR was run to estimate amplification. Table 27 shows the results of this quantification. Raw product extracted from droplets can be compared to results obtained in a previous successful active/inactive selection realized in the same conditions with rhqPCR measurements. When comparing measured DNA quantity in the before and after selection tubes in this mock experiment the DNA was amplified about 550 times in active droplets during the selection. This has to be compared with the "raw" data column as these samples were not purified or treated with dpNI. If one assumes 10% of the library to be active, a 550-fold amplification of these mutants would result in a global amplification of 55 times. In selection experiment droplets, amplification was in the same order of magnitude, between 50 and 60

times which seem to be consitent with a successful selection run.

In parallel, we digested the samples with the restriction enzymes DpnI, which cuts only methylated DNA, and this removes the original plasmids. Further PCR of the digested product with primers internal to the selection primers led to the amplification of a product with the expected size. All these elements taken together are pointing toward an amplification of some NBI genes in droplets.

Activity improvement in the selected library

Cloning of the selected genes back in vectors proved more challenging than anticipated. seem to be prone to generate primer dimers.

Addendum: Results presented on the defense day

After many trials, we managed to obtain a recloned library with measurable activity but still an important quantity of empty vectors. To evaluate the activity more accurately, we expressed and purified the enzymes from the library before and after selection and compared their activity at the same concentration with the activity of the wild-type. We can see on the

Figure 154 that we have a slight increase in activity after selection. Therefore, it seems that we indeed selected some active variants but with a low selection pressure. approaches like crystallography, NMR or functional studies and are usually base on saturated libraries.

In 2015, Romero and al. 10 proposed to use droplet-based microfluidics as a general tool for screening of enzyme libraries and beneficiate from the advantage they offer to run "in vitro" selections. In this situation multiple enzyme properties like thermo-resistance, catalytic speed, buffer composition tolerance can be mapped systematically. They proposed to use the results

for multiobjective protein engineering.

These methods based on sequencing currently possess several limitations. The first and most important one is the limitation in the size of the sequences that can be read. Illumina sequencers can read 10 million of sequence but of only 300 bp in average and can go to 500 by using pair-end techniques. The iontorrent PGM is also limited to 400 bp. Barcoding systems can be used to palliate this issue but necessitate to have read the sequences at least once 305 .

Methods able to provide long reads like PacBio or MinION have high error rates that are not directly compatible with the detection of point mutations in sequence. MinION has an average error rate of around 5-10%. PacBio has an average error rate of 13% per base that can yield up to 99.99% of sequence accuracy, using circular templates read multiple times (40X coverage).

Using such sequencers, fictitious mutations can be messed-up with intended ones. But the sequencer is not the only way of observing fictitious mutations. It is primordial when performing deep mutational scanning to try to avoid introducing unwanted mutations that would increase artificially the mutation rate and bias the data. To obtain high quality sequence, high fidelity polymerases should always be used for amplification and excessive or unnecessary PCR cycles should be avoided. Using the wild-type gene control at various stage of the process (from selection to sequencing) can allow to calibrate the mutation rate induced by the DNA manipulation and the sequencer internal error 306 .

To overcome the read size issue, one can sequence independently the different parts of the gene, as we did to analyse the ePCR libraries. Kowalsky and all 307 proposed a similar method to map the full-length of a protein using tiled regions. They introduce mutations only in one part of the enzyme, submit the library to selection and sequence only this mutated part.

Functional mapping by sequencing cannot be readily applied to any protein, because a selection or screening strategy need first to be set up. So far it has been mostly applied to a few model proteins, in many cases ligands because they can be easily selected for function (e.g. …).

Proteins with more complex function, such as enzymes, or with unknown function represent more difficult targets. Other limitations can be the cost of next generation sequencing, which is still quite high although it decreases steadily. Typical functional scanning requires many cycles and therefore many NGS experiments to use the full capacity of the sequencer to explore the biggest possible part of the landscape. Limitation in the number of reads reduce the portion that can be studied but as in general directed evolution experiments, transformation of organism with the genes is generally one big issue. Sequence-specific amplification bias during sample preparation is also affecting the results.

In one of the first comprehensive survey of fitness landscape, Jimenez and al. 308 focused on small RNA to have full coverage of the sequence space using deep mutational scanning analysis. They discovered that a limited neutral network is present, but most fitness peaks are evolutionary isolated. Drops in fitness appear mostly in the first mutation away from the peak species and additional mutations had less effects. Higher fitness peaks appeared more robust to mutations. Their results are encouraging in the sense that they show that fitness landscape study can provide great information. Could similar pattern be found for proteins? Because the structure and function emerge form very different set of chemical interactions between the building blocks (in particular, nucleic bases interact mostly by pair, which is not the case for aa) these results would probably not directly translate to proteins.

Deep mutation scanning can be applied to selections targeting for enzymatic efficiency but also in many contexts like in antibody-epitope mapping, antibody-antigen maturation. It has also been used for the fine tuning of computationally designed protein 307 . Whitehead and al.

used the method to increase affinity and specificity of influenza inhibitors 309 .

Despite the huge interest in functional mapping of proteins, these techniques are not perfect. Otwinowski and Plotkin identified bias inherent in the procedure of subsampling the fitness landscape 310 . Exploring a reduced sequence space may lead to underestimation of high fitness and overestimation of low fitness. Therefore, the measure of epistasis is distorted in the inferred landscape and predictions for the dynamics of adaptation are biased.

In directed evolution methods, following the evolution by deep mutational scanning can allow to identify important residues and help taking decisions for the next evolution cycle. From such experiments, Boyer and al. discovered rules that should help designing synthetic protein libraries for binding purpose. They could estimate the density of functional biomolecules in the sequence space and experimentally investigate the evolvability of a library 311 .

It is a safe bet that new improvement in the throughput of NGS sequencing and in vitro evolution protocols could improve the yield to better coverage of the fitness landscape and unleash the power of comprehensive and unbiased sequence-function mapping. Data-driven protein engineering could even outpace traditional physics-based methods in rational design. In particular, while many studies focus on the effect of point mutations, evolutionary data can also be used to infer epistasis information 312 . Such data can even be used to predict protein structure 313,314 . Co-evolutionary signals can allow to identify local native contacts 315,316 .

NGS sequencing of the selected libraries

While waiting for the activity tests of the recloned library, we performed NGS sequencing on the selected libraries. The two selected libraries were sequenced using a MinION nanopore sequencer. As explained above, MinION sequencing has an error rate that is quite high. We could have used ionTorrent sequencing, as for the library characterization. But, the NBI enzyme is quite large and we are interested in eventually finding correlations in amino acid substitutions. We thus expect that statistical analysis of the data can allow to overcome the high error rate of MinION reads and access to the pair coupling information.

MinIon sequencer proposes several kinds of chemistry. In the 1D chemistry, the single DNA strands are read separately. In the 1D 2 mode one strand is read and, just after, the other strand is engulfed in the same pore to provide a second read of the complementary sequence.

This is used to increase the read accuracy. The first PEN CSR experiment done with the wrong primers were done using the 1D chemistry. It yielded in a high error rate, estimated on the wildtype gene to about 10%, but ~2.5 million reads were obtained from which ~2 million sequences add sufficient quality. The 1D 2 chemistry was then used for the sequencing of the library selected with the right primers either neutrally or with heat shock. The error rate was decreased to 5% but fewer reads were recovered. Only ~200 000 double reads were obtained but 93% of them was of good quality. generate the inactive mutant is also in red and not permissive. In the binding site, we can see that some parts are more permissive than the other. These results seem to confirm the selection and that it is possible to find information in the noisy data recovered from the MinION. At present, the analysis of the data does not use the couplings between mutation on a given mutant but focuses of point mutations. In other words, this information could be obtained from truncated sequencing. However, we plan to extend this analysis to pair correlation soon. One hypothesis could be that some sequences are better amplified by the PCR than others, even if it seems surprising a single mutation can have a drastic effect of the amplification of a 2kB gene. Alternatively, some sequences could plateau in the PCR reaction at low concentrations, while other would reach higher saturating yields. Another hypothesis is that in the IPA-PCR conditions, the high quantity of magnesium could yield to increase mutations toward A or T, thereby creating a supplementary bias. Note that the NBI gene is AT rich (67% AT), so possibly the molecular biology protocols that we built for it are inherently biased toward AT rich sequences. This looks like a general effect, as a similar bias toward AT reach sequences was observed by Holliger team in their CSR experiment [START_REF] Ghadessy | Directed evolution of polymerase function by compartmentalized self-replication[END_REF] . In their case, for silent mutations, a decrease in GC content was observed. This bias was not observed for "coding" mutations. AT rich sequences promote PCR amplification by facilitating strands separation and destabilizing secondary structures. The bias, observed only for silent mutations suggested that this effect was not caused by the encoded codons, and that it was secondary to the main selection force, working on amino-acids mutations. The author this attributed it also to base effect on PCR efficiency. Overall, one has to remember that in the context of the PEN-CSR, like in other selection scheme, all the molecular steps present in the replication loop impact the evolutionary outcomes. Here for example, the gene can evolve simultaneously toward a better enzyme, toward a higher expression, and toward being a better template for PCR. The possibility to change the shape and properties of the selection function is one of the main interests of using molecular program for directed evolution, in contrast for example to microfluidic screening method which are restricted to simple YES/NO functions.

My

Unfortunately, few results were obtained on this aspect. In future works, more experiments to demonstrate and characterize other selection functions should be envisioned. The selection function we characterized and used for the mock library presents a rapid saturation, and a relatively small window for primer production. It is not applying very much pressure and just require a minimum of activity to pass the selection step.

Using a reporting probes to determine when a certain amount of primers is reached could be used to determine when the best mutants (in droplets) has produced enough primers and decide when to start the PCR step. Such system would allow to have more control on the selection but would require additional steps and monitoring of the reaction under microscope.

First tests in that direction showed that the introduction of a primer-reporting probe negatively affects the PCR, even at low concentrations. Other tests with loopact probe in droplets showed that heat diffusion in the microscope slide could also be an issue to have a system really reflecting what is happening in the PCR machine.

Although the selection function was characterized, we have to stress that is does not exactly represents what will happen in a real selection. This is because we used increasing concentrations to mimic increase in activity. To really characterize the selection function, different mutants with different activity due to their mutations could be used. This approach was started during the work, but activity recorded for the different mutant did not seem to be perfectly linked to the mutation, as different wild-type versions exhibited different activity in the assays. Furthermore, despite all optimization efforts, the IPA-PCR reaction in bacteria lysate remains a capricious system. Nevertheless, trying different selection functions and characterizing them to see the effect on library selection should be one important part of the next developments.

Modifications of the templates are not the only optimization allowing the reaction to perform in bacteria lysate. Some template sequences had increased sensitivity and required adjustment of the bacteria lysis process. The lysis of the bacteria was optimized using either rlysozyme, BugBuster or their combination and we showed that none of the solution can for sure fully lyse all bacteria. In addition, rlysozyme and Bugbuster both demonstrated inhibiting or interfering effect with reporting and/or molecular program, especially in the absence of bacteria lysate. Thus, a balance needs to be found between the lysis rate, quantity of released target enzymes, quantity of released toxic component, and total quantity or lysing agents. The non-homogenous lysis of bacteria can also introduce bias in the selection experiments which are benign in the context of a mock ON/OFF experiment but can have more critical effect for real libraries. Surprisingly, in the absence of any lysing agent it was still possible to run an IPA-PCR with success in lysate in bulk, but it is not clear what is the fraction of bacteria that excrete NBI enzyme upon temperature jump to 45°C in droplets. Furthermore, a mild lysis is working in the case of NBI, but for adaptation to other enzymes, balance between their releasing level and lysate toxicity could be completely different. The spontaneous leakage of NBI activity in the buffer can also introduce bias in selection and could explain with higher IPA incubation times yield to less efficient selection. Several observations during the project seem to show that the order in which elements are added in the reaction tubes is important for the reaction. In particular, when bacteria were added before the v11 trigger in some experiments, the detected primer production signal was really low in two repeated experiments. When adding the bacteria after assembly, the usual signal was retrieved.

The dynamics of the molecular programs adds to this toxicity problem. The incubation time for the IPA step is one way to leverage the flexibility of the selection function. For example, decreasing the released activity, while incubating for longer time could help to solve toxicity issues. However, in practice we observed that the available time window for IPA incubation is limited. First, something happening in the bacteria lysate prevents incubation over 45 min. Second, even for in vitro IPA-PCR, we recorded a failure for long IPA incubation times, e.g. for more than 1 or 2 hours, depending on the produced primers. The rationale behind these observations is not clear but could come from a slow degradation of enzymes or templates in the lysate or by appearance of byproducts such as primer-dimers. Differential production and degradation of the two primers leading to unbalanced primers concentration could also be an explanation. Even if we obtained positive results for the NBI selection experiment, more investigation needs to be done on this aspect. Better understanding of all these toxicity and molecular program issues would allow to propose a more robust selection system, easier to transfer between laboratories or adapt to other enzymatic activities. The limited time window can be a problem for more complex network requiring more time to produce primers, or for detecting lower initial activities in libraries. Pursuing investigation on the Bst polymerase used in the program could eventually help. Work to increase the program resistance could also allow to decrease more the droplet size and eventually increase the throughput of the experiment. But, limitations in small droplets generation speed could act conversely and finally restrain the throughput.

About the microfluidic device development, the current speed of droplet production with reasonable production time provide compartments in the range of ~10 [START_REF] Dill | The Protein-Folding Problem, 50 Years On[END_REF] . Because of the actual library transformation bottleneck (10 6 almost 10 7 in the best cases), further improvement of the device does not seem to be necessary. If the situation changes, devices with high number of parallel nozzles like the millipede 269 device reproduced by Rémi Sieskind in the lab could be envisioned for further improvement but would require adaptation for droplet size homogeneity.

One more important aspect than the throughput now would be to eventually improve filters, to prevent bacteria clogging or find a proper way to be sure of each flow contribution while using the very convenient pressure pumps. Issues linked to the encapsulation and DNA and/or protein capture by droplets interface should be better characterized. Even if a solution was found to retrieve the DNA, it is not sure how the used compounds Pluronic f-127 and yeast RNA act on the problem nor if their concentration is optimal.

In order to perform IPA and PCR in the same buffer, optimization was done to finally reach the IPA V0 buffer, with a total of 6 mM of Mg 2+ , in which the IPA can perform at satisfying speed despite the lower magnesium concentration and the PCR can still be run despite the high magnesium concentration. Even if the PCR is still working in these conditions, it is very certainly not performing as well as in its optimized buffers, with potentially a higher error rate. For example, in these degraded conditions, we observed specific amplification of a gene with primers having 5 bases mismatch in 3'. Linking the primers production to another amplification method, such as RCA, LAMP, RPA or other, with better buffer compatibility, could be an option. Sadly, except for RCA, based on the heat sensitive Phi29 polymerase (working at 37°C), specific primer-dependent isothermal amplification methods described in the literature are not able to amplify long DNA strand of 2 kb. As presented in the annexes, LAMP and RPA were tried at the beginning of the project with no success. Assay on the Bst 2.0 WS, also presented in annex, shows the important slow-down in strand production according to the length of the output and could explain difficulties of long template amplification. Adapting the program to work at lower temperature could allow to use Phi29 based amplification methods. Another option could be to evolve the Taq polymerase to work properly in higher magnesium concentrations.

One obvious next step for the PEN CSR system now address by Vasily Shenshin in his PhD project is to prove its ability to perform on enzymes not working directly on DNA. The Compartmentalized-Partnered Replication 80 is also based on adaptating CSR to various enzymes but, as the selection of the enzyme is happening inside the host organism, it loses the possibility to test the enzyme in vitro. In our semi in vitro/in vitro system the enzyme is tested in the droplet, outside of the bacteria. This allow to adapt the buffer and components used in the selection. Still, as everything happens in the same compartment, important restrictions apply: the buffer should still allow IPA and PCR to be run and it will also contain bacteria lysate (strongly interacting with the reaction even when highly diluted (10 000 times in the 10pL droplets). This is important as we should expect that the evolved enzyme will be specifically adapted to the selection buffer. Droplets fusion or display systems allowing to purify the protein and its gene and perform the selections in various buffers could partially solve this issue at the cost of new technological constraints. The IPA could be run in a first buffer and then the PCR in a new one, provided that produced primers stay with the corresponding gene. Displays systems also permit to use in vitro expression systems. In vitro systems can bypass the bottleneck of transformation and avoid issues linked to cytotoxic proteins, like nickases. At the beginning of the project, I tried to use RTS expression kit and a cell-free extract furnished by the Kiga Laboratory. I showed that it was not possible to perform PCR as soon as 2% or RTS mix or 5% of cell extract was added to a reaction, whereas the mixes could not be diluted more than 50% to keep producing proteins. Working with IVTT therefore requires more complex handling systems which are now developed by Rémi Sieskind as part of his PhD project.

During his internship, one of my interns, Nicolas Larrouy, tried to characterize the library activity diversity at the single enzyme level. To do so, he used a molecular program with a fluorescent reporter in droplets, with enzymes distributed in droplets with a λ below 1. With this assay he was able to detect single enzymes in a digital format, and retrieve the actual concentration of active enzymes in the commercial NBI stock. Analysis of the library using the same protocol was more complicated, but could in principle provide a unique way to characterize the distribution of activities in an enzymatic library, at the single molecule level (and therefore avoid many biases such as expression level). This opens up the possibility of using the PEN DNA molecular programs and droplets microfluidic with in vitro systems and eventually mass spectroscopy to directly have access to the selected protein sequence. The continuous decrease in DNA synthesis prices and the ability of IVTT system to use artificial tRNA could be combined to generate sequence variability at the level of the protein giving access to unprecedently reached throughput capacities 317,318 .

Even though the PEN CSR could beneficiate for further improvements, some conditions have been found to perform the selection of NBI in droplets. Next experiments will focus on improving the enzyme thermostability, its catalytic rate or change of the recognition site. This last point could be very challenging [START_REF] Karpinski | Directed evolution of a recombinase that excises the provirus of most HIV-1 primary isolates with high specificity[END_REF]319 because of the requirement to protect the bacterial DNA with methylase, but could be overcome with the addition of other methylases in the strain.

One issue to really take advantage of the throughput of our method will still be to find a robust cloning strategy.

Accumulation of sequences data during the evolution cycles, but also during neutral selections, has been initiated with Rocio Espada and will be used to look for amino acids pair correlation and epistasis effect in long sequence. Since NGS sequencing with MinION has a high error rate, a strategy to decrease this error rate will be essential to use full length data.

Circular amplification of templates before sequencing could be used to increase the number of read of the same sequence and increase sequences accuracy. Another aspect could be to work on the library construction and eventually design smart libraries, or work on focused regions to be able to use more accurate NGS systems. Another important point that Rocio is already trying to address is the wild-type sequence data pollution. In neutral selections, the wild type will always be selected, while mutated variants tend to be eliminated, on average. As shown by ePCR prediction software, with 2 mutations per genes, up to 20% of the library can be wildtype. After selection, theses sequences would represent a big part of the gene pool, while not giving any information for analysis. Decreasing the proportion of wild-type proportion in the initial library is then crucial.

NGS sequencing data, revealed an important bias in the selection method with selection toward A and T bases. This bias was also observed by Gadhessy et al. in 2001 but to a lesser extent, only on synonymous mutations. An explanation could be a bias during the PCR amplification that could be overtaken by mutants with better PCR-ability. In our case, no difference can be seen between synonymous and non-synonymous mutation. This could be because the bias is even stronger, or because our applied selection pressure is not strong enough.

Bias due to PCR amplification could provide another incentive to look for alternative methods

291

of amplification. But one can think that some conditions could be found in which these biases are reduced. Thermophilic organisms and Thermus acquaticus in particular do not generally possess an AT-rich genomes. In fact, Thermus acquaticus has a GC content of 68%, meaning that the Taq polymerase can efficiently replicate GC riche genes. Of course, genome replication in vivo has little in common with the PCR reaction 320,321 .

This worked hopefully exposed most of the problems one can encounter when trying to set up a PEN CSR evolution scheme in bacteria lysate and I hope it will help the researchers working on the next implementations the system.

in the designs simplified the practical microfabrication steps. The files obtained were then sent to a private company specialized in laser photoplotting (Selba SA, Switzerland), with instructions (resolution: 50800dpi, polarity: negative plotting, orientation: mirror plotting). The masks were received within 7 working days.

Mold fabrication

The no-step-molds were fabricated using the SU- 2) Spin-coating 5g of the adapted SU-8 photoresist dilution with corresponding speed and program for the target thickness (500 RPM for 10s, chosen speed for 30s).

3) Baking of the wafer using 65°C and 95°C hotplates following microchem instructions.

4) UV exposure of the photoresist coated mask using the MJB4 mask aligner with its UV-240 nm filter and the mask intercalated between the photoresist and the UV light source.

The insolation time is given by the power of the UV lamp and the required exposure energy given in the microchem protocol. In this work the exposure energy was divided by 10 to obtain the proper exposure time (the calculation done by the IPGG microfluidic platform engineer takes into account light absorption in the entire light path).

5) After exposure, baking of the wafer using 65°C and 95°C hotplates following microchem instructions.

6) Removing of the non-insolated photoresist from the mold using PGMEA solvent (SU8developper).

For the 1-step-molds, these steps were performed twice (one for each thickness) with an additional step of alignment before the second insolation. The SU-8 alignment crosses deposited on the wafer at the first step had to be aligned with the crosses of the second mask.

Careful removal of the fresh SU8 with SU8 developer using small Q-tip above the cross allow to make them visible for the alignment.

Chip fabrication

40g of Sylgard 184 PDMS mixed with 4g of Curing Agent were poured on the wafer laid on a circle-shaped aluminum foil. PDMS was then degassed in a vacuum chamber until complete removal of air bubble, before a curing step at 70°C for 2h. The crosslinked PDMS slab was then delicately detached from the mold and covered with florist plastic film. Holes of 1mm diameter were made at the inlet and 1.5mm at the outlet with a biopsy punch (Miltex).

Glass slides to be used for bonding were washed with acetone and isopropanol, rinsed with milliQ water and dried using dry compressed air. Dusts on the printed surface was also removed using dry compressed air in the gray room. Both glass slides and devices were then put in the oxygen plasma machine for surface activation. After exposing (part to be stuck upwards) to the oxygen plasma under a pressure of 300 mTorr for 1 minute, the functionalized surfaces were stuck together, forming covalent bonds. After bonding, the device was baked at 200°C during 5 hours in order to increase PDMS diffusion and facilitate droplet formation thanks to hydrophobic PDMS coating.

Slides cytoping

For droplet imaging under the microscope, thin slides (thickness 0.17 mm) were made hydrophobic by Cytop coating. 1 volume of Cytop was mixed with 10 volume of diluent. 300 µL were then poured on a 70x44 mm microscopy glass slide (40 µL for a 22x22mm cover slide) inside a spin-coater. The slide was spun at 2000 rpm for 30 s and then incubated at 180°C for one hour.

Microscope imaging

The droplet array images were all taken in home-made chambers. A microscopy slide (72x56mm) and a cover slide (22x22mm) were made hydrophobic thanks to cytop coating. To create spacers, a matrix of nine 1µL droplets of a 1/100 dilution of 10µm or 30µm Polybead microspheres (Polyscience) aqueous suspension at 0.025% solids (w/v) was deposited on the glass slide in an area corresponding to the cover slide. The glass slide was then put on a hot plate set up at 120°C until the water evaporated completely. 10 µL of emulsion were then deposited in the area and the cover slide was added on top and thoroughly displaced back and forth in order to spread the Polybead spacers. The chamber was then closed using epoxy glue (SADER 2-component glue) deposited all around the cover slide.

Droplet generation

For the droplet formation, flexible PTFE Tubing of 1.56 mm external diameter and 0.2 mm internal diameter were inserted in inlets and p200 tips were used to collect the droplet at the outlet. Oil phase was made by mixing HFE 7500 oil with 2% of surfactant. The flow in the channel was controlled using N2 gas pressure in the Fluigent MFCS™-EZ pressure controller.

In the final experiment, using the 2-step device (20/40 µm) generating ~26 µm droplets, oil phase pressure was set at ~500 mbar and ~400 mbar was applied to the aqueous inlet. Droplet generation was monitored under the microscope. In some experiments, syringe pumps were used instead of pressure pump to generate flows using 10 µL/min for oil and 1 µL/min for both water channels.

IPA in droplet with NBI scanning

For the commercial NBI concentration scanning in vitro, two aqueous solution were prepared. Both were containing 100 nM of Zenphex reporter, 1% of Hot Start Taq polymerase (NEB 5000U/mL), 0.1% of Bst 2.0 WarmStart® DNA Polymerase (NEB 8000U/mL), 1% of EvaGreen 20X, 200 µM of dNTPs, a dilution of pNBI+ plasmid, 20 mM of cv11r2p (aT), 10 nM of v11tor2p25 (ppT), 10 nM of v11tof2p25 (ppT) and 10 nM of v11 (T) in the nIPA buffer.

One of the two tubes was supplemented with 0.5% of commercial NBI (NEB 10 000 U/mL) and 1µM of dextran Alexa 647 (Thermofisher). The program used by Alexandre Baccouche to map the bifurcation diagram 130 was simplified and used to control the pressure of the two aqueous inlets to adjust the content of droplets. In order to keep monodispersity during the droplet formation pressure need to be balanced so that the total pressure applied on the aqueous channels is constant. Since the aqueous channels are identical, they have the same hydrodynamic resistance, and this guarantees that the total aqueous flow rate is constant. The first step of the protocol is to empirically find the maximum (Pmax) and minimum pressures (Pmin), corresponding to the pressure for which one channel invades or is invaded by the other one. The script starts from an equal pressure and consists of a loop in which the pressure is incrementally decreased in channel 1 while increased in channel 2 until the Pmin in channel 1 is reached. Conversely, the pressure is then decreased in channel 2 and increased in channel 1.

The number of increments can be changed and adapted to have the best NBI concentration

Oil (EA/Ran/ Jeffa 1000_HFE7500/ Novec 7500)

Fluorinated oil with 2% w/v surfactant, was prepared using either EA, RAN or Jeffa 1000 surfactants with 16 mg of surfactant for 500µL of oil. A small amount of surfactants was collected using a P1000 pipette's tip and weighed on a precision scale. A volume of Novec 7500 (previously HFE 7500), corresponding to the measured weight of surfactant, was added inside the tube. As the surfactant is really viscous, the pipet tip used to remove surfactant from the beginning was used to pipet directly up and down in the oil so that the surfactant was resuspended without losing a part of it on tips edge.

Emulsion breaking (PFO) (50µL emulsion)

½ volume of PFO (1H,1H,2H,2H-perfluorooctanol, Sigma-Aldrich) was added in 1 volume of emulsion. The mixture was vortexed ~10s at maximum speed and centrifuged for 1min in a benchtop mini-centrifuge. The upper phase (aqueous phase) was finally taken with a pipette. When the emulsion was not completely broken PFO was added progressively by ½ initial volume and the process repeated until the emulsion broke. In some experiments, various way of emulsion breaking was tried like electrostatic gun or freeze and thaw at -80°C, followed by centrifuge.

Molecular programs

IPA, primer production reporting

The IPA (Isothermal Primer Amplification) reactions were performed in IPAV0 buffer

Turn over and effect of magnesium concentration on isothermal amplification

Effect of magnesium concentration effects and first turnover reaction in bacteria was run with the x11 system. For the assay on magnesium concentration, the reaction was run un IPA buffer with various added concentration of magnesium and 1% of Nt.BstNBI (NEB 10 000U/mL), 1% of Bst DNA Polymerase Large Fragment (NEB 8 000U/mL), 10 nM of x11h (T), 40nM of cx11s3uP (aT), 40 nM of x11toON, 1x Evagreen dsDNA intercalating dye (Biotium). For the investigation of bacteria lysate effect, reaction were run in Smix buffer (Thermopol buffer 1X, NEB 3 buffer 0.5x, Synperonic 1% and 2 µM of Netrospin), 100 µM of dNTPs, 1x Evagreen, 0.05% of Bst A 3-5 mL overnight culture of a colony of the considered strain in LB medium (with 100 µg/mL of ampicillin if needed) was started the day before in an orbital shaking incubator (37°C, 250rpm).

2 mL of this culture were inoculated in 200 mL of LB medium (with 100µg/mL of ampicillin if needed), the bacteria were then incubated at 37°C, 250 rpm and the OD600 was regularly controlled until it reached 0.35-0.4 in cuvettes (or 0.6 in 14 mL culture tubes).

The culture was then dispatched in 50 mL tubes and centrifuged at 4000 rpm for 15 min at 4°C. The supernatant was discarded, and the pellets resuspended in a total amount of 20 mL of sterile iced cold MgCl2 (0,1 M) before they were gathered in one 50mL tube. This mixture was centrifuged at 3000 rpm for 15 min at 4°C, the supernatant then discarded, and the pellet resuspended in 20 mL of CaCl2 (0.1 M) before incubation on ice for at least 20 min. This suspension was then centrifuged at 3000 rpm for 15 min at 4°C, the supernatant was then discarded, and the pellet resuspended in 10 mL ice cold 85 mM CaCl2 15% glycerol before another centrifugation step at 2100 rpm for 15 min at 4°C. The supernatant was again discarded, and the pellet resuspended in 400 µL of ice cold 85 mM CaCl2 15% glycerol.

The mixture obtained was then aliquoted into sterile 1.5 mL tubes chilled on ice in advance and flash frozen in liquid nitrogen before long-term storage in a -80°C freezer.

During the project KRX strain (Promega) carrying the ple1 plasmid were made chemocompetent (KRX/ple1 bacteria)

• Electrocompetent cells 200 mL of glycerol 10% solution, 300 mL of LB-low salt, 4x 50 mL centrifuge tubes and 20x 1.5 mL tubes were prepared and autoclaved in advance.

A 5 mL overnight culture of a colony of the considered strain in LB-low salt medium (with 100 µg/mL of ampicillin if needed) was started the day before in an orbital shaking incubator (37°C, 250rpm). The glycerol solution, the centrifuge rotor and the centrifuge 50mL tubes were stored overnight at 4°C.

The OD600 of the overnight culture was checked to be between 4 and 5. In a sterile, 2mL of this culture were inoculated in 200 mL of LB-low salt medium (with 100µg/mL of ampicillin if needed), the bacteria were then incubated at 37°C, 250 rpm and the OD600 was regularly controlled until it reached 0.6-0.8 in cuvettes (or 1-1.3 in 14 mL culture tubes). The culture was rapidly chilled on ice for 15-20min and swirled from time to time. It was then split into 4x 50 mL sterile centrifuge tubes chilled in advance and all this was centrifuged at 8000 g for 5 min at 4°C. The supernatants were discarded, the 10% glycerol solution was put on ice. In a sterile hood, the four pellets were resuspended in 20mL of cold 10% glycerol solution each and the suspensions obtained were gathered in 2 tubes containing 40 mL each. They were centrifuged again at 8000 g for 5min at 4°C and the supernatants were discarded. Each pellet was resuspended in 30 mL of cold 10% glycerol solution before a new centrifugation step at 8000 g for 5min at 4°C. The supernatants were discarded, and each pellet were resuspended in 20 mL of cold 10% glycerol solution and the solution obtained were combined into one 40 mL solution that was centrifuged at 8000 g for 5 min at 4°C. After discarding again the supernatant, the pellet was resuspended in 2 mL of cold 10% glycerol solution and the dilution adjusted to obtain a final OD600 between 80 and 100 (0.8 to 1 for a 1:100 dilution). In a sterile hood, the TTAGGTCTCAGGCTGCTAACAAA. The PCR product was purified and used with the NEB® Golden Gate Assembly Mix (NEB) and the ligated product were transformed in bacteria.

Site Directed Mutagenesis

Site directed mutagenesis was used to generate inactive version of the NBI, Taq and KlenTaq gene using the Q5® Site-Directed Mutagenesis Kit (NEB). The manufacturer protocol was used with the following primers, on plasmids coding for the respective enzyme: gamS (on the pBADmod1_linker2_gamS from Addgene) was expressed and purified using the same protocol but grown in a Oneshot Top10 bacteria strain (Thermofisher), induced with 0.25% of L-arabinose for 5 hours.

After SDS page (NuPAGE Bis-Tris Gels, Thermofisher) elution fractions containing the proteins were assembled and dialyzed and concentrated in their respective 2x storage buffer usingSlide-A-Lyzer™ Dialysis Cassettes (Thermofisher) or Amicon® Pro Affinity Concentrator (Merck). 50% of glycerol was then added for conservation. Taq and KlenTaq polymerase were also purified using heat precipitation protocol adapted from 322 . After sonication in the Sonication buffer (40mM Tris-HCl pH 7.6, 300mM NaCl, 10mM Imidazole pH 8, 1mM DTT, 0.05% Triton), the lysed bacteria were centrifuge at 14 000 g for 10 min. We then added 10% of PEG 8000 in the recovered supernatant. The solution was heated at 70°C for 15 min before ice cooling for 20 min. Finally, the samples were centrifuge at 12 000 g for 10 min and the supernatant applied on a NuPAGE Bis-Tris Gels (Thermofisher).

Library generation

Error prone PCR (Mutazyme II)

For

KlenTaq:

Bacteria expressing either a wild-type or the inactivated version of the Klen-Taq polymerase was grown and induced at 37°C and 250 rpm for 2 hours with 0.1% of L-rhamnose.

After induction, the bacteria were pelleted in a centrifuge at 5000 g for 5min. They were washed (resuspended and centrifuged 5min at 5000 g) twice in a resuspension buffer (Tris-HCl ph 7.5 50mM, NaCl 100 mM). A solution was then prepared containing ThermopolDF buffer (NEB) (1x) with 1.5mM MgSO4, 400 μg/mL of BSA9000S (NEB), 200 μMof each dNTPs (NEB), 1ng/μL of Yeast RNA (Merck), 0.4% of Pluronic F-127 (Sigma-Aldrich) and the bacteria. We

Selectivity assay (rh primers)

The rhPCR was set up using DreamTaq buffer (1x Phylogenetic information can also give a lot of valuable inputs. In more fundamental studies, these focused mutations provide more insight about the structure-function relationships, the mechanisms of the enzymatic reaction or help to map the active site.

Target a region or few close positions

Targeting a small region of the protein with random mutations allows to explore more exhaustively a reduced fraction of a sequence space. The easiest way to do so is to use random oligonucleotides. Random oligonucleotide mutagenesis allows to incorporate random mutations into a specific region of a gene using a synthetic oligonucleotide cassette. The cassette is introduced by a simple restriction digest and cloning in a plasmid encoding the wildtype version of the gene. The mutation rate can be controlled by the length of the targeted region and the randomization of the synthetic cassette. The sequence space that can be studied is huge but generally restrained by the achievable library size. Besides, the limits of the studied region cannot exceed the oligosynthesis size limitations. Restriction in relative positions and number of randomized positions can also be problematic.

Site saturation mutagenesis

One of the most used focused mutagenesis methods is the site saturation mutagenesis.

This approach allows to mutate one or several chosen positions to all the other 19 amino acids. Recombination 325 (OSCARR) uses carefully designed mutagenic cassettes to introduce up to 10 randomized codons at focused regions with only 3% of remaining wild-type product.

In the OmniChange 326 protocol, up to five codons can be simultaneously saturated. The method is based on the chemical cleavage of phosphorothiolated nucleotides by ethanol-iodine.

PCR forward and reverse primers comprising 12 bases with phosphorothioate bonds and random sequences at the targeted positions are used to amplify up to 5 pieces of target plasmid.

After cleavage of the phosphorothioated bond by the ethanol-iodine, overlaps are created allowing to put the plasmid back together by simple hybridization of the mutated fragments.

The created nicks will be repaired in the bacteria. In a similar fashion 327 Seyfang and Jin manage to introduce up to 10 simultaneous randomized sites by using primers with randomized positions and external anchor primers. After a primer extension and ligation step filling the gap between primers, the mutant strand is retrieved by PCR amplification using primers matching the external added anchor primers. Classical restriction ligation cloning method is then used to transform cells. In the QuickChange Multi-Site directed mutagenesis (agilent) primers all binding to the same strand and comprising the mutations are used to amplify a template creating a circular single strand mutated version of it thanks to the common work of a polymerase and a ligase. Transformation into XL10-gold bacteria allows to retrieve the double stranded form.

Some techniques provide other way to reach any desired amino acids thanks to oligonucleotides synthesis. In the Incorporating Synthetic Oligonucleotides via gene

Reassembly 328 (ISOR) method, oligonucleotides encoding randomized substitutions are spiked into the fragmented gene during a shuffling step. As many as 50 positions can be randomized in a 300 amino acids protein. Gene assembly mutagenesis 329 is using exclusively synthetic overlapping oligonucleotides of ~40bp assembled by DNA polymerase extension. Oligos are encoding both strands with a junction offset by 20 bases. Hybridization and extension over several cycles lead eventually to full length genes that are amplified with outermost primers in a second PCR. Using oligos with a degenerate part can allow to build libraries with multiple positions mutated with no limit on the proximity and guarantee to have mutations at every targeted position. Synthetic oligos can also allow a great flexibility in the codon usage for a specific host organism and to remove and add easily any desired sequence in frame.

Furthermore, it has the advantage to not contain wild-type or template DNA in the final product.

But, the inherently error-prone assembly process is generating an important load of additional errors that requires a minimum 4-fold increase in the library size to be sure to have the desired mutants. Using PAGE purified oligonucleotides is recommended to decrease the error rate but can largely increase the cost of the library. Outside the cost of the numerous synthetic oligos that are necessary to assemble long DNA sequences, this assembly method is difficult to perform for long genes because of the increase of misspriming events restricting its use mainly to relatively short sequences. Alternatively, splitting the genes in several overlapping smaller sequences can be an option.

Combinatorial library comprising different mutation profile in various portion of the genes can be assembled using more complex system relying on microfluidics 323,330 . The different versions of the several parts of the gene are spread in different tubes and sequentially mixed in droplets thanks to microfluidic pumps. The assembly reaction is then run in droplet to give various combination of the distinct parts.

As increasing the number of saturated positions is exponential, the needed size of the library to explore all the landscape some protocols choose to sequentially mutate few positions at each cycle. In the Iterative Saturation Mutagenesis 331 (ISM) protocol one or few positions are assigned to multi-residue sites and are mutated with a user-defined randomization scheme to all amino acids or a defined subset. Selected mutants are then used as template to introduce mutations at other multi-sites. This is repeated between each cycle until the desired activity is achieved. Although it is not exploring all the possibilities of combination of these various multisites, improve catalytic properties such as rate, substrate scope, stereo-and regioselectivity and binding affinity have been achieved using this method.

Recombination methods

Another way to generate libraries is to recombine genes. Recombination can be interesting for various purposes. As a starting point a library generated from recombination of various enzymes can allow to have more plasticity in the sequence. Using homologous enzymes or members of the same phylogenetic or structural family allows to cumulate many mutations that have already been selected by the natural evolution and thus, beneficiate from the nature pre-evolved repertory. Such starting points have been shown to be more evolvable and more resistant to mutations introduction [START_REF] Porebski | Consensus protein design[END_REF] . Even spiking of some consensus position of the scaffold used to generate the completely in silico designed Kemp eliminase resumed in new possibilities of evolution [START_REF] Khersonsky | Bridging the gaps in design methodologies by evolutionary optimization of the stability and proficiency of designed Kemp eliminase KE59[END_REF] . Throughout an evolution experiments, recombination can also be used to look for synergy between beneficial mutations selected in previous cycles or to get rid of passenger mutations. Recombining the different selected mutants after an evolution cycle will allow to brew the beneficial mutations and obtain the best combination of them. Recombining the selected mutant with the wild type version of the gene provide a way to remove the passenger mutations. These mutations are selected just because they sit on the same gene than a beneficial mutation but do not have a significant impact on the enzyme activities.

To realize the different recombination is not an easy task. Homologous recombination with sequence identity over than 70% revealed to be easier to handle than Heterologous recombination of more distant sequences. Also, controlling the number and position of crossover between the sequences is quite tricky. But a large panel of recombination methods have been developed to enable both kind of recombination and address these specific issues.

Homologous recombination of genes with >70% sequence identity

The first DNA shuffling mechanism was proposed by W.P. Assembly (PCA) like the ADO method to assemble the overlapping oligos. Outer primers are then used to amplify the full-length templates. ADO is presented as the best method as it has a simpler protocol and can allow to construct high-quality and bias-free combinatorial libraries with careful design of the overlapping oligos. One interesting point of this method, like in the Gene Assembly mutagenesis, is the ease of introducing new mutations at desired spots. When the original set of strands is acquired, one can easily order a new modified one to introduce new mutations at desired position.

As we have seen, most recombination techniques rely on template switching to generate protein chimeras. As a consequence, they require a relatively high sequence identity. Moreover, the crossovers they create are biased towards regions of highest identities. Using oligonucleotide-based methods in a variation using ligation of single strand of unknown sequence can allow to mix heterologous sequences.

The natural mating possibilities of yeasts can also be an appropriate alternative to induce recombination in vivo. Cornish and colleagues demonstrated this apporach using donor cassette inserted in mating yeasts 218 . This can have many advantages presented in part 1.5.

Heterologous recombination

It can be interesting sometimes to shuffle sequence of structurally close enzymes with low identity. As it is not possible to use sequence homology for recombination, other methods xvii Golden Gate assembly is a molecular cloning method allowing to simultaneously and directionally assemble multiple DNA fragments into a single piece using Type IIs restriction enzymes and T4 DNA ligase.

based on digestion of the parental genes and ligation of the obtained fragments have been developed. The Sequence Homology-Independent Protein Recombination 342 (SHIPREC) allows the building of chimeras of two parental genes. The C-terminus of one of the two proteins is linked to the N-terminus of the second with a small linker containing a unique restriction site. The merged sequences are then randomly truncated by the DNAse I and S1

nuclease. Fragments corresponding to the size of the parental gene are then selected to ensure proper sequence alignment after a subsequent recircularization. Cleavage of the initially introduced restriction site provide a recombined sequenced ready to be cloned in an expression vector. The Incremental Truncation for the Creation of Hybrid enzYmes 343 (ITCHY) also address the recombination of two independent proteins. Two versions of this protocol exist. The first one is based on the time dependent digestion of the two parental genes by the exonuclease III, starting from the same unique restriction site placed between the two genes. The digestion is stopped at different time points creating fragment of diminishing sizes. The two remaining parts of the two genes are separated and the obtained fragments are then ligated. In the second option α-phosphorothioate dNTPs are incorporated randomly in the sequence during a PCR amplification of a vector comprising the two genes. This step is followed by a digestion by the same exonuclease III that will stop at every position where phosphorothiated bridges have been incorporated. An intramolecular ligation recircularizes the plasmid containing the new hybrid proteins. Both SHIPREC and ITCHY methods can generate chimeric libraries of all possible single crossover between two parental genes.

To increase the number of crossovers, the SCRATCHY 344 method adds a DNA shuffling step after ITCHY recombination. The ITCHY libraries are simply digested by the DNase I (like in the simple DNA shuffling method) and reassembled by a self-primed PCR, generating multicrossover hybrids of sequence independent proteins. On another side, the USER-friendly DNA recombination 345 (USERec) method can directly mix genes from near homology-independent parental genes. Here, up to 10 DNA fragments of 40-400 bp are amplified from the different templates using uracil containing primers encoding a minimal crossover sequence of less than 10 bases. This relatively small sequence constraint facilitates organization according to the structural or functional domains and is the reason why structurally similar but not necessarily sequence similar proteins can be combined using this method. The fragments are then mixed and incubated with the USER enzyme, thus generating single-stranded overlap between the fragments. After ligation and removal of the original template by DpnI digestion the full-length gene is PCR amplified and cloned in a vector, leading to very few parental versions of the genes.

In the Non-Homologous Random Recombination 49 DNA fragments are digested by the DNase I like in the classic DNA shuffling methods but then an intermolecular ligation is favorized using hairpins to generate full length chimeras. The use of ligation instead of PCR extension removes the need of sequence homology and allow to recombine different fragments. In the Sequence-Independent Site-Directed Chimeragenesis 346 (SISDC) consensus sequence are determined after alignment at the desired crossover location. Then a tag comprising restriction site and this consensus sequence are inserted into the parental genes. After restriction generated fragments are ligated thanks to the overlapping consensus part, creating recombinant full-length gene.

Domain recombination

To explore the effects of the different subdomains organization some researchers generate random circular permutation libraries. To do so, they introduce a linker between the N-terminal and the C-terminal part of the protein by ligation of the 2 extremities of the DNA sequence and then cut an equivalent peptide bond elsewhere in the sequence. This domains relocation technique can modify the protein ternary and quaternary structure to give birth to some functional enhancements 347 .

According to the number and positions of mutations to be recombined, it is not always recommended to use recombination methods. If mutations are close in the sequence, they have little chance to recombine by these techniques. For such purpose site directed mutagenesis is generally more efficient.

Box 2: Degenerate codons Degenerate codons are principally used in saturation mutagenesis library creation methods to decrease the complexity and the size of the nucleotide sequence space. They allow to screen for the 20 amino acids or a user-defined subset of amino acids without the need of using completely randomized 'NNN' codons, where N = A, C, G or T. Indeed, due to the redundancy of the genetic code the complete set of amino acids can be encoded using 'NNK' or 'NNS' codons, where K = G or T and S = C or G. Using such codons only 32 DNA sequences are needed to explore all the 20 amino acids possibilities at one position instead of 64 with fully randomized codon. It is particularly interesting for multi-site combinatorial saturation mutagenesis as the library size increase exponentially with the number of mutated sites. Full coverage of 5 saturated positions using 'NNN' codons would require a library size exceeding 10 9 mutants as with 'NNS' degenerate codons the size is reduced 32-fold leading to library around 3x10 [START_REF] Dill | The Protein-Folding Problem, 50 Years On[END_REF] . Some tricks have also been developed to decrease more this aa/DNA ratio. The "22c trick" and the "small intelligent libraries" use respectively 3 or 4 degenerate codons to encode the 20 amino acids with correspondingly 22 or just 20 codons. But these improvements still limit the number or position to be mutated to 5 before the library exceed a size of 10 7 that is usually the size that can be obtained when transforming bacteria. To further limit the library size and explore more position the 'NDT' codon, where (D = A, G or T) codes for a subset of carefully chosen 12 amino acids using 12 DNA sequences. This subset targets chemical variability in the aa side chain as it comprises non-polar, aromatic hydrophobic, hydrophilic and charged amino acids.

following value OD 0.1 ≡ 2 x 10 7 ml -1 could be found.

Anton could count 56 green and 30 red bacteria and 61 green and 21 red bacteria in two fields of view. In average it gives 59 live and 89 total bacteria per field of view. This corresponds to the relation OD 0.1 ≡ 1.4 x 10 7 ml -1 (live) and OD 0.1≡ 2 x 10 7 ml -1 (total).

Measured colony forming units concentration for KRX Taq+ and KRX pSB3K3 bacteria at OD600 cuv approximately 0.6 gave the following results: KRX Taq+, OD 0.1≡ 2.5 x 10 7 ml -1 and KRX PSB3K3, OD 0.1 ≡ 0.6 x 10 7 ml -1 .

We reached good agreement with the literature value that can be safely used as a reference. Therefore, OD600 cuv 0.1 ≡ 20 bact/nL is now the conversion value used for every bacteria concentration estimation.

Protocol: KRX pet14b-Taq-, bacteria were grown to OD 0.4 in LB + 100 mg/l of ampicillin and then induced with 0.1% of L-rhamnose for 3 hours. There medium with bacteria was filtered with 5 µm filter and the final OD600 cuv was 0.91. The suspension then was washed with the resuspension buffer and diluted to OD600 cuv 0.06. The cells were stained with 10 µM SYTO 9 (green, alive) and 60 µM propidium iodide (red, dead) for 15 min at room temperature.

The solution (OD 0.06, stained with SYTO 9 and propidium iodide) was injected to a square shape chamber with dimensions 9 x 9 mm and the volume 25 µl. Using a 60X lens, the number of bacteria per field of view was counted including free floating bacteria. The field of view has side size (inferred from the observed size of 10 µm beads) 151 ± 4 µm times.

Note about OD600

OD600 cuv, OD600 tub, OD600 pr: During the course of the project, I noticed that I was not paying attention to the fact that the optical density measurements are dependent on length of the optical path and that it was changing according to the used support. OD600 cuv refers to the OD600 in a classic cuvette with 1cm path and probably the one given in microbiology protocols. OD600 tub refers to the OD600 measured in a round-shaped culture tube. And OD600

pr correspond to the optical density measured in the plate reader. A simple conversion can be made using the following table :  iii. Design of reporting probes

Probes for detection and estimation of nbi activity

Several times during the project NBI activity needed to be assayed for instance to check the activity of expressed and purified enzyme, the activity contained in bacteria, the global activity of a library and comparison of all these different activities to the one of commercially available NBI. A simple activity reporting system were designed by my supervisor before I started working on the project. Nicknamed loopact, this hairpin loop is comprising the recognition site of NBI on the stem and a quencher and fluorophore at the 3' and 5' extremity.

Cut of the top strand by NBI enzyme allows to separate the quencher and fluorophore creating an increase in fluorescence we can follow in real-time PCR machines. This straightforward design needed to be adapted when working with bacteria. Adding phophorothiate bonds at the 3' and 5' extremity as well as in the short loop allowed to make it resistant to relatively high concentration of bacteria lysate. An equivalent loop, loopactnuc, with a modified restriction site was also designed to check the specificity of the reaction (Table 30).

Table 30. Different hairpin loop used to detect NBI activity and specificity.

In the second generation of loop, the stem was made longer and the loop part that might be the reason of the probes instability in lysate was made shorter. These probes were design to resemble the loopact reporting system that showed to be resistant in bacteria lysate up to some concentrations. This new loop design lead to very inefficient reporting of the primer's concentration, the delay between primer actual production and effective increase in fluorescence being quite enormous (Figure 162).

A third design that revealed to be the more efficient was also tested at the same time and is based on stable DNA duplexes. The idea behind this new design is to use reporting systems mimicking the molecular program. Since the protected templates used in the selection network seemed not to be degraded too much similar reporting probes should remain in bacteria lysate. For each primer to be detected, two complementary strands were therefore designed each of them having a fluorophore or a quencher ending in very high proximity when the strands are bound. Produced primers should have higher affinity with one of the two strands to displace the other bound one. Then polymerase extension should stabilize the complex preventing the initial bounded strand to come back. In this design we are also drawing the polymerase activity, but the effects revealed to be very less important than in the case of the first molecular beacons. The first implementation of this design (v1) was not very responsive, so the size of the initially bound strand was shortened to reduce the stability of the duplexes. In the second implementation (v2) instantaneous detection of primers concentration was achieved with few burdens added on the molecular program (Figure 160, Figure 161). But, only one of the reporting system (p17) displayed resistance to reasonably high concentration of the bacteria lysate.

iv. The Poisson's Law

The Poisson law is a discrete probability distribution that describe statistical experiments where a given number of independent events classified in successes or failures and occurring in a fixed interval of time or space have a known probability of success (λ)

proportionally linked to this fixed interval providing that the probability of a success in an extremely small interval is virtually 0. The Poisson law therefore describe well the droplet encapsulation process as we have a given number (N) of independent objects (cells, genes, …)

that can be encapsulated (success) or not (failure) in a number (Nd) of generated droplets with a fixed volume with the probability λ = 𝑁 𝑁 𝑑 ⁄ and could not be encapsulate in droplets smaller than their own size. According to the Poisson law the proportion of droplets containing n objects is:

𝑃(𝑛, 𝜆) = 𝜆 𝑛 𝑒 -𝜆 𝑛!

Where n is the number of objects per droplet and λ is the average number of objects per droplet.

The Poisson law describe particularly well the encapsulation of objects in microfluidic system where droplets are monodispersed and have a fixed size but can also be applied somehow for emulsion generated by other means as long as the distribution of droplet size is not too wide.

On fig mhh you can see encapsulation of fluorescent bacteria in droplets. After counting the number of droplets with 0, 1, 2 or 3 cells you can see that the repartition in the droplet actually match with a Poisson distribution.

How to deal with λ?

If one droplet contains two or more variants of the gene to be evolved, they can end up being all selected the same way, leading to false positive selection or loss of some interesting mutants since all genetic molecules are copied or selected without distinction in each compartment but as a function of the combined phenotypes. To avoid this problem one can choose to use a very low value for λ, meaning that the average number of bacteria or genes per droplet will be largely below 1. This would guarantee to have very few droplets containing two or more mutants but will drastically increase the number of empty droplets and therefore the to one, would generate at least 26% of droplets with 2 or more than 2 mutants. All compartmentalized selection processes are therefore highly dependent on the λ value. matching, the DNA/RNA duplex is stable enough for the RNase H2 to cut the primer strand just before the RNA base freeing an extendable 3' end. The primer extension therefore depends on the nucleotide base. The RNase H2 is thermophilic and can be used in a PCR reaction in combination with the Taq polymerase or equivalent polymerases as long as buffer compatibility is respected. The process is represented in Figure 164

rh qPCR for mock section assays

For the experiment described in this thesis, the first generation was used to discriminate between the wild type active gene and the inactivated one during a qPCR reaction. To run the rh qPCR some calibration experiments had to be made to set the right concentration of RNase H2. For each sample 2 qPCR reactions were run with a different rh primer matching the original or the mutated base. This allowed to access separately to the quantity of each version of the gene in the sample. To achieve the best performance, IDT recommend to use blocked-cleavable primers for both forward and reverse primers therefore, forward and reverse primers were both ordered as rh primers with a common primer for the two version of the gene and two complementary primers each recognizing either the wild-type or the mutated gene.

rh qPCR calibration

To set up the rh qPCR, first the quantity of RNAseH2 must be adapted. To do so, various concentrations of the enzymes are used with the target gene and compared to a normal PCR with equivalent normal primers. The reaction should start at the same time with the rh primers and the classic primers. In parallel amplification of the mutated gene should also be performed with various concentration of RNAseH2. The optimal concentration corresponds to the one where amplification of the target gene starts as soon as the one with classical PCR and the amplification of the mutated gene is not starting or after an important number of cycles allowing to distinguish between at least 3 order of magnitude difference in DNA concentration (~10 cycles).

For the KlenTaq gene (KT), a first trio of primers were ordered and tested. After optimization and trials, they did not manage to give a proper signal amplification and rh qPCR obrained results were confusing. We therefore, ordered the only other possible trio of primers. As the RNA base has to be placed exactly on the mutation, and the rh primer has a specific design, there is not much possibilities. For this second version of rh primers, concentration of RNAse H2 ranging from 0.25 to 1mU/µL were tried as recommended in IDT protocol. Due to the relatively high melting temperature of the primers, a two-step protocol was used with 20 s at 95°C and 40 s at 60°C which is optimal for RNAseH2 and polymerase activity to work together. As we can see in Figure 165, no big difference was obtained for wanted and unwanted amplification unless for the KT-(inactive gene) at very low RNAse H2 concentration. We also noticed that the behavior was different according to the gene version/primers association. Finer resolution of the RNAse H2 range from 0 to 0.2mU/µL were tried and revealed that very precise concentrations as low as 0.02 and 0.03mU/µL was ideal for respectively detection of KT+ (wild-type gene) and detection of KT-. Smaller or bigger concentrations were not furnishing enough differences between ON and OFF detection. Figure 166 shows the differential amplification of KT+ and KT-genes with 0.2 or 0.3 mU in 10µL. This very narrow range of RNAse H2 was not very convenient and the obtained PCR curves were not as nice as classic PCR curve, but the systems allowed to determine initial and final quantities of KT+ and KT-genes after PEN CSR.

For the NBI case, similar optimization was performed this time with a 3-step PCR (95°C 10s, 52°C 20s, 68°C 20s). The optimal RNAse H2 concentration was found to be 0.75mU/µL and yield to a very good qPCR assay. All reaction was run with the DreamTaq buffer to avoid potential issue due to bacteria component remaining after selection or with bacteria component for the initial quantity measurements. The DreamTaq buffer was supplemented with 1.5 mM of magnesium to reach the 3mM recommended in IDT protocol.

Notes on rhqPCR experiments in IV, p.238

Looking at the results for measured initial fractions or bulks one can wonder what is causing the differences between the theoretical fractions and the measured ones. The rhqPCR is a rather accurate method. Especially in the case of the NBI genes, rh primers shows a very good differentiation between the two genes. Contrary to the KlenTaq gene where the calibration was more complicated. The error calculations on Klentaq rhqPCR show a clear difference between the situation were bacteria lysate, even in low quantity, is present or not. For bulks or before selection measurements errors on DNA concentrations are between 3.5 and 11.4% whereas for DNA extracted from droplets they are between 0.1 and 2.8%, reaching 9.5% for droplets initially containing the higher bacteria concentration. It can also be noticed that the error rate seems to increase with bacteria concentration. Bacteria lysate is clearly interacting with the rhqPCR and causing defect in the method. In a general manner when performing qPCR with bacteria it is not possible to observe signal amplification with already relatively low bacteria concentration generally because the genomic DNA is binding to the intercalating fluorescent reporting dye. With rhqPCR working with bacteria concentrations over 10 bact/nL is impossible and as exemplified by the variations in the results their lysate components are still influencing the outcome of the reaction even when greater dilutions are used. In the framework of these experiments original bulk or before selection tubes were always diluted at least 10 times or more according to the initial bacteria concentration. Annoyingly, when low bacteria concentration is used in the initial sample, diluting it too much also prevents good evaluation of DNA quantity by rhqPCR.

vi. Preparing electrocompetent cells

We first used NEB online protocol to prepare electrocompetent cells. The resulting cells had a competency of ~10 7 cfu/µg for the pNBI_vide plasmid which is not high enough for our experiments. Furthermore, electroporation did not happen as planned. When using electroligation product with 2000 V and 5 mS, the electroporator could only apply 220V and displayed 654 mS. Fillipo Caschera, postdoc in my laboratory at this moment advised me to contact Erik Carlson from North Western University. I told him what was happening with the cells and he sent me his protocol that is similar to the NEB protocol. He also told me that the issue with electroporation was certainly due to salt present in too high quantity in the cells. I was already using the SOB medium which only contains 2.4 g/L MgSO4, 0.5 g/L NaCl and 0.186 g/L KCl compared to the 10 g/L of the classic LB (Miller) medium. So, I first tried his protocol keeping the SOB medium. As presented in Table 32 I could reach an efficiency of ~10 8 cfu/µg for pNBI_vide this time but with the same issues on electroporation voltage. I then tried with the LB low salt which contain only 0.5 g/L of NaCl. This time an electroporation test was done with water only and performed with the right programed voltage and time. But again, when adding the plasmid, only 940 V were applied to the cells. I then performed several tests with water, elution buffer or nothing with the cells prepare with LB or SOB and all were functioning giving ~1650 V with ~5mS for 1700 V set in the program. Trying again with the plasmid on thaw bacteria from -80°C I did not have this electroporation issue anymore. It seemed that the stay at -80°C of the prepared competent cells fixed the issue.

An adaptation of Erik Carlson protocol described in the material and methods is now used in the lab to make electrocompetent T7 express cells.

In the protocol we use and most protocols to prepare competent cells, it is generally advised that cells should be kept on ice most of the time, handle in cool environment, and centrifuge in refrigerated machines. But in 2016, Tu et al. claim that room temperature electrocompetent bacterial cells improve DNA transformation and recombineering efficiency 350 . We tried their protocol with my intern Nicolas Larrouy and also to add glycine in the growing culture both for cold or room temperature preparation as it could make cells more porous and increase electroporation efficiency. Preparation with 1% glycine in the culture medium did not yield to any transformant and cells prepared at cold temperature had higher transformation efficiency.

vii.

Alternative amplification method to

PCR: LAMP

To address these gene amplification problems, we also thought of using another amplification method. We chose to investigate the possibility of using the isothermal Loopmediated AMPlification (LAMP) [31], [32] (fig. 10). The LAMP uses 4 primers FIP, BIP, F3 and B3 to amplify a sequence thanks to the strand displacement activity of the Bst polymerase.

The FIP and BIP primers are made of two gene specific sequences separated by a linker. The first sequence serves to initiate the first stage of amplification and the second one creates a loop that allows the later stage of amplification. The primers B3 and F3 bind just near the first sequence and launch the amplification by soliciting the strand displacement activity of the polymerase. By elongating the B3 and F3 primers, the bst polymerase releases the strand containing the loop primers. Then FIP and BIP primers can pursue the elongation and create long continuous strand with multiple copies of the amplified sequence. Restriction site can be introduced to recover the wanted sequence. The LAMP is running in a buffer that seems more compatible with the IPA buffer since it contains 4 to 8mM of magnesium and the remaining components (KCl, (NH4) 2 SO4, detergent) have the same concentration than the thermopol buffer used to make the IPA mixture. The LAMP can detect a few copies of a gene and is very specific because of it need of 4 primers containing 6 specific sequences. One other advantage of the LAMP is that it has a higher biocompatibility than PCR [33]. But this method has also some important drawbacks. It is designed for quick biological diagnosis and has until now only be used for the amplification of short sequences with a maximum size of about 300pb. By reading literature, it appears that longer amplification is possible but has a lower yield in the time usually used for the reaction that must be short [31]. So maybe by increasing the incubation time the yield can increase. The other problem of this method is the design of the primers. They need to fulfill very specific requirement for the melting temperature of each specific sequence.

The melting temperature has to be adapted to the running temperature but also between the recognition sequences. Thus, it is very difficult to place the primers exactly where you want.

LAMP primers were first designed according to the specification described in [31]. This method was first preferred because we wanted to control exactly what we would amplify. After several attempts it turns out that only inter or intra primers reactions (primer dimers) were happening. So, we tried other primers designed using the PrimersExplorer V4 (http://primerexplorer.jp/e/). The software gave several primers possibilities but is programmed to propose short target sequences. 3 groups of primers were chosen according to their 3' and 5'ΔG values and their location, one in the beginning of the gene (L#2), one in the middle (L#23)

and one at the end (L#104). By mixing the different primers we would thus be able to check the amplification of longer sequences. Additionally, the TTTT linker usually used in the FIP and BIP primers was replaced by the restriction site of EcoRI so that we can digest the amplified sequences back to monomeric amplicons.

The amplifications using the software designed primers were successful for each of the 3 groups of primers after one-hour incubation and also for the combination of the F and B primers of L#2 and L#104 after 3 hours of incubation. A digestion with EcoRI was performed at 37°C for one hour and then during 3 hours with 2 times more enzyme. In both case a slight band could be seen at approximatively 300pb even for the long sequence, but a lot of the amplified DNA remained uncut. This is probably due to the temperature of 37°C that is not high enough to unpack the DNA. Actually in [34], [35] As shown in figure 1 all the primers combination generated amplification. But when we look at the gel on figure 2 we see that both type of reactions produce parasites products.

Generally, results are better in the absence of EvaGreen dye. Type T reactions seem to give better results and allow the amplification of amplicons of up to 1500pb. S reaction didn't give encouraging results even for the shortest amplicon. RPA (Recombinase Polymerase Amplification) T reactions give generally better results than RPA S reactions. Up to 1500 bp can be amplified using RPA but optimization need to be done for cleaner results.

Illustrations

Figures

  run in test tubes and not in droplets OD600 cuv, OD600 tub, OD600 pr: see annex Bacteria counting CSR: Compartmentalized Self-Replication CPR: Compartmentalize Partnered Replication PEN DNA: Polymerase Exonuclease Nickase Dynamic Network Assembly PEN CSR: Programmable External Network Compartmentalized Self-Replication

Figure 1 .

 1 Figure 1. The various roles of proteins in bold with some examples to illustrate them.

  structures are stabilized primarily by hydrogen bonds. α-helix and the β-sheets follow a precise organization pattern. The α-helices have a righthanded-spiral conformation in which every backbone N-H group forms a hydrogen bond with the backbone C=O group of the amino acid located three or four residues earlier in the protein sequence. β-sheets are composed of strands connected laterally by at least two or three backbone hydrogen bonds. They form a twisted and pleated sheet. β-turns allow rapid directional change in the structure.

Figure 2 .

 2 Figure 2. The 20 common amino acids and their properties.

Figure 3 .

 3 Figure 3. The different levels of structure of proteins. The primary structure consists of a chain of amino acids. Repetitive motifs are forming the secondary structure. The tertiary structure is the combination of these motifs in space. The quaternary structure is the combination of several monomers as subunit to form a bigger construct.

Figure 4 .

 4 Figure 4. Protein-folding energy landscape. Funnel shaped energy landscape with many high-energy unfolded structures and few lowenergy folded structure. Folding occurs via alternative microscopic trajectories. (taken from 7 )

Figure 5 .

 5 Figure 5. Enzymatic catalysis. A. Enzymes decrease the energy activation barrier and stabilize the transition state. B. Models proposed for the interaction between the enzyme and the substrate.

Figure 6 .

 6 Figure 6. Beginning of protein engineering timeline. (adapted from 11 )

Figure 7 .

 7 Figure 7. De novo design of ligand biding protein (adapted from[START_REF] Damborsky | Computational tools for designing and engineering biocatalysts[END_REF] ) Interactions of the substrate with amino acids thought to be necessary for the catalysis are placed into a suitable scaffold. Binding sites with pre-organized organization and high complementarity are selected.

Figure 8 .

 8 Figure 8. Mimicking natural selection via directed evolution. The steps of natural evolution are reproduced at the molecular scale. Diversity is generated at the level of the gene sequence. The mutants' proteins, the corresponding phenotypes, are then expressed and tested for their capacities to perform the desired action. Selected mutants' genes are retrieved and either recombined or mutated again to run a new cycle of evolution.

  the phenotype-genotype linkage has been created from one of the techniques presented above, various methods can be used to apply the selection pressure and create the artificial evolutionary loop. Two different approaches, screening and selection, in two different contexts, in vivo and in vitro, are presented in D. While in vivo experiments mimicking natural selection by linking the protein activity directly to the survival or reproduction of the host (a.k.a. complementation) have very high throughput, they lack the flexibility provided by in vitro systems. In vitro complementation would thus represent an ideal solution for efficient evolution experiments, offering the best possible throughput and level of control. Contributing to the development of such a method is the goal of this PhD project.

  . Obexer et al. also used 3 rounds of DE to improve an artificial aldolase to reach 30 times the capacity or their computationally designed enzyme 73 . DE led to the apparition of an unanticipated catalytic tetrad that emerged near their designed hydrophobic pocket. Other methods use data collected by sequencing during a DE run and try to predict the best possible path toward an improved enzyme. In the ProSAR-driven enzyme evolution 74 , statistical analysis of the protein sequencefunction relationship is used between evolution cycles to determine where to introduce the next mutations. In other approaches, rationally-designed scaffold proteins are used as starting point for DE experiments 38 . Development of a suitable screening or selection method is one of the biggest obstacles when starting a DE experiment. The possible solutions, presented with more detail in the next part, depend on the nature of the target and the desired improvements. Future advances in the field will certainly pursue the development of versatile techniques facilitating high-throughput experiments. The growing importance of microfluidic DE platforms is a promising illustration.

Figure 9 .

 9 Figure 9. Evolution of the number of publications on Protein engineering and Directed evolution. DE publication represents only 3.9% of the total protein engineering publications.

Figure 11 .

 11 Figure 11. Selection methods for directed evolution. Different methods can be used for the selection of the mutants: Affinity selection based on the affinity between a target and the mutant protein, Phage Assisted Continuous Evolution (PACE) where the replication of the phage is dependent on the activity of the target mutant protein, the target protein can also be or made essential for the host organism survival, in the Compartmentalized Self-Replication (CSR)-like methods, the replication of the gene by PCR in droplets is linked to the activity of the studied protein.

Figure 12 .

 12 Figure 12. Directed Evolution Methods efficiency assessment. Acronyms refer to the Table 2 Selection methods: PACE: Phage Assisted Continuous Evolution, IVC: In vitro Compartmentalization, CPR: Compartmentalized Partnered Replication, CSR: Compartmentalized Self-Replication, OF&AC: Organismal Fitness and Auxotroph Complementation, Display: all displays methods. Screening methods: CSD: Cell Surface Display, IVC: In vitro Compartmentalization, ILC: Isolated Liquid Culture, CSM: Colonies on Solid Media.

Figure 13 .

 13 Figure 13. Hamiltonian path problem encoded with DNA by Adleman. In 1994, Adleman encodedthe travelling salesman problem with short 20 bp DNA strands. The problem asks if, given a set of cities and paths connecting them, one can find a so-called "Hamiltonian" path starting at a given city (here 0), passing through each other city exactly once and ending at a final city (here 6). 20 bp DNA strands are used to represent cities. Strands that match 10bp of the 3' end of a city strand (City A) followed by 10bp of the 5' beginning of another city (City B) are used to represent paths between cities.

of 2

 2 meters long total DNA strand 2nm thick are folded into a 6µm ball. Microsoft recently encoded 200MB of data consisting of 35 files containing high-definition video, audio, images, and text in synthetic DNA strands and some of their researchers are actively involved in the fields of DNA computation.

Figure 14 .

 14 Figure 14. Parallel between silicon-based computers and biological chemical reaction networks. DNA can be seen as a data storage device encoding a molecular program in its sequence. Ensuing information allow to generate chemical reaction networks governing decisions at the cell level, in an analogy to processors in silicon-based computers.

  carbon. This creates a single DNA strand oriented according to the sugar position from 5' to 3' or inversely. Nucleobases composing the nucleotides used in DNA can be a purine or a pyrimidine. Purines have two aromatic cycles, while pyrimidines possess only one. The purines used in the DNA are the adenine (A) and the guanine (G). Pyrimidines are the cytosine (C) and the thymine (T). A DNA sequence can therefore be written with these 4 letters. The four bases form hydrogen bonds thanks to their nitrogen and oxygen groups, preferentially combining a purine to a pyrimidine. A and T are paired with 2 hydrogen bonds and C and G with 3. Thanks to these base pairing capacities, complementary single DNA strands form a double stranded right-handed helix in solution, which is additionally stabilized by π-stacking interactions between the base pairs. This structure was discovered in 1953 by Watson and Crick, who gave their names to the observed standard base pairing rules. Two matched complementary strands form an anti-parallel duplex due to the orientation of the DNA backbones.

Figure 15 .

 15 Figure 15. Structure of the DNA. Nucleotides are the building blocks of DNA molecules. Four different bases A (Adenine), T (Thymine), G (Guanine) and C (Cytosine) are used in the DNA molecule. A and G are purines, possessing two cycles, whereas T and C are pyrimidines, possessing only one cycle. Nucleotides are bond by phosphodiester bonds to form a single strand of deoxyribo-nucleic acids oriented from 5' to 3' according to the orientation of the deoxyribose carbons. Two single DNA strands can assemble by Watson-Crick specific base pairing via hydrogen bonds. 2 hydrogen bonds can be made between A and T bases, 3 between G and C. Stacking phenomenon between overlaying aromatic cycles also stabilize the double strand complex. Strands are paired in an anti-parallel fashion that can be schematized with an arrow on the 3' side, the starting point for polymerase elongation. The classical DNA duplex has a right double helix B shape with a diameter of 2nm.

Figure 16 .

 16 Figure 16. DNA origami. On the left, the principle of DNA origami formation is schematized. Using a long scaffold DNA strand and many synthetic short staple strands it is possible to shape 3D DNA structures at will by programming the DNA folding. Structures such as a nanoscale smiley (here observed by atomic force microscopy) can be obtained. (Image taken from Folding DNA to create nanoscale shapes and patterns). On the right, an origami DNA nanocage loaded with protein payload in its open state. Toehold are used to guide the cage formation. Aptamers on the extremities allow to lock or open the structure when bind by an antigen. Such system could be used to deliver drug specifically to particular cell types. (Image borrowed from A logic-gated nanorobot for targeted transport of molecular payloads89 .)

Figure 17 .

 17 Figure 17. DNA-only molecular computing systems. Top: representation of two most widely used toehold systems. The toehold-mediated strand displacement is an irreversible mechanism where one strand replaces another thanks to its higher affinity to the template. The toehold-exchange strand displacement mechanism is a reversible mechanism where the strands can replace one another. The shared region between the sequences is called a toehold (region number 2). Bottom left: DNA nanotweezers able open and close by strand displacement are represented. Bottom right: the mechanism of hybridization chain reaction (HCR) is described. Hairpins with partial sequence complementarity are mixed with an initiating strand. This strand will open one of the hairpins leading to exposure of the sequence complementary to the other hairpin. A chain reaction is therefore started where hairpins open and attach to each other forming long DNA strands that can be observed on agarose gels.

Figure 18 .

 18 Figure 18. Molecular programing hybrid systems inspired from biology. On top, the Reverse-transcription and Transcription-base Autonomous Computing System (RTRACS) is presented. This system allows to generate several RNA output strands from an initial input RNA using various templates and several enzymes; a reverse transcriptase, a RNAseH, a DNA polymerase and a T7 polymerase. At the bottom, the genelets systems is schematized. In this system, an activator strand completes a T7 promoter sequence enabling the production of several output RNA strands. Produced RNA strands can interact with other nodes by binding and inhibiting their activator strand.

Figure 19 .

 19 Figure 19. Oligo strand production in the PEN DNA Toolbox. Amplification scheme based on the repeated extension and nicking of one small trigger strand on a short DNA duplex.

Figure 20 .

 20 Figure 20. Different modules of PEN DNA Toolbox. The different modules that can be connected in the PEN DNA Toolbox. From top to bottom: Autocatalysis allows exponential amplification of the input strand. Activation allows linear production of an output strand different from the input strand. Inhibition is realized by a strand able to bind a template but prevent elongation, with higher affinity than the original input. Deactivation is based on the use of a pseudotemplate that modifies the input strand so that it cannot trigger its template strand anymore. Degradation is the simple depolymerization of unprotected strands by an exonuclease. Detection allows to transform external signals into input manageable by the DNA based molecular program.

Figure 21 .

 21 Figure 21. Computer assisted design of PEN DNA Toolbox networks. Three different computational approaches developed to help network topology design or verification and template sequences optimization are presented.

Figure 22 .

 22 Figure 22. Chemical modifications of template strands. Various modifications can be introduced in the molecular templates. From the left to the right: Addition of a quencher or a fluorophore, generally used to monitor the reactions. Can be added on the extremities or inside of the molecule. Phosphorylation on the 3' side is used to prevent elongation of the strand by polymerases. Deoxyinosine base can bind to any other base. Methylation of some bases can make the strand resistant to some endonucleases. Random bases in the sequence, can be used to make a process unspecific or to slow down the reaction by decreasing number of exactly matching sequences. RNA bases can be added to have specific RNA related enzyme activity. Deoxyuridine can be introduced to be used in combination with uracil-DNA glycosylase creating abasic sites. It can also mask nbi sites on the output part of templates or in pseudotemplates used in the deactivation module. Substitution of phosphodiester bonds by phosphorothioate (PTO) bonds, prevents cut by the exonucleases. Spacers can be added between the sequence and added element to avoid steric clashes that could prevent strand polymerization completion. It can also be used to concatenate sequences. Biotin at the extremity, in combination with streptavidin in the medium. Biotin streptavidin interaction will prevent strand degradation by polymerases. It can also be used to attach template on a streptavidincoated support.

Figure 23 .

 23 Figure 23. Different fluorescence reporting strategies for PEN DNA Toolbox reactions monitoring. Intercalating dyes (pink box) can be used to follow the global quantity of DNA. Nucleobase quenching (yellow box) can allow to follow input binding on the templates. This technique as the advantage of not putting any additional load on the network. Annealed strands (green box), random coiled (orange box)or hairpin loop reporters (blue box) can detect target strands in the reaction. All these methods rely on separating a quencher and a fluorophore in presence of the target strand. According to the design the reporting can be made irreversible by for instance adding restriction endonucleases like in the irreversible reporting method describe in the dark blue box. Similar design can be done for annealed strand. These kinds of reporting are adding a load to the network because they use or capture transiently a part of the produced input. Furthermore, when stabilization by the polymerase is required, additional load appears due to enzyme activity sharing.

Figure 24 .

 24 Figure 24. Linking enzyme activity to gene replication. Scheme of the different approaches of compartmentalized selection.

Figure 25 .

 25 Figure 25. Scheme of some possible ways to adapt molecular programs so they can be triggered by non-DNA dependant enzymes.

Figure 26 .

 26 Figure 26. Possible selection functions. Different shape of selection function can be imagined. Continuous selection function (green) would select according to enzyme activity, sigmoidal selection curve (red) would select equally all the mutant reaching the threshold activity, delayed continuous selection function (black) would allow to get rid of poorly active mutants and selection of the other mutants according to their activity.

F.

  The nicking enzyme Nt.bstNBI a. Presentation of the enzyme a.1 Discovery of Nt.BstNBI and other nicking enzymes We want to apply this compartmentalized self-replication method to the evolution of the Nicking Enzyme Nt.BstNBI (NBI). This enzyme was first isolated from the thermophilic organism Bacillus stearothermophilus in 2000 by New England Biolabs researchers 136 . Nicking enzymes, or nickases, are restriction endonucleases that can recognize a specific sequence, like classical restriction endonucleases, but contrary to them, cleave only one of the two DNA strands, thus leaving a nick in the sequence. Nt.bstNBI recognizes the sequence 5' GAGTC 3' and nicks preferentially the top strand 4 bp downstream (fig. F.1). Nt stands for Nicking top strand and Nb would respectively be used to name a nickase cutting the bottom strand. NBI was one of the first discovered single polypeptide nickases, along with N.BstSEI discovered in 1996 by researchers of SibEnzymes in Russia. Before them, proteins cleaving only one strand had been reported, but these were forming complexes with other proteins to realize this action. These nicking proteins are generally involved in DNA replication, DNA repair, DNA relaxation, or other DNA-related metabolisms. For instance, the gpII protein of the bacteriophage fI generates a nick at the replication origin to initiate rolling-circle amplification of the phage genome. Another one, called MutH, is involved in mismatch repair and initiate the correction mechanism by creating a nick in the sequence near the mismatch when interacting with MutL and MutS in E. Coli. After the discovery of Nt.bstNBI in the NB strain of B. stearothermophilus and N.BstSEI in the SE strain of the same species, other similar enzymes have been discovered. In 2001 N.BspD6I was isolated in the laboratory of N. I. Matvienko from strain D6 and N.Bst9I was isolated from strain 9 by Dedkov et al. These four enzymes, despite coming from different strains of b. stearothermophilus were actually all isoschizomers viii , recognizing exactly the same sequence and cutting at the same 4bp distance of the recognition site. A first simple explanation could come from the fact that nickases can fragment DNA only if two opposite viii Isoschizomers are pairs of restriction enzymes specific to the same recognition sequence.

Figure 27 .

 27 Figure 27. Presentation of the nicking enzyme Nt.BstNBI. Nt.bstNI (NBI) was discovered in 2000.Its recognition site is GAGTC and when bound it can cut the top strand 4 bp further. One particularity of NBI is the presence of three negatively charged residues in its catalytic site, contrary to classical restriction enzymes that usually possess one positively charged residues. The origin of NBI was proposed to either i) come from an homodimeric enzyme having lost its dimerization capacity or ii) be the large subunit or a heterodimeric restriction enzyme. The second option seems to be the more probable. Like classical restriction enzymes, NBI is part of a Restriction-Methylation (R-M) system. The related methylase recognize the palindromic site GASTC and methylate the adenosine. Restriction enzymes with the same recognition site have been used to better understand NBI particularities.

  restriction endonucleases: Mly1 (from Micrococus lylae) and Ple1 (from Pseudomonas lemoignei). These two enzymes recognize the same sequence but cut both strands 138 and, similarly to NBI, work with a single type of polypeptide.Type IIS restriction endonucleases are a sub-category of the Type II restriction endonucleases themselves being part of 4 distinct types of endonucleases. Most restriction endonucleases are components of restriction modification (R-M) systems usually found in prokaryotic organisms or in some algae viruses. Their main function is to protect the organism against foreign DNA, like bacteriophage genomes. But, other functions, such as maintenance of species identity, generation of genetic variation or, in the case of viruses, prevention of other viruses' infection, have also been attributed to these R-M systems. Some even consider them as selfish DNA elements. R-M systems were first discovered in 1953 when some bacteria strains demonstrated lower susceptibility to bacteriophage infections. This protection is conferred by the concurrent actions of a restriction endonuclease and the associated methyl transferase forming the R-M system. The methyltransferase or methylase recognizes the same site than the restriction enzyme and can catalyze the transfer of a methyl group from the cofactor S-adenosyl-L-methionine (AdoMet) to specific recipient bases, depending on the enzyme. Two major types of methylases exist. The first one methylates the fifth carbon of the cytosine to form a 5-methylcytosine. The second one targets the exocyclic amino group of either the cytosine or the adenine to form respectively the 4-methylcytosine or the 6-methyladenosine.

  Other similar systems are based on the simple cleavage of foreign modified DNA. In that case the DNA of the organism is protected by not being modified. Restriction enzymes being part of such systems are considered as the type IV of restriction enzymes. They recognize and cut methylated DNA. One well-studied representative, McrBC, consists of two subunits.Other types are all part of the R-M systems.Type I restriction endonucleases consist of an assembly of 3 different subunits interacting in general with two asymmetrical bi-partite recognition sites. They cut the DNA approximately in the middle of these two sites. Type III RE contains only 2 subunits, interacts with two head-to-head asymmetrical arranged recognition sites and cuts the DNA close to one recognition site. Finally, Type II is the most studied, as it is heavily used in molecular biology to create recombinant DNA. Type II restriction endonucleases differ from the other types in that they are composed of a single type of polypeptide of 30 to 100 kDa and do not require triphosphate nucleotides (ATP or GTP) as cofactor. They cleave DNA within or close to their 4-8 bp recognition sites. As many as 11 subtypes of the type II restriction endonuclease exist.Distinctions are based on the multimerization state, the type of recognized sequences (palindromic, asymmetric, double), number of cuts that are realized, and so on with overlapping categories. A sub-class, type IIS, is distinguished for enzymes that typically recognizes an asymmetric sequence and cuts the DNA on one side outside of the recognition sequence at a defined distance 139 . They can be monomeric, homodimeric or heterodimeric but they multimerization state is not always known. They can be divided in five classes according to this multimerization states and their action mode140 . Nicking enzymes such as NBI are sometimes considered by researchers as unconventional type IIS restriction endonucleases.a.2 Nt.BstNBI StructureThe comparative study of MlyI and PleI with Nt.bstNBI showed respectively 32.1% and 33.1% similarities in the entire sequence. Protein sequence alignments revealed a conserved Nterminal domain with ~34% identity corresponding probably to the recognition site and a less conserved C-terminal domain with ~30% identity attributed to the cleavage domain. This domain organization is similar to the most studied type IIS FokI restriction enzyme which possess only 26.1% sequence identity with NBI. Structural similarities between type II and IIS enzymes suggest for a conserved catalytic core with three essential charged residues, two acidic amino acids D or E and a positively charged lysine (K) corresponding to the following motif: P-D/E-Xn-D/E-X-K. Alanine scanning ix of some positions based on this information shows that the aspartic acid 456 and the glutamic acids 418 and 469 are essential to the catalytic activity but not to the DNA binding capacity of the enzyme. Gel filtration studies showed that MlyI ix Alanine scanning is used to determine the contribution of a specific residue to the stability or function of given protein by mutating it into an alanine, a non-bulky and chemically inert residue. would dimerize in presence of DNA contrary to NBI, suggesting a sequential process for DNA cleavage requiring a first nick on one DNA strand and further cleavage on the other strand by a dimerized unit. NBI was therefore thought to have lost partially its dimerization capacities. In 2008, Kachalova et al. showed that nicking endonucleases are probably not mutated restriction endonucleases but more likely originate from the large subunits of heterodimeric endonucleases. An open reading frame encoding a small protein of 186 aa with formerly unknown function and adjacent to the N.BspD6I gene was expressed and purified. Experiments

Figure 28 .

 28 Figure 28. Structural representation of NBI. The structure of NBI was represented using USF Chimera. In A the catalytic residues are represented in cyan and the recognition loops in blue. In B the surface representation of NBI with electrostatic potential coloring. Blue is positive, red is negative.In could be interesting to target the blue part that seems to interact with DNA to increase the velocity of the reaction. In C, Superposition of NBI, Ple1 and Mly1 modelled structure with active site residues. Blue: NBI, Purple: Mly1, Beige: Ple1. These two enzymes are mesophilic and work at 37°C so we can compare their overall structure to try to find the part responsible for the higher thermostability of NBI and try to enhance it. Molecular dynamics simulation at different temperatures could be used to find the residue that are destabilized in Mly1 and Ple1 but not in NBI to apply site directed mutagenesis. But error prone PCR can also be a simpler option that can be allowed thanks to the high throughput of the method. This structural information can help in the directed evolution process.

  homologous recombination. This induced recombination has low amount of non-homologous end-joining events and reduced cell toxicity upon expression. Nickases are also used to study gene modification mechanisms. By introducing gaps in the sequence, researchers can monitor DNA repair, recombination and translocase activity mechanisms 151 . The ability of nickases to create long single strands was also used to improve pyrosequencing xi sample preparation protocol 140 . In the field of nanodevices, molecular motor able to transfer a cargo oligonucleotide along a DNA track has been designed using nickases 152 . The track is constituted of three short DNA strands each having one half annealed on a one of the three domains of a base DNA strand. The other non-complementary half can bind the cargo strand possessing the uncleavable part of the nicking site. When the cargo binds on one floating DNA strand part, the nickase will cut the top of the track strand. The short cut part leaves and the cargo strand is then attracted by the next floating strand. The cargo can transfer at a speed of 0.1nm/s. Outside their use in the PEN DNA toolbox, nickases receive attention from the DNAcomputing community where it is sought they could solve some computing issue, or have already been used to generate fluorescent readout 153 . Nicking endonucleases are not yet widely used in research and medical diagnosis. This can be explained by the relatively low variety of recognition sites and of course their relatively recent discovery. But, as more and more applications are developed, they appear as new essential tools that should add to polymerases, classical restriction endonucleases, ligases and other similar enzymes in the molecular biologist toolbox. The growing number of nicking endonucleases of different specificities will likely promote their wider use in the future.

  Monomeric restriction enzymesIn 2004, Zhu and al. carried out directed evolution experiments on BsaI. BsaI is a monomeric type IIs restriction endonuclease with two active sites. After introducing random mutations in the sequence, they then recombined the c-terminal and n-terminal wild-type domain in order to introduce mutations only in on one half of the protein 156 . They obtained two variants Nb.BsaI and Nt.BsaI able to nick respectively the top and bottom strand with the same recognition sequence as BsaI. Amino acids substitutions equivalent to the retained one were then introduced in BsmBI and BsmAI, generating other nicking enzymes. Despite the fact that they obtained both top and bottom strand nicking activity for BsaI, their method was not strand specific and for instance only top-strand nicking variants were found for BsmAI.

  Heterodimeric restriction enzymes Using heterodimeric restriction endonucleases, it was possible to create nicking enzymes by inactivating the catalytic site of one or the other subunit 158 . Inactivation of one or the other subunit gave nicking enzyme cutting on one or the other strand. Nicking variants of R.BbvCI were engineered this way. • Other approaches Protozanova et al. used pseudo-complementary peptide oligonucleotides (PNA) to mask the DNA from cleavage by the ple1 restriction enzyme while not preventing the enzyme from binding to its recognition site 159 . PNA are binding strongly the natural DNA at the level of the cutting site of pleI bending the DNA. The resulting interactions led to apparition of a nick at a distant site that the usual one. Researchers also engineered variants of CRISPR Cas9 xii genome editing system itself to act as an RNA guided nickase. Inactivation of one of the two nuclease domain in the Cas9 protein resume as a CRISPR Cas9 system with a nickase function 160 . Paired nicking using CRISPR Cas9 nickases resulted in a reduction of off target activity and enhanced genome editing 161 .

Figure 30 .

 30 Figure 30. Different methods to engineer new nicking enzymes.

Figure 31 .

 31 Figure 31. Workflow of the Directed evolution cycle using molecular programs, Programmable External Network directed evolution cycle Mutations are introduced in the target gene and the obtained library is cloned and expressed in bacteria. Single bacteria are then encapsulated with the molecular program and lysing agents in monodisperse ~ 20µm diameter water-in-oil droplets using a microfluidic device.Droplets are then placed in a PCR machine to undergo a 45°C incubation followed by a classic PCR protocol except for the buffer. This incubation allows production of primers in each droplet according to the encapsulated mutant activity. The PCR yield is dependent on the reach primers concentration and therefore of the activity of the enzyme. Finally, the droplets are broken to retrieve the amplified DNA, corresponding to a library enriched with the best mutants. The selected genes can then be used to start a new cycle of evolution or to analyze the selected mutations and/or selected mutants.

1 .

 1 Presentation of the molecular program 1.1 The different templates and enzymes used fo IPA

Figure 32 .

 32 Figure 32. Different parts of the molecular program used for selection of nickase activity.

Figure 33 .

 33 Figure 33. Interactions between the different modules for the IPA. A. Schematic representation of the different nodes used in the molecular network. A first block allows to convert NBI activity in a trigger strands, which are then used in a second block to trig the production of primers. B. More detailed presentation of the templates and how they interconnect.

Figure 34 .

 34 Figure 34. Shapes of primer production curves depending on the network topology and the concentration of the different components. T: Trigger, TtoP: Trigger to Primer or Primer Producing Template (ppT), aT: autocatalyst, sT: source template, pT: pseudotemplate

Figure 35 .

 35 Figure 35. Prediction of primers production by molecular program with various design, as a function of NBI activty. All circuits contain 20nM of each primer producing Template (ppT), 40nM of autocatalyst (aT) and 10 nM of trigger strand (T) or 2nM of source template (sce). Pseudotemplates (pT) and exonuclease effects (RecJ) are also predicted. At the bottom right representation of the network in the DACCAD software. On the bottom left, PCR yield according to NBI activity in a perfect PCR were all primers would be incorporated. Bottom right: representation of the network in DACCAD.

Figure 36 .

 36 Figure 36. Estimation of primers production rate via dNTPs consumption. The Isothermal Primer Amplification (IPA) reaction was performed in the nIPA buffer in presence of an intercalating fluorescent dye (Evagreen) with various concentration of dNTPs, aT 20nM, ppT 10nM, T 10nM and followed in a real-time PCR machine. 10µM, 25µM and, 50µM of dNTPs are consumed resepctively in 140, 225 or 390 min at which times, the curve is reaching a plateau. This would give an average primer production rate of 6.4nM /min, assuming if all the dNTPs are consumed equally by all reactions of the molecular program.

Figure 37 .

 37 Figure 37. Primer production rates in nIPA buffer. Production curves and rates calculated for A) p25f2 (forward) and B) p17r3 (reverse) primers. Light lines are production curves for reactions with different concentrations of probes added (50,100,150 or 200 nM) for different concentrations of NBI. Dark lines are fitted to the initial linear section of the experimental curves. C shows the values of fited slopes. (Results obtained from Giselle MacGallum internship report)

Figure 38 .

 38 Figure 38. Recording of primers production speed in vitro according to NBI concentration using a double stranded molecular probe. 50nM of the molecular probe dsp17r3_F/dsp13r3_Q was added to an IPA reaction in the IPAV0 buffer in vitro with various quantity of NBI enzyme, aT 40nM, ppT 20nM, T 20nM. Fluorescence increase, followed in a real-time PCR machine, indicates binding of the produced reverse primers p17r3 on the probes. Primer production speed increases up to 0.3% of NBI and saturates above that concentration.

Figure 40 .

 40 Figure 40. Effects of pseudotemplate and triggering mechanism on primer production. (aT 40nM, ppT 20nM) Using a source template rather than the trigger strand v11 to start the reaction allows a faster production of primers. Effects of the pseudotemplate are not the same if used in combination of the trigger strand or source.pT has almost no effect on the IPA reaction run with a source while it is delaying the production when a trigger is used. Measurement done with 50nM of reporter.

Figure 41 .

 41 Figure 41. PCR of NBI gene with Taq in thermopol buffer + 20 mM NaCl with various concentration of additional Mg 2+ . Left Full length (~2kb), Right short fragment (780bp). PC is the positive control in thermopol buffer.

Figure 42 .

 42 Figure 42. The effect of Magnesium concentration on isothermal production of oligonucleotides through EXPAR. A 11 bases oligo (X11) is amplified by the dual repeat template Cx11 and can bind to a second template x11toON to produce a third oligo. X11toON bears a TAMRA dye. When x11h bind on x11toON (and is subsequently extended by the bst polymerase) the fluorescence of TAMRA decreases through a nucleotide-quenching effect. This is an undirect way to follow the x11h amplification, sometimes more practical than the use of dsDNA intercalating fluorescent dyes. The curves correspond to various Mg 2+ concentration added to the classic PCR thermopol buffer. We can see that the amplification kinetics is badly affected below +2 mM of Mg 2+ .

Figure 43 .

 43 Figure 43. PCR of full length NBI gene with Taq for various annealing temperatures and various forward primers. Reverse primer isp17r3 in IPA buffer.

Figure 44 .

 44 Figure 44. Linking the NBI activity to the amplification of its gene. Left: The IPA PCR process is performed using the selection program and targets the 780 first bases of the gene with 17 base primers and various NBI concentration. EvaGreen real-time fluorescence measurement of the PCR step (after 30 minutes at 45°C for the IPA stage). PC primers =Positive control with NBI replaced by 500 nM of primers.Right: Similar realtime PCR after 30 minutes IPA generating primers of 25bp.

Figure 45 .

 45 Figure 45. IPA-PCR of the 780 first bases of the gene in vitro with various NBI concentration before and after optimization. Before: short length 17 pb primers, IPA buffer, Bst Large Fragment. After: 25 bases long primers, nIPA buffer, Bst 2.0 WS.

Figure 46 .

 46 Figure 46. Scheme of the NBI concentration droplet-scanning experiment. Two premixes are prepared, containing the molecular program, PCR components and PCR reporters (Intercalating dye EvaGreen and specific reporting probe Zenphex). One of them is supplemented with 0.5% NBI and a fluorescent dextran dye (Alexa 647). The premixes are then mixed in a microfluidic chip allowing to generate droplets with various NBI concentration (from 0 to 0.5%.) thanks to variation in the pressure control of the two inlets. The fluorescent dye is used as a barcode to retrieve the concentration of NBI in each droplet. After IPA-PCR in emulsion, droplets are observed with a fluorescent microscope and fluorescence of the barcode and the PCR reporters are recorded for analysis.

Figure 47 . 3 .

 473 Figure 47. Droplet scanning of NBI concentration for in vitro IPA-PCR by Giselle MacCallum. The experiment was performed exactly as in Figure 48 except for the use of more surfactant in the oil phase (4% instead of 2%).

Figure 48 .

 48 Figure 48. Shape of the selection function in vitro in nIPA buffer. Droplet-scanning (see Figure 46Figure 47) of NBI concentration from 0 to 0.5% for in vitro IPA-PCR. Top: Fluorescence of the barcode indicating NBI concentration and PCR yield observed with a TaqMan probe, in each droplet. Bottom: Channel signal merging and result of image analysis on 13 000 droplets.

Figure 49 .

 49 Figure 49. Observation of GFP carrying bacteria lysis in droplets under the fluorescent microscope. Bacteria expressing the fluorescent GFP were encapsulated with rlysozyme and with or without Bugbuster. We can see that the small green points corresponding to intact bacteria are disappearing when the bacteria are lysed. Some bacteria seem to survive even after an overnight. More bacteria resist the overnight without BugBuster.

Figure 50 .

 50 Figure 50. Surviving (intact) bacteria as a function of time in one microscopic frame, with (top) or without (bottom) the lysing agent BugBuster at 10%. The incubating temperature is switched to 45°C at the time indicated on the plot. Fit with a single exponential decay and baseline provide a good result without Bugbuster, whereas a sum of exponential decay is necessary to obtain a good fit in the presence of Bugbuster.

Figure 51 .

 51 Figure 51. Degradation of protected specific or unspecific loopact probe by bacteria lysate with or without lysing agents. Either NBI expressing bacteria or bacteria expressing the Taq polymerase are mixed with 250nM of specific (pink-purple) or unspecific (blue) loopact probes in presence or absence of lysing agents at 45°C.

Figure 52 .

 52 Figure 52. Effect of rlysozyme and BugBuster on the in vitro IPA-PCR reaction with 1% commercial NBI. Positive Control (PC) are run in absence of lysing agents. When BugBuster is used in combination with rlysozyme, no product is observed.

Figure 53 .

 53 Figure 53. Effect of rlysozyme and Bugbuster in NBI activity release (measured by loopact digestion rate). A. Activity assay of NBI either commercial or released from bacteria (~100 bact/nL) with 0.5% on rlys or 0.5% of rlys and 10% of BugBuster. B. Effect of BugBuster concentration on NBI activity release by bacteria (50bact/nL) in presence of 0.2% rlys. C. Effect of increasing concentration of rlys on NBI activity release by bacteria (50 bact/nL). D. Effect of BugBuster concentration on 0.05% of commercial NBI.

Figure 54 .

 54 Figure 54. Effect of heat shocks in bacteria lysis and NBI activity release. A. Activity assay of bacteria at various concentration without any lysing agent with several heat shocks at 50°C for 1min. B Activity assay of bacteria at 70 bact/nL with 1 min heat shock at various temperature. No extra release of activity is observed after the heat shock.

Figure 55 .

 55 Figure 55. NBI activity release from bacteria according to rlysozyme concentration, temperature and pre-incubation at room-temperature. A. Speed of the loopact probe degradation by NBI released from bacteria (50 bact /nL) at 45 or 50°C according to rlysozyme quantity. B. A few curves of the probe degradation in A. C. Degradation rate of the protected loopact probe by NBI enzyme released by bacteria (50 bact/nL) with very little rlysozyme (1.10-7 dilution) and with or without a 1'30 heat shock at 50°C.

Figure 56 .

 56 Figure 56. Activity assay of wash supernatant. A. Protected loopact activity assay. B. Primers production reporting.

Figure 57 .

 57 Figure 57. Protected loopact activity assay in various solutions. Wash (1 or 2): In 1/10 th supernatant of washing solution; 2h RT: in 1/10 th supernatant of resuspension solution after 2 hours at room temperature; After droplet generation: In 1/10 th of the supernatant of the reaction premix after the time of droplet generation; Bact: Bacteria suspension at 50 bact/nL = ~1/100 th of the original solution. NBI comm: commercial NBI.

Figure 58 .

 58 Figure 58. PCR on pNBI plasmid from bacteria with the HS Taq in the nIPA buffer.

Table 8 .

 8 Resistance to bacteria lysate for different commercially available PCR polymerase (including a number of Taq derivatives), in their recommended buffers.

Figure 59 .

 59 Figure 59. Gel electrophoresis of PCR on pNBI plasmid contained in various concentration of bacteria with different commercially available Taq polymerases in their respective buffer.

Figure 60 .

 60 Figure 60. Increasing Mg concentration in a PCR run with OneTaq polymerase.

Figure 61 .

 61 Figure 61. Test of Bst 3.0 and Vent(exo-) in the IPA-PCR reaction and various buffer optimization tests. A. In vitro IPA-PCR with the new tested enzymes Bst 3.0 and vent (exo-). B. Table of enzyme performance according to the buffer concentration in KCl or Mg 2+ . C. PCR with the OneTaq polymerase at various KCl concentration. D. Loopact probe assay at increasing concentration of Mg 2+ . E. Comparison of BSt 2.0 and 3.0 perfomance in increasing Mg 2+ concentrations in IPAV0 buffer. The reaction is a turnover. An autocatalyst is producing a strand detected reversibly by a reporter. Once the dNTPs are over, the reaction cannot continue, the exonuclease degrade the remaining produced strand and the fluorescent signal go back to its low level. This experiment allows us to estimate the speed of the reaction and therefore here the speed of the polymerization by Bst 2.0 or 3.0. F. In vitro IPA-PCR reaction with Vent (exo-), Bst 2.0 and Bst 3.0 in their optimized buffer. (+90mM KCl for Bst 3.0, IPAV0 buffer for the others).

Figure 62 .

 62 Figure 62. Test of the bst evolved by John Milligan from the Ellington lab in a turnover and IPA-PCR reaction with bacteria. Left: Turnover reaction to estimate the concentration of John's bst to use. Right: IPA-PCR in increasing concentration of bacteria with protected templates.

Figure 63 .

 63 Figure 63. Test of two new polymerase in the IPA-PCR reaction. A. Primers production assay for the different enzymes at various concentration (0.5, 1 or 2%) to estimate the enzyme concentration to use in a IPA-PCR reaction. B IPA-PCR using the different polymerases for the IPA step with increasing concentration of bacteria expressing active version of the NBI enzyme, and a control with the inactive variant (50(-)).

Figure 64 .

 64 Figure 64. Initial trials to follow IPA-like turnover reactions with bacteria lysate. The templates are protected with a biotin and phosphorothioate. A. Simple molecular program with an aT strand containing a fluorophore at its 5' extremity. Thanks to nucleobase quenching, it is possible to observe when the templates are saturated by the produced trigger. Addition of the exonuclease in the reaction allow to follow when the reaction has consumed all the available dNTPs and give an idea of the reaction speed. The reaction was performed with 100 nM of aT either with no bacteria or with ~250 bact/nL with or without rlys 0.025%. B. Another reporting system based on a random coil reporting probe was used to follow the trigger production of another aT template (see Material & Methods).

Figure 65 .

 65 Figure 65. First sign of an IPA-PCR almost taking place in bacteria lysate. The IPA-PCR was run in various concentration of bacteria with 100nM of aT and 50nM of ppT and T, rlys 0.25% in nIPA buffer.

Figure 66 .

 66 Figure 66. Tests of different conditions to perform IPA-PCR with various concetration of bacteria lysate. The ratio between aT and each ppT or the T strand is always kept constant (2:1). 40nM of aT means that 20nM of each ppT and 20nM of the trigger strand was also added to the IPA reaction. 1. The gamS protein was used in combination with 20nM of aT for various concentration of bacteria on the left with 0.05% of rlys in the nIPA buffer. On the right, various concentration of biot MP templates have been added in 50 bact/nL lysate with 0.25% rlys. 2. Trials to use biot or unbiot templates for IPA-PCR with 50 bact/nL on the left. On the right increasing bacteria concentration for 40nM of biot aT and corresponding amount of biot ppT. 3. Increase of bacteria concentration and IPA incubation time with 50nM of biot aT. All the reactions were performed in the nIPA buffer.

Figure 67 .

 67 Figure 67. Effect of molecular program concentration on the IPA-PCR reaction in bacteria or in vitro. Left: IPA-PCR in nIPA buffer, rslys 0.03% for increasing concentration of templates in presence of bacteria lysate of cells not expressing any enzymes supplemented with 0.3% NBI. Right: Mg 2+ was added to the new IPA buffer and in vitro IPA PCR reactions were run for either 10 or 100nM of aT and corresponding concentrations of other templates with 30 or 45 min of IPA incubation.

Figure 68 .

 68 Figure 68. Degradation of protected biot molecular templates in bacteria lysate. 1µM of Biot or unbiot templates were incubate in different concentration of bacteria lysate with or without streptavidin in nIPA buffer. Then an electrophoresis on native PAGE gel with 40% acrylamide was done to observe the evolution of their quantity. Red boxes indicate primers position.

Figure 69 .

 69 Figure 69. Buffer optimization in bacterial lysate. A. Lysate assay of NBI activity in nIPA (4 mM Mg 2+ ) or in the IPA V0 buffer (6mM Mg 2+ ) B. PCR of either a short piece of the NBI gene of the full-length gene in nIPA or IPAV0 buffers. C. IPA-PCR in various bacteria concentration in IPAV0 buffer. The same experiment in nIPA failed.

Figure 70 .

 70 Figure 70. Following the production of primer p17r3 in increasing concentration of bacteria lysate. Primer p17r3 production was followed using 50nM of the dspreportp17r3F_Q reporting system in IPA V0 buffer. On the right, increase in fluorescence reveal the production of the primer or the degradation of the probe by the lysate. On the left, maximum rate of fluorescence increase.

Figure 71 .

 71 Figure 71. p17r3 primer production by commercial NBI or commercial NBI in presence of bacteria lysate expressing an inactive version of the enzyme. Top: Speed of primer p17r3 production according to commercial NBI concentration. Bottom: Primer p17r3 production in increasing amount of inactive bacteria lysate with 0.5% of NBI. Reactions were run in the IPA V0 buffer.

Figure 72 .

 72 Figure 72. Effect of rlys and streptavidin on primer production according to the buffer. Primer p17 r3 or p25 f2 production was recorded with the corresponding molecular beacon reporting probes in vitro in either IPAV0 or nIPA buffer.

Figure 73 .

 73 Figure 73. IPA-PCR in supernatant of bacteria lysate. The bacteria were incubated with rlys 0.2% at RT for 15 min in a rotating tube (400 rpm) and then centrifuged at 16 000 g for 20 min at 4°C. The supernatant was retrieved and added to the reaction mix at concentration equivalent to 50 to 200 bact/nL. Additive were tried in this experiment and simultaneously in another experiment, with the whole bacteria lysate. On this second experiment no product was obtained.

  increasing incubation time. The results of this experiment presented on Figure 75 are very interesting. They show that using the old templates, it is not possible to obtain PCR products for IPA incubation longer than one hour. This observation suggests that bacteria toxicity is possibly not the only reason for limitation of the IPA-PCR process in high bacteria concentration. In complementary experiment, incubation of the primers with or without the molecular program for various amount of time in vitro all yield to normal PCR amplification.

Figure 74 .

 74 Figure 74. New selection primers and templates PCR and IPA-PCR assay. Left: PCR with various combination of the new envisioned primers. Right: IPA-PCR with new templates encoding the forward primer T7 and the reverse primer T12.

Figure 76 ,

 76 Figure 76, old and new templates were first pre-incubated with bacteria lysate and used, after removal of bacteria lysate activity with a 5 min at 95°, in an in vitro IPA-PCR reaction. The difference between the old and new templates was obvious. Compared to the reaction without incubation the intensity of the band for new templates was greatly reduced.The modifications used for protection of the new templates were not exactly the same as the first ones. Bioteg was added instead of biotin and a spacer was used instead of the addition of some extra bases between the biotin and the template sequence. In order to check if these modifications could explain the low resistance to lysate a new version with the same modification than the initial template were ordered. As the new version also failed in IPA-PCR reaction in bacteria lysate, it was concluded that the sensitivity was sequence dependent.With this in mind, old and new ppT pairs were mixed to perform an IPA-PCR reaction in lysate. Results presented in Figure77show that only one of the two new templates is highly sensitive to bacteria lysate. FwdT7 ppT worked correctly in combination with the old reverse ppT. Other ppT coding for different reverse primer sequence were ordered and unfortunately none of them could work in lysate conditions, despite the fact that they were all performing well in vitro. Possibly the region of the plasmid we are trying to target is rich in sensitive sequences.

Figure 75 .

 75 Figure 75. In vitro IPA-PCR with various IPA incubation times for various combination of templates. Old and new templates have been mixed in in vitro IPA-PCR reaction with increasing IPA incubation time in order to see if the new templates need more times to be produced in quantity allowing PCR reaction.

Figure 76 .

 76 Figure 76. Investigating new templates resistance to bacteria lysate. Left: IPA PCR with increasing concentration of active or inactive bacteria showing weak amplification with the new templates. Right: In vitro IPA-PCR using old or new templates pre-incubated in cell lysate for various amount of time. O = old templates, n= new templates.

Figure 77 .

 77 Figure 77. IPA-PCR with 100 bact/nL for various combination of ppT and either 15 or 30 min of IPA incubation time.

  show that after a 1 min heat shock from 45°C to 59°C all the IPA-PCR reaction work for a 15 min IPA incubation. But, only half of them managed to perform the PCR after 30 min of IPA and none of the tubes exposed to a heat shock was able to perform the PCR after a 45 min incubation. It was therefore decided to run the reaction with an IPA incubation of 15 min for the new templates. For the new templates, only mild 1-or 2min heat shocks yielded to a PCR product. Heat shocks seemed not to be a satisfying solution.Finally, I remembered that bacteria were releasing NBI activity even in the absence of rlys at 45°C. I thought that decreasing or even removing the rlys from the reaction may release fewer toxic components from the bacteria, while delivering enough NBI activity for IPA-PCR to take place. On Figure78left, we can see that indeed without rlys it was possible to perform an IPA-PCR in 100 bact/nL with the new templates, but this required at least 30 min of IPA incubation. Repeating these experiments, some tubes actually succeeded in amplification using new templates at original rlys concentration of 0.25% but all tubes could perform well with 30min of IPA incubation without rlys. It is possible that a part of the variability in results comes from the bacterial batches, age or storage conditions.

Figure 78 .

 78 Figure 78. Effect of rlys on IPA-PCR using new templates. Left: IPA-PCR with 100 bact/nL with 0.25% of rlys or without rlys for 2 different new templates or the old reverse template with the same FwdT7 ppT. Right: IPA-PCR with or without 0.25% of rlys for various concentrations of bacteria with and an IPA incubation time of 15 or 30 min.

Figure 79 .

 79 Figure 79. Effect of 1-or 2-min heat shocks on IPA-PCR reaction in bacteria. Top: 1min Heat shock for IPA-PCR reaction in 100 bact/nL with FwdT7 ppT and the old p17r3 ppT with various IPA incubation time and 0.25% rlys. Bottom: 1-or 2-min heat shock for IPA-PCR reaction in 100 bact/nL with FwdT7 ppT and the new RevT12 ppT for 15 min IPA incubation time at 0.5% rlys.

Figure 80 .

 80 Figure 80. Summary of bacteria lysis for IPA-PCR. Applying 45°C is enough to release NBI from the bacteria, higher temperatures are not increasing the release but use of rlys can help by increasing membrane degradation. Biotinylation of templates and protection with streptavidin allows to prevent the program degradation by exonucleases present in the lysate. Some unknown toxic components can also interfer with the program or the Taq polymerase. Taq polymerase resisting this effect have been found.

Figure 81 .

 81 Figure 81. Inactive mutant amplification in bacteria lysate. Left: IPA-PCR with 30 min incubation IPA and 20nM of v11 trigger with bacteria carrying the WT active or inactive version of NBI. Amplification is observed in the inactive case. Right: Decreasing concentration of the trigger seem to prevent the self-replication of inactive gene (the range indicates IPA incubation time).

Figure 82 .

 82 Figure 82. Effect of v11 (trigger) or ssv11 (sceT) on the primer production in vitro.

Figure 83 .

 83 Figure 83. Comparison of primer production speed using either v11 (T) or ssv11 (sceT) as initiating template with various bacteria concentration.

Figure 84 . 3 . 5

 8435 Figure 84. Comparison of IPA-PCR yield when using either 20nM of v11 (T) or 2nM of ssV11 (sceT) in 100 bact/nL of inactive and active bacteria with various IPA incubation time.

Figure 85 .

 85 Figure 85. Using the source template ssv11 as initiating template for IPA-PCR in active and inactive bacteria. Different concentration of ssv11 were tried in IPA-PCR reaction at 50 or 100 bact/nL for inactive and active bacteria. No amplification is occurring for the inactive bacteria even at high sceT concentration.

Figure 86 .

 86 Figure 86. Primer p17r3 production in inactive bacteria lysate with increasing concentration of commercial NBI. A: Reporting of primer p17r3 production in 100 bact/nL with the dsreportp17r3F_Q probe. B: Primer accumulation after 15 or 30 min of IPA according to NBI %. C: Fitting of the primer production speed with Michaelis-Menten model. The WT bacteria activity correspond to ~0.3% of added commercial NBI.

Figure 87 .

 87 Figure 87. Yield of IPA-PCR in inactive bacteria lysate with increasing amount of active commercial NBI. Top: Gel electrophoresis of IPA-PCR reaction product diluted 5 times. Bottom: Quantification of IPA-PCR reaction product with the TapeStation analyzer from the LBC laboratory for sample diluted 55 times.

Figure 88 .

 88 Figure 88. PCR with selection primers (p17r3 and p25f2) for a range of low primers concentration.

Figure 89 .

 89 Figure 89. Measurements of PCR product quantity by qPCR for various primer concentration. Left: Measured number of gene copies. Right: Amplification yield.

  throughputs have been greatly improved since the beginning in 1993 and now one can find directed evolution methods able to select or screen more than 10 12 variants at each evolution cycle. But despite these great improvements the throughput stays way to small compared to the sequence space of a given protein. I like the comparison done in "Advances in generating functional diversity for directed protein evolution" 172 . If you assume the height of the Mount Everest represents the sequence space of very small 10 amino acid peptides, that is about 10 13 possible amino acid sequences. A screen of one million variants would only represent an impressive uphill climb of 0.86 mm. It is not even possible to adapt it to the size of our protein NBI as the sequence space of 20 604 is too hyper-astronomically large to be computed. These numbers are not even considering the underlying genetic codes as the 20 amino acids can be encoded by 64 codons increasing again the complexity of the sequence space. For this reason, it has become very critical throughout the evolution of the field to pay particular attention to the library design. Smart libraries trying to focus on the most promising part of the sequence space, more likely to yield positive results, replaced the fully randomized library and are now the trend.The goal of an evolution experiment is to move around the fitness landscape either to find the best possible fitness or just to get more information on its structure and how this affects structure-function relationship. The fitness landscape can be presented like on the Figure90. It attributes a fitness value to all possible sequences. The representation of the landscape in 2D

Figure 90 .

 90 Figure 90. Representation of a putative fitness landscape and the different problem arising from its shapes.

Figure 91 . 1 . 1

 9111 Figure 91. The different types of mutations that can be observed.

  techniques or would require repeating an astronomically huge number of cycles. For instance, a mutation from AUG (Met) to UGG (Trp) is very unlikely. The Figure 92 shows the achievable amino acids starting from the glutamic acid encoded by the GAA codon. It has been calculated that in average a single base substitution in a codon can only lead to 5.7 of the 19 possible amino acids changes 192 . The Figure 93 borrowed from "The Mutagenesis Assistant Program" paper 193 showsthe achievable amino acids substitutions for the P450 BM-3 heme domaine using either a nonbiased random mutagenesis method or an epPCR method using the Taq polymerase. This clearly demonstrates the inability of any random mutagenesis technique to access all the sequence space and the particular bias due to the use of a given one.

Figure 92 .

 92 Figure 92. Accessible amino acids starting from the codon GAA according to the allowed number of mutations in that codon. We can see that only 6 amino acids can be reached when only one mutation is introduced in the codon as it is the case in error prone PCR.

Figure 93 .

 93 Figure 93. Statistical analysis of amino acid substitution patterns of P450 BM-3 heme domain generated either by a theoretical non-biased mutagenesis method (on the left) or an epPCR using the Taq polymerase (on the right). Original amino acids sit on the Y axis while the X axis shows the substitution pattern for each amino acid. Substitutions are colored according to their probability to happen, red for the lowest probability and blue for the highest. The values on the right Y-axis represent the fraction of amino acids with different amino acid side-chains. Borrowed from MAP 193 .

the

  Combinatorial Libraries Enhanced by Recombination in Yeast (CLERY) 214 PCR reassembled full expression vectors are self-cloned and recombined in vivo in the host organism. Furthermore, this recombination technique prevents the apparition of new mutations, that are recurrent in vitro, thanks to the natural mutagenesis repair apparatus of the yeast.

Figure 96 .

 96 Figure 96. SDS PAGE gel of purified NBI with (Left) or without (Right) co-expression of ple1.Samp=After sonitcation, Sup= Supernatant aftersonication and centrifuge, Pe= Pellet, FT= Flow Through, W=Wash, E=Elution, LB= bacteria from culture

Figure 97 .

 97 Figure 97. Comparison between purified NBI protein obtained in KRX/ple1 bacteria with or without induction of the ple1 methyltransferase.

  McrCB, McrA and Mrr restriction systems. As explained in the patent, the methylase can be as toxic as the nickase if transformed in an inappropriate strain. Indeed, McrCB, McrA, Mrr systems can cut DNA that is methylated at unusual positions. As pleI is methylating the DNA of the host cell a new way, the SOS response is triggered 220 , and these enzymes start cutting the bacterial genome. This discovery

Figure 98 .

 98 Figure 98. IPA-PCR with 50 bact/nL of KRX/ple1/pNBI containing an unexpected insert. IPA-PCR reaction was run with various concentration of the v11 trigger with 50 bact/n and primers p25r2 and p25f2. After gel electrophoresis, the PCR product is higher than the expected size of 0.6 kb. This experiment shows the apparition of the IS5 intron in the bacteria strain and testify for its absence of effect on the expression level and activity of the enzyme since bacteria lysate can still perform the IPA-PCR reaction.

Giselle

  McCallum cloned the Green Fluorescent Protein (GFP) in the pNBI plasmid either in a fused NBI-GFP protein (pNBI-CemGFP, pNemGFP-NBI), or for segregated expression under the same promoter (pNBI-T7-GFP). The fusion of GFP with NBI was tried on the N-terminal (NBI-CemGFP) or the C-terminal side (NemGFP-NBI). These constructions were made to explore the expression variability by looking at the GFP intensity but also to test the effect of the fused GFP on the protein activity. Expression of a fused (or segregated) NBI-GFP for selection experiment could allow us to control the bacteria encapsulation better because we could visualize the cells more easily. Eventual inactivating effects of the fusion could also be used for a better expression of a non-toxic NBI activable by protease cleavage. Under the fluorescent microscope, we noticed that NBI-T7-GFP and NBI-CemGFP bacteria had strange filamentous shape. The bacteria seemed to not divide anymore after induction, according to their average size and the number of replication cycles expected in the course of the induction.

Figure 99 .

 99 Figure 99. Effects of inducer quantity, induction OD600 and induction time on NBI activity in bacteria. NBI expression in KRX ple1/pNBI bacteria was induced at various starting OD600 (0.4, 0.6 and 1) with various quantity of L-rhamnose (0, 0.02%, 0.04%, 0.1% [recommended]) during 30 min to 3 hours. Evolution of the OD600 for the bacteria induced with the recommended 0.1% L-rhamnose concentration are given next to the corresponding activity values.

Figure 100 .

 100 Figure 100. Microscope observation of T7 express/ple1 bacteria expressing either the wild-type or an inactivated version of the NBI enzyme. Bacteria expressing the active (wild-type) version of the nicking enzyme form long chain of not dividing bacteria

Figure 102 .

 102 Figure 102. Expression level and NBI activity according to induction time and concentration for T7 express cells.

Figure 103 .

 103 Figure 103. Effect of the culture condition and temperature on the bacteria shape and NBI activity. A. NBI-GFP expressing bacteria grown in autoinduction medium (AIM) at 37°C. B. Lysate loopact assay and primer production assay results for bacteria expressing wild-type NBI and grown in various conditions. C. Microscope observation of bacteria expressing NBI, induced with various IPTG concentration and at different temperatures.

Figure 105 .

 105 Figure 105.NemGFP-NBI expression in bacteria. Distribution of pNemGFP-NBI bacteria after induction of NemGFP-NBI expression. A shows size distribution, B shows distribution of mean fluorescence per cell and C shows a lack of correlation between size and mean expression level.

Figure 104 .

 104 Figure 104. Single cell nbi expression level. Bacteria expressing wildtype NBI are encapsulated in droplets in the presence 10% of BugBuster and the fluorescent NBI reporter loopact. After the temperature jump, the bacteria lyse and NBI is released in the droplet. The NBI activity level in the bacteria is monitored via the initial slope of reporter digestion and fluorescence increase. a. typical microscopic image after segmentation of the droplets. b. The droplets tend to move during the timelapse experiment. Tracking allows to reconstruct their spatial trajectories and build the time series of reporter cleavage in each droplet (shown in c). d. Histogram of the initial slopes and gaussian fit with fixed ratio according to the observed λ.

  The first cloning strategy was a classical restriction cloning. Two different restriction sites SalI and BamHI were inserted in the vector just before the His-tag. Using two different enzymes ensure the proper insert orientation in the recombinant plasmids. It is generally recommended not to use enzymes with star activity, which is not the case of SalI and BamHI if used in the NEB 3.1 buffer. Extra bases were added on the extremities next to the recognition/cutting site of the enzyme for efficient digestion. Several protocols were tried, using either the original plasmid vector or a PCR product of the vector. To try to have the maximum quantity of DNA for the transformation, using a PCR product of the vector can allow us to have more DNA, because it is difficult to recover a lot of this very low copy plasmid via miniprep or even midiprep. As supercoiled miniprepped DNA transformed more efficiently in bacteria, we took the step of treating all the obtained PCR product with DpnI restriction enzyme. DpnI is cutting methylated DNA, therefore removing the original template DNA plasmid.Experiment done by Iaroslava Golovkova when she tried to clone her library of Taq polymerase exemplify the issue. Despite decreasing the quantity on initial template in her mutazyme II reaction she kept on having only wild type sequence in her library. After my suggestion of digesting the PCR product by DpnI she recovered libraries with 19 mutations per gene.

4 .

 4 Library characterization by NGS sequencing NGS sequencing Next generation sequencing (NGS) technology give access, in a single instrument run, to thousands to billions of individual reads from a mixture of DNA molecules. First emerging in 2006 and originally principally used for genome-scale sequencing, the number of applications and methods of this technology has increased at an exponential pace 236 . NGS data are used in various research applications like genomics, transcriptomics, single cells analysis, population migration study, cancers study, but also for clinical purposes in personalized medicine applications. Thanks to NGS, curious individuals can even have their genome sequenced and get some information about their ancestors. Because of the rapid evolution of the instruments, chemistries, and techniques, NGS machines are changing very regularly and their associated analysis algorithms and sample preparation protocols follow. It is thus very challenging for researchers or clinicians to keep up with the rhythm. Nowadays, four technologies are mainly used; Illumina, Pacific Biosciences, Iontorrent and Oxford nanopores technologies. The four methods use different mechanisms, leading to different pros and cons. Depending on the purpose of an experiment, one method will be preferred over another. Illumina and Iontorrent systems are both limited in the reads length, while the limitations of fourth generation technologies, such as PacBio and Oxford Nanopore systems, are more related to the relatively high error-rate of the base-calling process. The four technologies are presented in Figure 106. • Illumina: In the Illumina technology, the samples are clonally amplified to create locally DNA clonal clusters (also called polonies) on a chip. A polymerase reaction is then primed on the single stranded DNA cluster. During the polymerization, blocked fluorescent nucleobases are alternatively flowed to the chip, so that only one nucleotide can be added at a time. Fluorescent imaging of the probe allows to detect which fluorophore was integrated in each cluster.

1

  000 $. A minion sequencer can generate approximately 10-20 Gb of data per run and an average read length of ~6 kb. Here also, high error rates of 5-10% are recorded. GridION and PromethION platforms combining several chips are now proposed to increase the throughput.NGS sequencing techniques differ in the way they detect bases, and their dependency, or not, to clonal DNA amplification. They all have their pros and cons according to the targeted applications. Long reads of PacBio or MinION sequencer can be combined with the high accuracy of the numerous reads of Illumina or Iontorrent sequencers to assemble more easily sequenced genomes. Error rates, reads length and throughput of the different machines are classically compared by practitioners for sequencing but other metrics, more difficult to access, such as cost per run, cost per base, run time, sample preparation time and costs, platform error bias (insertion, deletions, homopolymers) should also be considered and may influence the decision236,238 . In our application, using reads with low accuracy is not indicated as all the template DNA have almost the same sequence with few differences, there is no direct mean to find a consensus sequence by combining the reads. It is therefore not possible to detect mutations among sequencer errors. Because of the length of NBI gene (1800 bp) high-accuracy methods require that we split the gene in several parts. This prevents the possibility to access full length information.

Figure 106 .

 106 Figure 106. The four main methods of Next Generation Sequencing in 2018. A: Ilumina, B: IonTorrent, C: Oxford Nanopore Technologies, D: Pacific Biosciences. Adapted from Coming of age: ten years of next-generation sequencing technologies.

Figure 107 .

 107 Figure 107. Mutational bias observed in the sequenced libraries. The different mutations type and their frequency in the different sequenced libraries compared to Mutazyme II kit prediction and the sequenced wild-type. Mutations recorded in the wild-type correspond to mutations introduced by sample handling and sequencing errors.

Figure 108 .

 108 Figure 108. Mutation rated according to initial template quantity.

Figure 109 .

 109 Figure 109. Repartition of the reads for each part of the gene in the different experiments

Figure 110 .

 110 Figure 110. Activity test of the libraries compared to the wild-type NBI. The activity of the libraries and the wild-type (NEB+4) are assessed by the protected loopact probe, directly form the lysate, for 50 bact/nL. In the inset, the initial slope for each sample is presented.

Figure 111 .

 111 Figure 111. Activity assay of 93 picked clones. 93 clones were picked randomly in the library generated from 200 ng of template DNA. After growth and induction, their activity was assayed with a protected loopact probe and normalized according to the optical density. Comparison with recorded inactive mutant activity allowed to detect inactive clones. The wild-type sequence is circled in purple. Red points were selected for further tests.

Figure 112 .

 112 Figure 112. Activity test and primer production rate of 13 picked clones. Activity of 13 clones picked from the Lib 200 was assessed with the protected loopact probe at 50 bact/nL as well as their primer production rate at 200 bact/nL (lower bacteria concentration did not allow to reach the 50 nM plateau in the timescale of the experiment). Variations with respect to the WT sequence are given in the table inset.

90 . 1 .

 901 Deviation from the original chemical distribution are the following (with initial values in parenthesis): Stop codons: 7.39 % (0.00%); Charged: -8.49 % (32.63 %); Neutral: 0.27 % (25.32 %); Aromatic: -2.26 % (11.04 %); Aliphatic: 3.09 % (31.01 %). epPCR will most strongly affect the amino acid content of the NBI gene decreasing charged and aromatic residues and increasing aliphatic residues. The Figure 113 shows the amino acids substitution mapping.III. Droplet generation Different methods of droplet generationMutants present libraries need to be compartmentalized to keep the genotype and phenotype together during the selection experiment. Water-in-oil emulsions allows to create appropriate reaction compartments. Emulsions are made of two immiscible liquids. The first liquid called the disperse phase is scattered in the other liquid called the continuous phase. In

  volume ratio of the two phases are other parameters influencing the size. Droplets produced with such systems are in the µm range 245 .• ElectrosprayRecently,Sharma and al. proposed to use electrospray systems used in mass spectrometry to generate sub-femtoliter droplets. In certain condition, when an electric force is applied on the surface of a liquid at a capillary nozzle outlet, a jet elongates into a fine filament breaking up into fine droplets smaller than the orifice diameter. Using a prototype head with super-fine electrostatic-based inkjet technology plunged in oil, they manage to produce water in oil droplets with average sizes of 1.3 μm and 1.5 μm at a rate of 10 5 per second. The analysis of droplets size dispersion gave a coefficient of variation between 11 and 15%. Parameters affecting droplet size and monodispersity are electrical parameters such as voltage and frequency, orifice diameter size, and fluid properties. The produced droplets were used to express fluorescent proteins using cell-free expression systems 246 .

Figure 114 .

 114 Figure114. Some examples of droplets generation methods. A. Sonicatiion B. Membrane emulsification. In the direct mode, the disperse phase in directly pushed through the membrane. In the premix mode, the two phases are mixed in a first rough emulsion before going through the membrane. C. Self-assembly of fatty acids. Upon pH variation fatty acids assemble and form vesicles. Aggregation of the fatty acid in a first step allow to accumulate fluorescent proteins through crowding effects. D. Electrospray. This system uses the electrospray systems of mass-spectrometry and an inkjet tip to generate droplets by immerging the disperse flow in an oil bath.

  in conditions where flows are completely laminar. The laminar regime offers predictability of the fluids flows. Such flows allow to control the transport of the molecules or particles they contain. Another consequence of the laminar regime is that fluids flowing in contact of each other mix only by diffusion.

  detect small amount of target molecules or cells by reducing the reaction volume thus increasing locally the concentration of the target and allowing amplification of a signal 259 . As analytes are encapsulated in discountinous concentration corresponding to 0 or 1 molecules per droplets, no calibration is required. Another advantage of digitalization is the limitation of contamination effects. In bulks, unspecific amplification of analogs can yield to wrong estimation, separation in droplets prevent coordinated effects of these analogs. Droplets can also be combined with other instruments like FACS to analyze reactions or perform sorting experiments like compounds screening. Addition of electronic and usage of laser beam on microfluidic chips can allow to precisely manipulate the droplets 260 . Controlled merging, mixing, reaction triggering and fluorescence monitoring in droplets are possible 261 . Many directed evolution experiments for enzymes are based on droplets microfluidics. Because of the high capacity of spatiotemporal control for large-scale parallel experimentation it offers and the possible on chip sorting of the positive mutants

Figure 115 .

 115 Figure115. Microfluidic chip fabrication. First the design of the mold is printed on a mask. Photosensitive resin (photoresist) is then poured regularly on a surface. According to the nature of the resin, the mask will cover or exhibit the channels part when put above the resin. Positive or negative resins can be used. When a negative resin is exposed to the UV light crosslink reactions and polymerization happen, rendering the resin harder and difficult to dissolve by developer. The opposite is true for positive resins. 1. The resin is exposed to a U.V. light under the mask so that channel parts react. 2. The remaining part are then removed with chemical agents (developer). This results in a mold with protruding channels parts. 3. Liquid PDMS mixed with its curing agent in then poured on the mold and will take the shape of the channels. 4-5. After baking to make it solid, the obtained carved PDMS is bound to a surface, often a glass surface to create the channels. The mold can be reused many times, but PDMS chips are typically used only once. Tubing are then connected to the inlet and outlet of the chip thanks to holes in the PDMS.

  Co-flowAs shown in Figure116, in the co-flow or coaxial flowing systems, two concentric channels with parallel flows are used to generate droplets. The inner channel or capillary comprise the disperse phase and emerge inside the outer channel comprising the continuous phase 267 . When entering the continuous phase, the stream of disperse phase is subjected to a hydrodynamic force generating the droplets.• T-junctionIn the T-shaped devices, the two phases are flowing in two orthogonal channels and forming droplets at their meeting point. Droplet formation cycle starts by the growth of the droplet at the entry point of the disperse phase into the continuous phase. The pressure gradient, shear forces and interfacial tensions elongates and distort the droplet until the tail of the disperse phase become too thin and eventually breaks267,268 . Several models have been proposed to describe this droplet generation method and can be used to predict the droplets size and generation rate.

Figure 116 .

 116 Figure 116. Several ways of producing droplets in microfluidic chips. A. Co-flow B. Step-emulsification C. Flow-focusing D. T-junction. Adapted from cited papers.

  Several types of oil have been tried to produce emulsions. Silicone oil, mineral oil, mixture of several oils or fluorinated oil have been reported 268 . Among them, fluorinated oils are widely used in droplet-based biological applications. Most organic compounds are insoluble is these oils ensuring that exchanges between compartments are prevented. Another advantage of these oils is their biocompatibility and ability to solubilize gases. They are compatible with cell cultures. Besides, droplet generated with fluorinated oil are the only examples were cells survival and proliferation has been observed 273 . Furthermore, fluorinated oils have higher chemical and thermal stability and can withstand elevated temperature. Their viscosity is also lower than the other oils, reducing fluidic resistance in the microchannels 274 .Surfactants are not only involved in the stabilization of droplet interfaces by surface tension decrease but also in the biocompatibility and molecular exchange between droplets 275 . Surfactants motion, exchange properties with the bulk phase and its response to deformations control the rheological properties of droplets interfaces but also the behavior of the whole emulsion273 . In miniaturized assay, interfacial effects become predominant. Adsorption or denaturation of proteins at the water/oil interface or surfactants interactions with cell membrane need to be avoided. To use droplets as microreactors, stability, exchanges control and biocompatibility must be addressed at the same time. This typically relies on the surfactant and interface properties. Fluorosurfactants derivatives based on perfluoropolyeters (PFPE) with hydrophilic head groups are widely used for fluorinated oil emulsions. Some of them have shown good biocompatibility. Biocompatibility is usually evaluated by protein adsorption on interface, the less the better. Using derivatives of perfluorooctanol (PFO), a surfactant with a short fluorinated tail, it was demonstrated that modification of the head groups influences biocompatibility. Carboxylic groups would increase protein adsorption while short ethylenoxide chain enhance biocompatibility. Currently, the most promising fluorosurfactants are copolymers made of perfluoropolyether and polyethylenoxide. These copolymers reduce interactions with cell membranes and have good biocompatibility273 . But available surfactants formulations are often kept secret. Several surfactants were used during this project according to availability.

Figure 117 .

 117 Figure 117. Different regimes of droplet production. This figure extracted from 267 depict the three usually observable regime in droplet production; squeezing, dripping and jetting.

Figure 118 .

 118 Figure 118. Kim's device A. Filter of the microfluidic device. B. Cross-junction of the device. C. Droplet generation. D. Device design.

Figure 120

 120 Figure 120 Geometry of the device nozzle compared with the inspiring device from Shim and al. On the left, original design of Shim and al. 219 . On the right, first designed device with 10x10 µm nozzle.

Figure 119 .

 119 Figure 119. Different types of filters. A. First filter design. The blocks are all of the same size and the space is directly reduced to ihiuhµm between them. B. Second generation of filters with decreasing size restriction to prevent clogging at the filter beginning. C. Modification of the filter connection to the inlet and following channel to decrease the fluidic resistance and prevent the clogging of the filter at its entrance.

Figure 121 .

 121 Figure121. Evolution of droplet generation device designs. Throughout the project, the design was adapted to be more convenient and answer the problematic of bacteria encapsulation. The device was reduced to be able to pack more devices on a single mold. Filters were adapted to be prevent clogging and finally hydraulic resistances were added to prevent back flows during pressure adjustment and transitory regimes. Another design used spiral channels to position the bacteria regularly in the flow before encapsulation 267 was envisioned but it was not tested because of its important size.

Figure 122 .

 122 Figure 122. Troubled encountered with filters design. Several views or the problems encountered with filter design. On the top, dusts are blocking the flow entry in the channel because of the sudden size reduction of the channel width and height. At the bottom, the last row of plots filters is too small and not properly lithographied.

Figure 123 .

 123 Figure123. The final device design. The final device is a 2-step device. The first layer comprising the nozzle and the filters has a height of 20µm. The second layer with the remaining part of the device has a height of 40µm. Marks are added for the alignment. The device was compacted so that 8 devices fit on 50x60 mm rectangle that can be directly bounded to a microscopy glass slide of 72x56mm. Plots were added to the inlet and outlet to avoid bonding of the PDMS ceiling on the glass. Filters were connected to the inlets with a wide channel to avoid having dusts clogging and to decrease the global resistance of the section. In the main part, channels are 50µm wide.

Figure 124 .

 124 Figure 124. Tridimensional representation of the final device. Filters and nozzle are below the level of the remaining part of the device to better control the droplet size and decrease the device global resistance.

Figure 125 . 3 .

 1253 Figure 125. Anthony and Alex's Device. Taken from 130

Figure 126 .

 126 Figure 126. Analysis of droplets generated by different devices. A. The final device with a step of 20µm and the remaining channels at 40µm. The droplet radius is 13.8±0.4 µm. B.A similar device without step, and a height of 20µm. The droplet radius is 24.4±0.6 µm. C. A device resembling the Millipede device 211 reproduced and modified by Rémi Sieskind with a step at 5µm and the remaining channels at 50µm. The droplet radius is 11.2±2.5 µm.

4 . 4 . 1

 441 Solving issues relative to encapsulation of the selection process Coalescence during PCR in dropletsDuring the PCR amplification step of our selection method, droplets are submitted to elevated temperature and quick temperature changes. These temperature fluctuations can favor the demixing of the emulsion by destabilizing the droplets interfaces, leading to droplet merging. This phenomenon clearly depends on the size of the droplets, with larger droplet more prone to merging during PCR.Surfactants used to stabilize the emulsion interfaces mitigate merging during PCR.Different amounts of surfactants are added in the fluorinated oil, according to the need of the experiment. Some teams reported successful PCR with 2% of EA surfactant (Raindance) in the fluorinated oil, for droplets of 30µm in diameter 268 . In the lab I started working with HFE 7500 fluorinated oil in Japan and then used the European equivalent Novec 7500. Several surfactants were used with these oils upon their availability. Jeffa 1000 provided by Valérie Taly's laboratory was first used in Japan and was later replaced by EA (also lent by Valérie Taly's lab) or by RAN surfactants in Paris. No significant difference was observed between the different surfactants.It is interesting to note that commercially available surfactants compatible with fluorinated oils are quite rare and sometimes difficult to obtain. The EA surfactant for instance can just be bought with the raindance digital PCR machine, the jeffa is a labmade secret recipe and the RAN surfactant is sold only by a small and secretive company. Surfactants represent a commercial interest as they can be made proprietary and can allow or facilitate droplet-based applications that cannot be done without the appropriate one. It is therefore difficult to know the exact composition of the used surfactants and to anticipate its effect. In a general manner a lot of experimental tips for successful droplet PCR are difficult to find in literature and are mainly transmitted by informal discussion between laboratories. That is how I was by advised by Deniz Pékin whom I met in a conference in Bordeaux, to try to decrease the PCR machine slope in order to reduce the shock of heat variation in droplets to decrease the merging.PCR cycles are indeed promoting merging of the droplets. The narrow monodispersity observed in the previous part is generally lost at the end of the PCR lost. Experiments were run to assess the stability of emulsion produced with Novec 7500 and RAN surfactant with various PCR ramp slopes. The results are presented in the Figure128and resume in the Table

4. 2

 2 Capture of the DNA by water oil interface Another way of increasing droplet stability during PCR is to increase the quantity of surfactant. Dany Chauvin from the LBC laboratory advised me to use up to 4% of surfactant in the oil. Increasing the surfactant concentration did not show a clear effect in the first tests. However, when trying to reproduce one of my experiment, Ellie (Giselle McCallum), an intern in the lab, did observed some interesting behaviors. Droplets containing a high quantity of commercial NBI did not yield to gene amplification in IPA-PCR process although plasmid λ was ~30 (Figure 127).

Table 19 .

 19 Figure 127. Measurements of in vitro IPA-PCR gene yield using a fluorescent probe in droplets. A scan of NBI concentration in droplet was realized using a fluorescent reporter to follow the NBI quantity in each droplet. Between the two graphs, some droplets supposed to have high quantity of NBI enzyme but with no amplification at all are filtered out.

Figure 128 .

 128 Figure 128. Observation of droplet size distribution after PCR in droplets with various slope for temperature change.

Figure 129 .

 129 Figure 129. Tests realized to recover DNA from droplets. The many options tested and their conclusion are presented.

Figure 130 .

 130 Figure 130. Observation of biofilm formed by bacteria at the level of the filter. Biofilms form in the filter and extend in the channels. They reduce the flow by increasing the resistance. Bacteria can also attach on the channel walls.

Figure 131 .

 131 Figure131. Encapsulation of bacteria in droplets. Bacteria are encapsulated for IPA PCR. On the left, we can distinguish bacteria in the channel. On the right, the reaction component with rlysozyme lysing agents are mixed with the fluorophore Alexa 647 to check the contributionof both channels. This is in order to ensure the same contribution of both solutions to the droplet that are generated in the output channel (top).

Figure 132 .

 132 Figure 132. Purification and activity assay of the wild-type and inactivated Taq polymerases. Top: SDS PAGE of different fraction of purification of the inactive (left) and wild-type (right) version of the Taq polymerase. Bottom: PCR assay of the two purified enzymes.

Figure 133 .

 133 Figure 133. General scheme of CSR. (1) A repertoire of polymerase genes is cloned and expressed in E. coli. Spheres represent active polymerase molecules. (2) Bacterial cells containing the polymerase and encoding gene are suspended in reaction buffer containing flanking primers and dNTPs and segregated into aqueous compartments. (3) The polymerase enzyme and encoding gene are released from the cell, allowing self-replication to proceed. Poorly active polymerases (white hexagons) fail to replicate their encoding gene. The offspring polymerase genes are released and recloned for another cycle of CSR." (taken from the original paper)

Figure 134 .

 134 Figure134. Screening of expression condition to increase Taq expression. Top: SDS PAGE of heat purified Taq polymerase. Cix correspond to the tested condition with i= 1 or 2 for first or second clone and x= 1: L-rhamnose 0.05%, Induction OD 0.5; 2: : L-rhamnose 0.05%, Induction OD 0.7, 3: Lrhamnose 0.1%, Induction OD 0.5; 4: L-rhamnose 0.1%, Induction OD 0.7; 5: L-rhamnose 0.2%, Induction OD 0.; 6: L-rhamnose 0.2%, Induction OD 0.7

Figure 135 .

 135 Figure 135. Self-replication of wild-type KlenTaq with various bacteria concentration.

Figure 136 .

 136 Figure 136. Different mock libraries

Figure 137 .

 137 Figure 137. Assays to measure proportion of active and inactive clones.

  List of the references comprised in the tables: #1: 47 , #2: 290 , #3: 291 , #4: 292 , #5: 293 , #6: 262 , #7: 294 , #8: 76 , #9: 77 , #10: 46 , #11: 266 , #12: 295 , #13: 296 , #14: 10 , #15: 202 , #16: 297 , #17: 298 , #18: 299 , #19: 300 , #20: 301 , #21: 165 , #22: 302 , #23: 79 , #24: 80 , #25:[START_REF] Skirgaila | Compartmentalization of destabilized enzyme-mRNA-ribosome complexes generated by ribosome display: a novel tool for the directed evolution of enzymes[END_REF]

Figure 138 .

 138 Figure 138. Enrichements values from literature depending on the initial fraction (p). Numbers correspond to the experiments described in the Tables.

Figure 139 .

 139 Figure 139. Theoretical prediction of change in frequency upon one round of selection in a variety of selection dynamics. Left λ = 0.5, Right λ = 2

Figure 140 .

 140 Figure140. SQI scores calculated for the data in Figure138. Lines connect data point originating from the same report and done with a single value of λ (indicated next to the line). The plot legend refers to the reference number. Disks are used for protocol using screening and bulk (nonmonodisperse) emulsions; diamonds, for protocols using screening and microfluidic (monodisperse) emulsion; Stars, for protocols using selections in bulk emulsion. The dotted line at SQI =1 represents the theoretical maximum efficiency. The general trend now indicates that lower initial fractions negatively affect the process, in line with experimental knowledge. Protocols get less efficient when one uses lower initial fractions of active mutant. It is also clear that screening protocols with polydisperse emulsions perform worse than the monodisperse case.

Figure 141 .

 141 Figure 141. ON/OFF mock experiment selection scheme.

Figure 142 .

 142 Figure 142. Difference between active and inactive mutant for self-replication. Effects of coencapsulation.

Figure 143 .

 143 Figure 143. rhqPCR workflow used to determine quantity of active and inactive gene

Figure 144 .

 144 Figure 144. Self-replication of KlenTaq depending on bacteria concentration. First gel: Various concentration of KT+ or KT-bacteria. Second gel: Bulk experiments for mock selections 10% active fraction at various λ. Third gel: Results in bulk or in droplet for 10% active fraction et various λ.

Figure 145 .

 145 Figure 145. Increasing concentration of bacteria in self-PCR reaction.

Figure 146 .

 146 Figure 146. Comparison of theoretical obtainable final active fraction compared to the obtained data according to the model. Baret = Baret et al. definition, lnsh = linear with sharing, lnwosh = linear without sharing, cosh = cut-off (ON/OFF) selection with sharing, cowosh =cut-off (ON/OFF) selection without sharing. With 0.1 or 0.2 as initial fraction.

Figure 147 .

 147 Figure 147. Recovery of the correct wild-type and inactive mutant sequence. A. Colony PCR of 10 clones from each inactive or active gene transformation. The correct band height is 600bp, higher bands indicated the presence of the insert. B. Evaluation of picked clones growth behavior. C. Picked clones activity assessment using the protected loopact probe.

  rhqPCR was used to detect the originally introduced mutation or the wild-type sequence on AD_pNBI+_1, NEB_pNBI+_4, AD_pNBI-_1 and NEB_pNBI-_1. They all amplified at the corresponding time for their corresponding primers. After sequencing of the two genes confirmed the mutation on the inactive gene and the integrity of the active wild-type gene, the clones AD_pNBI-_1 and NEB_pNBI+_4 were chosen as inactive and active clone respectively.The inactive gene has approximately 5% of the active gene activity in the same expression conditions. The fig. shows the increase in activity when more bacteria containing the active mutant are added, contrary to the inactive version.

Figure 148

 148 Figure 148 Verification of negative mutant expression and inactivity. On the left, an SDS PAGE gel with eluted inactive NBI at the right position. On the right, assessment of the mutant inactivity with the loopact probe. The inactive mutant demonstrates a light activity. % refers to the quantity of purified and dialyzed enzyme.

2. 2

 2 Selection efficiency assessment leading to modification of the selection program Several mock experiments have been realized to evaluate the selection efficiency of the PEN CSR. The results of 14 different experiments are represented in Table

Figure 149 .

 149 Figure149. rhqPCR on some picked clone plasmids. AD_pNBI+_1, NEB_pNBI+_4, AD_pNBI-_1 and NEB_pNBI-_1 were tested by rhqPCR for the presence or absence of the inactivating mutation. Active (+) clones amplified before the inactive (-) ones in presence of the corresponding ON primers. In the contrary they amplify after the inactive clones in presence of the OFF primers detecting the inactivating mutation.

Figure 150 .

 150 Figure 150. Activity assessment of the chosen AD-1 and NEB+4 clones. Activity of the selected AD-1 and NEB+4 clones is assessed via the protected loopact probe. Increasing bacteria concentration are tested. There is no activity for the AD-1 clone even when the concentration of bacteria increases.

Figure 151 .

 151 Figure 151. Scheme of mock experiments workflow. Bacteria containing either the active or inactive version of the NBI gene are encapsulated in droplets with the molecular program and rlysozyme lysing agent. The bacteria and rlysozme are introduced via different channel and mixed at the moment of droplet formation. For each experiment, a value of λ as well as a starting fraction of active bacteria was selected. The resulting emulsions undergo the IPA-PCR selection process in a PCR machine. After emulsion breakage, recovered PCR products are analyzed by rh qPCR to measure the final fraction of active genes.

Figure 152 .

 152 Figure 152. Expected outcome of IPA PCR in droplets carrying the active, inactive or coencapsulated active and inactive NBI expressing bacteria. Active version of NBI is able to successfully produce primers from the molecular program while the inactive version cannot. After the IPA, when the PCR starts droplets containing active bacteria have primers and the active gene is amplified whereas nothing happens in droplets containing inactive variants. If active and inactive bacteria are co-encapsulated, primers produced by the active enzyme will also serve to amplify the inactive gene resulting in a shared amplification of both genes (with lower yield of each).

  Re-cloning of selection products was performed many times and led each time to a library composed almost exclusively of empty vectors, with no measurable activity. A second cloning strategy, using Gibson Assembly instead of the classical restriction cloning, was designed by Rémi and Rocio while I finally started writing this thesis. Cloning tests made by Rocio on the wild-type gene seem to indicate a good cloning efficiency with only 1% of empty vectors. But when the selected libraries where transformed a lot of empty vectors appeared again this time maybe because of the insertion of some of the numerous primer dimers formed during the PCR, instead of the correct genes. The cloning needs to be done again with gel purification of the PCR product. It is interesting to note here that the product obtained from the IPA-PCR reaction

3. 3

 3 Next generation sequencing of the selected library NGS for deep mutational scanning Next generation sequencing methods presented in part II are more and more used in combination of directed evolution experiments to access to high-resolution sequence-function relationship mapping. In these experiments, researchers perform selection or screening experiments on a library of mutants and analyse the content of the libraries by sequencing before and after the selection. The comparison of these data allows to determine key characteristics of the protein structure or function. Fowler et al 304 were the first to introduce the method in 2010 and many teams followed. Deep mutational scanning, based on multiple readsof the same sequences with a 10X to 1000X cover to evaluate fitness, is thought to be a powerful tool to better understand how an enzyme's sequence relates to its biological and chemical properties. The comprehensive view of an enzyme functional landscape should help to explore molecular basis of protein function. This approach can be complementary to more traditional

Figure 154 .

 154 Figure 154. Measured relative activity of the library before and after selection compared to the wildtype NBI enzyme after his-tag purification.

  PhD supervisor, Yannick Rondelez and Rocio Espada (Post doc in the laboratory) analyzed the sequencing data. The Figure 155 represents the selection coefficient of amino acids at each position. Black point indicates the original wild-type amino acid. On the color scale, redindicates the strongly rejected mutations while blue denotes the strongly selected ones. Only mutations with sufficient sampling in the libraries, either before or after selection are represented. Note that many amino acids substitutions are not directly accessible from libraries created by ePCR, when the closest codons are more than one mutation away. Substitutions with no -or not enough-data are thus in gray. It can be noticed that, as expected, introduced stop codons are generally red, therefore strongly rejected. A good correlation between the results from neutral and heat shock selection can also be observed (Figure159). Testifying that same inactivating mutations have been discarded in both selections. Some stretches of sequence are globally red and seem to indicate low permissivity regions.

  Figure 155. Selection coefficients of amino acids by position for the neutral and heat shock selection. Black point: WT; red: strongly rejected

Figure 157 .

 157 Figure 157. NBI structure with permissivity. Red: Low permissivity positions (conserved regions), Blue : High permissivity positions (easily mutated) Remarkable position in the catalytic site are highlighted. As expected, most are not permissive. (done with UCSF Chimera)

  Mutation rates, arranged by base substitution, in the libraries before and after selection are presented on the Figure158. The results are similar for synonymous and non-synonymous mutations. They show that mutations are generally eliminated by the selection. This is expected and is in agreement with the observation made by Romero and al.[START_REF] Romero | Dissecting enzyme function with microfluidicbased deep mutational scanning[END_REF] . It reflects that the wildtype gene and/or sequences with low mutation rate are preferentially selected. It is more surprising that some mutations types are completely rejected, even if they were frequent in initial library, especially the AT->GC modifications, and even more dramatically for A->G and T->C mutations. As it does not seem possible that the gene sequence can have any influence on the IPA process and NBI activity hypothesis are targeting issues with biased PCR amplification.

Figure 158 .

 158 Figure 158. Mutation rate observed by mutation types. Yellow: initial library; Brown: Neutral selection, Blue: heat shock selection

Figure 159 .

 159 Figure 159. Permissivity by positions in the neutral selection (NS) versus the heat Shock selection (HS). The permissivity for a position corresponds to the mean of the selection coeficients for each amino acid at this position. If mutations are generally accepted on a position the permissivity is positive, if mutations are generally rejected at a position the permissivity is negative. In theory, several positions should be equally accepted and/or rejected in both selection (red line) and since the heat shock selection is more restrictive, some position accepted in the neutral selection should be rejected in the heat shock selection (points under the red line).

  coverage.Zenphex reporter is an oligonucleotide sequence with wearing a HEX dye quenched by a ZEN quencher (IDT). Its sequence is complementary to one part of NBI gene. It binds on the gene during elongation by Taq polymerase. When the Taq polymerase reached the reporter, it will use its exonuclease activity to digest the reporter. The quenching is removed, and fluorescence increases. Unfortunately, this reporter interacts with the PCR and necessitates an extension of the annealing time: 95°C for 3 min, x times (95°C for 5 sec, 58°C for 1 min30 and 68 °C for 30 sec). The HEX dye being very closed to Evagreen fluorescence we tried to design another dye with different fluorescence but the fluorescence increase was too small to be used in our experiments.Encapsulation of bacteriaFor bacteria encapsulation, according to the experiments, several aqueous solutions were used with always one containing the bacteria at various concentrations and one containing the lysing agents rlysozyme (Merck 27-33 KU/µl) and/or BugBuster® Protein Extraction Reagent (Merck) at indicated concentrations. Encapsulation was done for GFP expressing bacteria for lysis assay, and NBI expressing bacteria for loopact assay and selection experiments.

(

  1x) (or another IPA derived buffer if indicated) with 200 µM of each dNTPs (NEB), 0.1% of Bst 2.0 WarmStart® DNA Polymerase (NEB), different molecular probes and various concentration of the following molecular program templates: cV11r2p (aT), ppT v11top17r3 and V11top25f2 and either ssv11 (sceT) or v11 (T). In one experiment pv11 (pT) was also added. Between 10 and 200 nM of each primer reporting probes, generally 50nM were used (see annex Design of reporting probes). For assay with bacteria lysate, 6µg/mL of Streptavidin (NEB 1mg/mL), and various amounts of bacteria (between 20 and 1000 bact/nL) were added. The mixture was then incubated at 45°C in a CFX96 Real-Time PCR machine (Bio-Rad) for various amount of time. All the oligonucleotides were ordered to Biomers and are listed below. sp18-*C*C*ANGACTCNGGGTAGACTCTCGAG*A*T*A*T*CTCGAGAGTCTACC cv11r2p biotin-*A*A*CCANGACTCNG-CCANGACTCN*G*A*-bioteg A*C*CATGGCACCACCACCA -CCANGACTCN*G*A*-biotin pv11 (biot) biotin -*T*T*T TTCCAAGACUCAGA*T*T* -bioteg

  2.0 WarmStart® DNA Polymerase (NEB 8000U/mL), 1.2% of ttrecJ/140 (laboratory stock), 5 nM of x11 (T), 50 nM of cx11BUP (aT), 30 nM of x11toivBBMN5, 50 nM of streptavidin (NEB), and various concentration of bacteria or 2% of commercial NBI. The turnover using a random coiled reporter, were run in the Smix buffer (Thermopol buffer 1X, NEB 3 buffer 0.5x, synperonic 1% and 2µM of netrospin) added with 3mM of DTT (Sigma-Aldrich), Evagreen dsDNA intercalating dye 1x (Biotium), 50µM of dNTPs (NEB), 200nM Streptavidin (NEB), 2.5% of BSa9000S (NEB, 20mg/mL), 2% of nt.BstNBI (NEB), 0.2% of Bst 2.0 WS (NEB), 0.07% of the lab ttRecJ stock, 100 nM of F6ctocBUP (aT) and 50 nM of the rcF6cBQ-Cy5 reporter. The mixture was then incubated at 45°C in a CFX96 Real-Time PCR machine (Bio-Rad) for various amount of time. All the oligonucleotides were ordered to Biomers and are listed below. -phosp x11toivBBMN5 biotin-AAT TAC TCA GCC AAG ACC TTG GAC TCTG-BMN5 F6ctocBUP biotin-TACCGACUCAC TACCGACTCAC-phosp rcF6cBQ-Cy5_1 TTT TG-DDQII-TAMeC CGA CUC ACA-Cy5 (MeC methylcytosine)

3 .

 3 C*ANGACTCNGGGTAGACTCTCGAG*A*T*A*T*CTCGAGAGTCTACC v11top17r3 CCATGGCACCACCACCA -CCANGACTCNG v11top17r3 biot biotin-*A*C*CATGGCACCACCACCA -CCANGACTCN*G*A*-biotin v11top25f2 A*A*T*TACTTCGCGTTATGCAGGC-CCANGACTCN*G*A*_biotin v11toNSelect_Rev4 biotin*-sp18-*A*A*CGGGTCTTGAGGGGTTTT-CCANGACTCN*G*A*_bioteg v11toNSelect_Rev6 biotin*-sp18-*T*T*TTTGactagtagcggccg-CCANGACTCN*G*A*_bioteg v11toSelectRev_T4 biotin*-sp18-*T*A*ACCCCTTGGGGCCTCTAA-CCANGACTCN*G*A*_bioteg v11toNSelect_Rev2 biotin*-sp18-*a*C*TAGCATAACCCCTTGGGG-CCANGACTCN*G*A*_bioteg Expression of the proteins in bacteria Competency making • Chemocompetent cells (for 16 aliquots of 50µL) 20 mL of MgCl2 at 0.1 M, 20mL of CaCl2 at 0.1 M, 11 mL of CaCl2 at 85 mM mixed with 15% Glycerol, 210 mL of LB medium, 1x 1L erlen, 5 x 50 mL tubes and 16 x 1.5 mL tubes were prepared and autoclaved in advance.

  cells were then aliquoted (50 µL, 100 µL or 200 µL) into 1.5mL tubes chilled in advance and stored at -80°C.• psB3K3_empty/pNBI_videIn order to clone the library or the NBI gene only, without the histidine tag, a new version of the psb3k3 (pNBI_vide) already including the histidine tag and new restriction cloning site was constructed. To do so, a 200 bases DNA strand containing the his tag and new restriction sites (OligoprpNBIvidev3_hismodif) was ordered to IDT and 20 ng was amplify by PCR with 500 nM of primers SpeIprpNBIvide (ttaatACTAGTCAAAAAACCCCTCAAG) and XbaIprpNBIvide (taatctagaTAATACAACTCACTATAGGG) with the Q5 polymerase. The PCR product and pSB3K3 plasmid were then simultaneously digested by 20 U of XbaI (NEB) and 10 U of SpeI (NEB) in the cutsmart buffer (NEB) for 1h30 at 37°C. After gel purification with a Qiagen gel purification kit, ~300 ng of plasmid was dephosphorylated with 3U of Shrimp Alkaline Phosphatase (rSAP, NEB) in the cutsmart buffer. The plasmid was then precipited with ethanol and 50 ng of vector was ligated with 5ng of oligo in T4 DNA ligase buffer with 800 U of the T4DNA ligase (NEB) for 2 hours at room temperature. After an inactivation at 65°C for 20 min, the ligation product was transformed in chemocompetent NEB5α High Efficiency cells (NEB). Colony PCR on the six obtained clones revealed the correct insertion of the oligo, but only pNBIvide_4 had the desired orientation according to sequencing results.Ethanol precipitation: 0.3 M sodium acetate + 2 volume of ethanol 100% at -20°C for at least 20 min, followed by 2-3 min in a tabletop centrifuge, removal of the supernatant and new addition of 2 volumes of ethanol 70%, centrifuge and evaporation 1h under vacuum. was cloned by Ruochi Hsu and Kazuaki Amikura before I started working on the project. They ordered the gene to IDT with some modification described in part II and cloned in the pSB3K3 low copy vector.• p14b-Taq pet14b vector (Novagen) was modified with the following protocol: site with cut plasmid. Restriction assays and sequencing confirmed the insertion of the new site. The Taq polymerase gene was amplified from the PakTaq plasmid (Addgene) with the following primers: 5'CATATGAATTCGGGGATGCT 3' and 5'ACTAGTCTATCACTCCTTGGC 3' to add Nde1 and Spe1 restriction site on the gene. The classical restriction cloning protocol described for pNBI_vide was run to introduce the Taq gene into the modified pet14b vector using SpeI and NdeI restriction enzymes in NEB 3.1 buffer. • p14b-KlenTaq KlenTaq was cloned by Iaroslva Golovskova in a modified version of the pet14b plasmid via Golden Gate (GG) Assembly. Pet14b was mutated to not contain any restriction site needed in the GG Assembly. A codon optimized version of the KlenTaq gene was ordered to GeneArt. Klentaq was then amplified by a PCR using the Q5 polymerase and following primers: KlenTaq_Clon_Fwd TTAGGTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACAT and KlenTaq_Clon_Rev TTAGGTCTCAAGCCGGATCAAGCTT. The vector was also amplified by PCR using the Q5 polymerase and the following primers: Pr_Et14b_Fwd_GG TTAGGTCTCATTTCTAGAGGGAAAC and Pr_Et14b_Rev_GG

  CemGFP, pNemGFP-NBI, pNBI-T7-GFP The fusion or co-expressed version of NBI with GFP were cloned by Giselle MacGallum using NEBuilder® HiFi DNA Assembly Cloning Kit (NEB) with the 5µL of plasmid at 80-100ng/µLor 20-80ng of PCR product and 5µL of sequencing primer at 5µM were mixed together in 1.5mL tube and brought to a GATC collection box. 37°C and 250 rpm overnight. Then the preculture was transferred in 200 mL of LB medium with same concentration of antibiotic until the OD600tub value of the medium reached 0.5 at 600 nm. Production of the T7 polymerase being inducible by rhamnose in the KRX strain, expression of protein was then induced with 0.1% of L-rhamnose (Sigma) at 37°C for 3.5 hours or other specified time and temperature. Bacteria were then centrifuged 2 min at 9000 rpm and pellets were frozen at -80°C until use. Pellets of bacteria were resuspended in 2 mL of Sonication Buffer per 100 mg of pellet (40mM Tris-HCl pH 7.6, 300mM NaCl, 10mM Imidazole pH 8, 1mM DTT, 0.05% Triton). Bacteria were sonicated 2 times during 1 min with pauses of 1 min (pulse-on 1'', pulse-off 1'', amplitude 30%) with a Brandson sonifier 250. The amplitude was adapted for smaller volume. After a centrifugation of 10' at 10000 rpm supernatant was put in contact with washed (2x milliQ, 2x Sonication buffer) Ni-NTA beads (Clontech) and incubated with mild rotation for ~1 hour at 10°C, using a ratio of 1 mL of Ni-NTA beads for 20mL of suspended bacteria. Then purification of the protein was performed by gravity. Briefly, bacterial lysate incubated with the beads was transferred in a plastic 2mL column (Clontech) and rinsed with 10 mL of Washing Buffer 1 (40 mM Tris-HCl pH 7.6, 300 mM NaCl, 40 mM Imidazole pH 8, 1 mM DTT, 0.05% Triton) and 10 mL of Washing Buffer 2 (40 mM Tris-HCl pH 7.6, 300 mM NaCl, 40 mM Imidazole pH 8, 1mM DTT). Elution was realized with 5mL of Elution Buffer (40mM Tris-HCl pH 7.6, 300 mM NaCl, 300 mM Imidazole pH 8, 1 mM DTT). Fractions of 500uL were collected. A small volume of solution was conserved at each step for SDS-PAGE. The same protocol was used for NBI nickases and Taq polymerases. Buffers were adjusted during the experiment, with replacement of the Washing Buffer 1 by Sonication buffer, and reduction of Imidazole concentration to 20mM in Washing Buffer 2.

  Storage buffer 1x: gamS: 50 mM Tris HCl pH 7.5 (Sigma-Aldrich), 100 mM NaCl (Sigma), 1 mM DTT, 1 mM EDTA, 2% DMSO. NBI: 10 mM Tris HCl pH 7.5 (Sigma-Aldrich), 50 mM KCl (Sigma-Aldrich) Taq: 10 mM Tris HCl pH 7.5 (Sigma-Aldrich), 100 mM KCl (Sigma-Aldrich)

  using OD600 measurement. The ratio of active over inactive bacteria was 1:10. The premix was then injected in a a microfluidic device under pressure control to generate droplets of ~24μm of diameters, with bacteria at indicated λ, in fluorinated oil (Novec 7500 (Sigma-Aldrich), 2% EA (Raindance)). The size of the droplets is controlled focusing step junction at the nozzle. The collected droplets were then transferred in a PCR tube, and the PCR was initiated.

Figure 161 .

 161 Figure 161. Time delay for different concentration of primers detection by either 50nM of the v1 or v2 of double strand primer reporting system with or without Bst 2.0 WS polymerase.

Figure 162 .

 162 Figure 162. Loopp17 probes reactivity assay. 50 to 200nM of primer p17 triggering the loopp17 probes have been mixed with 50nM of probes in the IPAV0 buffer with (pink curves) or without (blue curves) the bst 2.0 w.s polymerase.

Figure 163 .

 163 Figure 163. Isolation of GFP expressing bacteria in droplet. Droplets with 0, 1, 2 or 3 bacteria have been counted in 3 different snapshots. The distribution of cells in droplets follows a poisson distribution: 𝑝(𝑥) = µ 𝑥 . 𝑒 -µ /𝑥! with µ =0.70 is the average cells per droplets.

Figure 164 .

 164 Figure 164. rh-qPCR principle.

  . Two generation of rhprimers finishing with different 3' extremities exist. The first and second generation (GEN1 and GEN2) architecture is shown in the first panel of the Figure 164. They both comprise the final functional primer sequence and different 3' blocking extremity. The first one can be written as rDDDDMx and the second as rDxxDM with r being an RNA base, D a matching DNA base, x a C3 spacer and M a mismatch DNA base. Inserting a mismatched base near the end of the blocked primers ensures maximum effectiveness of end block. The first one is used for standard genotyping applications and for multiplexed amplification and requires low levels of RNase H2 enzyme. The second one is most appropriate for rare-allele detection or for applications where extremely high fidelity of template amplification is desired but requires up to 100 times more RNase H2 than the first generation.

Figure 165 .

 165 Figure 165. Calibration of rnaseH2 quantity. Left: Amplification of KT+ gene with either ON or OFF rh primers, Right: Amplification of KT-gene with either ON or OFF rh primers. Dot lines represent the PCR with classical primers. Increasing rnaseH2 concentration are indicated by darker color lines.

Figure 166 .

 166 Figure 166. Calibrated of rnaseH2 quantity. Rh ON_V2 primer detect the KT+ gene while rh OFF_V2 primer detect the KT-gene. Indicated values next to KT+ or KT-are the quantity of DNA in ng. Left: 0.3mU of RNAse H2 was used in 10µL reactions. Right: 0.2mU of RNAse H2 was used in 10µL reactions

  Figure 167. Principle of loop-mediated isothermal amplification (LAMP) method. (a) Primer design of the LAMP reaction. (b) Starting structure producing step. Borrowed from Tomita et al..

Figure 168 .

 168 Figure 168. Left: LAMP amplification of NBI gene using L#23 primer group at various temperature. Right: LAMP amplification of NBI gene with L#2 primer group and a combination of F primers of L#2 and B primers of L#104 for a hypothetical full length amplification. As well as the products digested by EcoRI.

  RPA is a new isothermal amplification method using recombinase, polymerase and SSB protein. The currently commercialized product can amplify DNA strand of up to 500pb. The company TwistDX that sale the RPA reactions component sent us some new types of reactions that could amplify up to 2000pb. S reactions for 500 to 1000 pb and T reactions for 1000 to 2000pb. Here we tried to amplify the GFP gene with 4 sets of primers amplifying 750, 1009, 1500 or 2000 pb on pGFPS1 plasmid.

Figure 1 .

 1 Figure 1. RPA amplification followed in Real Time PCR machine. Reaction was stopped after 1 hour for S reaction for amplicon of 750 and 1009 pb.

Figure 2 .Figure 170 .

 2170 Figure 2. RPA amplification on gel

FIGURE 8 .

 8 FIGURE 1. THE VARIOUS ROLES OF PROTEINS IN BOLD WITH SOME EXAMPLES TO ILLUSTRATE THEM. ........................................... FIGURE 2. THE 20 COMMON AMINO ACIDS AND THEIR PROPERTIES. ..................................................................................... FIGURE 3. THE DIFFERENT LEVELS OF STRUCTURE OF PROTEINS. ........................................................................................... FIGURE 4. PROTEIN-FOLDING ENERGY LANDSCAPE. .......................................................................................................... FIGURE 5. ENZYMATIC CATALYSIS. ................................................................................................................................. FIGURE 6. BEGINNING OF PROTEIN ENGINEERING TIMELINE. ............................................................................................... FIGURE 7. DE NOVO DESIGN OF LIGAND BIDING PROTEIN.................................................................................................... FIGURE 8. MIMICKING NATURAL SELECTION VIA DIRECTED EVOLUTION.................................................................................. FIGURE 9. EVOLUTION OF THE NUMBER OF PUBLICATIONS ON PROTEIN ENGINEERING AND DIRECTED EVOLUTION.. ....................... FIGURE 10. SCREENING METHODS IN DIRECTED EVOLUTION.. .............................................................................................. FIGURE 11. SELECTION METHODS FOR DIRECTED EVOLUTION.. ............................................................................................ FIGURE 12. DIRECTED EVOLUTION METHODS EFFICIENCY ASSESSMENT. ............................................................................... FIGURE 13. HAMILTONIAN PATH PROBLEM ENCODED WITH DNA BY ADLEMAN. .................................................................... FIGURE 14. PARALLEL BETWEEN SILICON-BASED COMPUTERS AND BIOLOGICAL CHEMICAL REACTION NETWORKS. DNA CAN BE SEEN AS A DATA STORAGE DEVICE ENCODING A MOLECULAR PROGRAM IN ITS SEQUENCE.. .......................................................... FIGURE 15. STRUCTURE OF THE DNA. ........................................................................................................................... FIGURE 16. DNA ORIGAMI. ......................................................................................................................................... FIGURE 17. DNA-ONLY MOLECULAR COMPUTING SYSTEMS. ............................................................................................... FIGURE 18. MOLECULAR PROGRAMING HYBRID SYSTEMS INSPIRED FROM BIOLOGY. ................................................................ FIGURE 19. OLIGO STRAND PRODUCTION IN THE PEN DNA TOOLBOX ................................................................................. FIGURE 20. DIFFERENT MODULES OF PEN DNA TOOLBOX. ............................................................................................... FIGURE 21. COMPUTER ASSISTED DESIGN OF PEN DNA TOOLBOX NETWORKS.. .................................................................... FIGURE 22. CHEMICAL MODIFICATIONS OF TEMPLATE STRANDS.. ........................................................................................ FIGURE 23. DIFFERENT FLUORESCENCE REPORTING STRATEGIES FOR PEN DNA TOOLBOX REACTIONS MONITORING. .................... FIGURE 24. LINKING ENZYME ACTIVITY TO GENE REPLICATION. ............................................................................................ FIGURE 25. SCHEME OF SOME POSSIBLE WAYS TO ADAPT MOLECULAR PROGRAMS SO THEY CAN BE TRIGGERED BY NON-DNA DEPENDANT ENZYMES. ....................................................................................................................................... FIGURE 26. POSSIBLE SELECTION FUNCTIONS. .................................................................................................................. FIGURE 27. PRESENTATION OF THE NICKING ENZYME NT.BSTNBI. ....................................................................................... FIGURE 28. STRUCTURAL REPRESENTATION OF NBI.. ........................................................................................................ FIGURE 29. NICKING-BASED MOLECULAR BIOLOGY APPLICATIONS. EXPAR ALLOWS EXPONENTIAL AMPLIFICATION OF A TRIGGER STRAND.. ........................................................................................................................................................ FIGURE 30. DIFFERENT METHODS TO ENGINEER NEW NICKING ENZYMES. .............................................................................. FIGURE 31. WORKFLOW OF THE DIRECTED EVOLUTION CYCLE USING MOLECULAR PROGRAMS (PEN CSR) ................................ FIGURE 32. DIFFERENT PARTS OF THE MOLECULAR PROGRAM USED FOR SELECTION OF NICKASE ACTIVITY. ................................. FIGURE 33. INTERACTIONS BETWEEN THE DIFFERENT MODULES FOR THE IPA. ..................................................................... FIGURE 34. SHAPES OF PRIMER PRODUCTION CURVES DEPENDING ON THE NETWORK TOPOLOGY AND THE CONCENTRATION OF THE DIFFERENT COMPONENTS. ................................................................................................................................ FIGURE 35. PREDICTION OF PRIMERS PRODUCTION BY MOLECULAR PROGRAM WITH VARIOUS DESIGN, AS A FUNCTION OF NBI ACTIVTY. ................................................................................................................................................................... FIGURE 36. ESTIMATION OF PRIMERS PRODUCTION RATE VIA DNTPS CONSUMPTION. ...........................................................

FIGURE 37 .

 37 FIGURE 37. PRIMER PRODUCTION RATES IN NIPA BUFFER. ............................................................................................... FIGURE 38. RECORDING OF PRIMERS PRODUCTION SPEED IN VITRO ACCORDING TO NBI CONCENTRATION USING A DOUBLE STRANDED MOLECULAR PROBE.. ....................................................................................................................................... FIGURE 39. MEASUREMENTS OF PRIMERS PRODUCTION BY DSP17R3 PROBES IN IPAV0 BUFFER ACCORDING TO V11 TRIGGER CONCENTRATION.. .......................................................................................................................................... FIGURE 40. EFFECTS OF PSEUDOTEMPLATE AND TRIGGERING MECHANISM ON PRIMER PRODUCTION. ....................................... FIGURE 41. PCR OF NBI GENE WITH TAQ IN THERMOPOL BUFFER + 20 MM NACL WITH VARIOUS CONCENTRATION OF ADDITIONAL MG 2+ . .......................................................................................................................................................... FIGURE 42. THE EFFECT OF MAGNESIUM CONCENTRATION ON ISOTHERMAL PRODUCTION OF OLIGONUCLEOTIDES THROUGH EXPAR.. ................................................................................................................................................................... FIGURE 43. PCR OF FULL LENGTH NBI GENE WITH TAQ FOR VARIOUS ANNEALING TEMPERATURES AND VARIOUS FORWARD PRIMERS. ................................................................................................................................................................... FIGURE 44. LINKING THE NBI ACTIVITY TO THE AMPLIFICATION OF ITS GENE. ....................................................................... FIGURE 45. IPA-PCR OF THE 780 FIRST BASES OF THE GENE IN VITRO WITH VARIOUS NBI CONCENTRATION BEFORE AND AFTER OPTIMIZATION.. ............................................................................................................................................. FIGURE 46. SCHEME OF THE NBI CONCENTRATION DROPLET-SCANNING EXPERIMENT............................................................ FIGURE 47. DROPLET SCANNING OF NBI CONCENTRATION FOR IN VITRO IPA-PCR BY GISELLE MACCALLUM.. ........................... FIGURE 48. SHAPE OF THE SELECTION FUNCTION IN VITRO IN NIPA BUFFER. ........................................................................ FIGURE 49. OBSERVATION OF GFP CARRYING BACTERIA LYSIS IN DROPLETS UNDER THE FLUORESCENT MICROSCOPE.. .................. FIGURE 50. SURVIVING (INTACT) BACTERIA AS A FUNCTION OF TIME IN ONE MICROSCOPIC FRAME, WITH (TOP) OR WITHOUT (BOTTOM) THE LYSING AGENT BUGBUSTER AT 10%.R. ......................................................................................................... FIGURE 51. DEGRADATION OF PROTECTED SPECIFIC OR UNSPECIFIC LOOPACT PROBE BY BACTERIA LYSATE WITH OR WITHOUT LYSING AGENTS. ....................................................................................................................................................... FIGURE 52. EFFECT OF RLYSOZYME AND BUGBUSTER ON THE IN VITRO IPA-PCR REACTION WITH 1% COMMERCIAL NBI.. ........... FIGURE 53. EFFECT OF RLYSOZYME AND BUGBUSTER IN NBI ACTIVITY RELEASE (MEASURED BY LOOPACT DIGESTION RATE) ...........FIGURE 54. EFFECT OF HEAT SHOCKS IN BACTERIA LYSIS AND NBI ACTIVITY RELEASE.. ............................................................ FIGURE 55. NBI ACTIVITY RELEASE FROM BACTERIA ACCORDING TO RLYSOZYME CONCENTRATION, TEMPERATURE AND PRE-INCUBATION AT ROOM-TEMPERATURE. .............................................................................................................. FIGURE 56. ACTIVITY ASSAY OF WASH SUPERNATANT. ..................................................................................................... FIGURE 57. PROTECTED LOOPACT ACTIVITY ASSAY IN VARIOUS SOLUTIONS. . ........................................................................ FIGURE 58. PCR ON PNBI PLASMID FROM BACTERIA WITH THE HS TAQ IN THE NIPA BUFFER. ................................................ FIGURE 59. GEL ELECTROPHORESIS OF PCR ON PNBI PLASMID CONTAINED IN VARIOUS CONCENTRATION OF BACTERIA WITH DIFFERENT COMMERCIALLY AVAILABLE TAQ POLYMERASES IN THEIR RESPECTIVE BUFFER. ............................................................. FIGURE 60. INCREASING MG CONCENTRATION IN A PCR RUN WITH ONETAQ POLYMERASE. ................................................... FIGURE 61. TEST OF BST 3.0 AND VENT(EXO-) IN THE IPA-PCR REACTION AND VARIOUS BUFFER OPTIMIZATION TESTS. .............. FIGURE 62. TEST OF THE BST EVOLVED BY JOHN MILLIGAN FROM THE ELLINGTON LAB IN A TURNOVER AND IPA-PCR REACTION WITH BACTERIA. ..................................................................................................................................................... FIGURE 63. TEST OF TWO NEW POLYMERASE IN THE IPA-PCR REACTION.. .......................................................................... FIGURE 64. INITIAL TRIALS TO FOLLOW IPA-LIKE TURNOVER REACTIONS WITH BACTERIA LYSATE.. ............................................ FIGURE 65. FIRST SIGN OF AN IPA-PCR ALMOST TAKING PLACE IN BACTERIA LYSATE. ............................................................ FIGURE 66. TESTS OF DIFFERENT CONDITIONS TO PERFORM IPA-PCR WITH VARIOUS CONCETRATION OF BACTERIA LYSATE.. ......... FIGURE 67. EFFECT OF MOLECULAR PROGRAM CONCENTRATION ON THE IPA-PCR REACTION IN BACTERIA OR IN VITRO. .............. FIGURE 68. DEGRADATION OF PROTECTED BIOT MOLECULAR TEMPLATES IN BACTERIA LYSATE. ................................................ FIGURE 69. BUFFER OPTIMIZATION IN BACTERIAL LYSATE. ................................................................................................ FIGURE 70. FOLLOWING THE PRODUCTION OF PRIMER P17R3 IN INCREASING CONCENTRATION OF BACTERIA LYSATE.. ................ FIGURE 71. P17R3 PRIMER PRODUCTION BY COMMERCIAL NBI OR COMMERCIAL NBI IN PRESENCE OF BACTERIA LYSATE EXPRESSING AN INACTIVE VERSION OF THE ENZYME.. .............................................................................................................. FIGURE 72. EFFECT OF RLYS AND STREPTAVIDIN ON PRIMER PRODUCTION ACCORDING TO THE BUFFER.. ................................... FIGURE 73. IPA-PCR IN SUPERNATANT OF BACTERIA LYSATE. ........................................................................................... FIGURE 74. NEW SELECTION PRIMERS AND TEMPLATES PCR AND IPA-PCR ASSAY. .............................................................. FIGURE 75. IN VITRO IPA-PCR WITH VARIOUS IPA INCUBATION TIMES FOR VARIOUS COMBINATION OF TEMPLATES. . ................. FIGURE 76. INVESTIGATING NEW TEMPLATES RESISTANCE TO BACTERIA LYSATE. ................................................................... FIGURE 77. IPA-PCR WITH 100 BACT/NL FOR VARIOUS COMBINATION OF PPT AND EITHER 15 OR 30 MIN OF IPA INCUBATION TIME. ................................................................................................................................................................... FIGURE 78. EFFECT OF RLYS ON IPA-PCR USING NEW TEMPLATES. .................................................................................... FIGURE 79. EFFECT OF 1-OR 2-MIN HEAT SHOCKS ON IPA-PCR REACTION IN BACTERIA. ....................................................... FIGURE 80. SUMMARY OF BACTERIA LYSIS FOR IPA-PCR. ................................................................................................

FIGURE 54 .

 54 FIGURE 37. PRIMER PRODUCTION RATES IN NIPA BUFFER. ............................................................................................... FIGURE 38. RECORDING OF PRIMERS PRODUCTION SPEED IN VITRO ACCORDING TO NBI CONCENTRATION USING A DOUBLE STRANDED MOLECULAR PROBE.. ....................................................................................................................................... FIGURE 39. MEASUREMENTS OF PRIMERS PRODUCTION BY DSP17R3 PROBES IN IPAV0 BUFFER ACCORDING TO V11 TRIGGER CONCENTRATION.. .......................................................................................................................................... FIGURE 40. EFFECTS OF PSEUDOTEMPLATE AND TRIGGERING MECHANISM ON PRIMER PRODUCTION. ....................................... FIGURE 41. PCR OF NBI GENE WITH TAQ IN THERMOPOL BUFFER + 20 MM NACL WITH VARIOUS CONCENTRATION OF ADDITIONAL MG 2+ . .......................................................................................................................................................... FIGURE 42. THE EFFECT OF MAGNESIUM CONCENTRATION ON ISOTHERMAL PRODUCTION OF OLIGONUCLEOTIDES THROUGH EXPAR.. ................................................................................................................................................................... FIGURE 43. PCR OF FULL LENGTH NBI GENE WITH TAQ FOR VARIOUS ANNEALING TEMPERATURES AND VARIOUS FORWARD PRIMERS. ................................................................................................................................................................... FIGURE 44. LINKING THE NBI ACTIVITY TO THE AMPLIFICATION OF ITS GENE. ....................................................................... FIGURE 45. IPA-PCR OF THE 780 FIRST BASES OF THE GENE IN VITRO WITH VARIOUS NBI CONCENTRATION BEFORE AND AFTER OPTIMIZATION.. ............................................................................................................................................. FIGURE 46. SCHEME OF THE NBI CONCENTRATION DROPLET-SCANNING EXPERIMENT............................................................ FIGURE 47. DROPLET SCANNING OF NBI CONCENTRATION FOR IN VITRO IPA-PCR BY GISELLE MACCALLUM.. ........................... FIGURE 48. SHAPE OF THE SELECTION FUNCTION IN VITRO IN NIPA BUFFER. ........................................................................ FIGURE 49. OBSERVATION OF GFP CARRYING BACTERIA LYSIS IN DROPLETS UNDER THE FLUORESCENT MICROSCOPE.. .................. FIGURE 50. SURVIVING (INTACT) BACTERIA AS A FUNCTION OF TIME IN ONE MICROSCOPIC FRAME, WITH (TOP) OR WITHOUT (BOTTOM) THE LYSING AGENT BUGBUSTER AT 10%.R. ......................................................................................................... FIGURE 51. DEGRADATION OF PROTECTED SPECIFIC OR UNSPECIFIC LOOPACT PROBE BY BACTERIA LYSATE WITH OR WITHOUT LYSING AGENTS. ....................................................................................................................................................... FIGURE 52. EFFECT OF RLYSOZYME AND BUGBUSTER ON THE IN VITRO IPA-PCR REACTION WITH 1% COMMERCIAL NBI.. ........... FIGURE 53. EFFECT OF RLYSOZYME AND BUGBUSTER IN NBI ACTIVITY RELEASE (MEASURED BY LOOPACT DIGESTION RATE) ...........FIGURE 54. EFFECT OF HEAT SHOCKS IN BACTERIA LYSIS AND NBI ACTIVITY RELEASE.. ............................................................ FIGURE 55. NBI ACTIVITY RELEASE FROM BACTERIA ACCORDING TO RLYSOZYME CONCENTRATION, TEMPERATURE AND PRE-INCUBATION AT ROOM-TEMPERATURE. .............................................................................................................. FIGURE 56. ACTIVITY ASSAY OF WASH SUPERNATANT. ..................................................................................................... FIGURE 57. PROTECTED LOOPACT ACTIVITY ASSAY IN VARIOUS SOLUTIONS. . ........................................................................ FIGURE 58. PCR ON PNBI PLASMID FROM BACTERIA WITH THE HS TAQ IN THE NIPA BUFFER. ................................................ FIGURE 59. GEL ELECTROPHORESIS OF PCR ON PNBI PLASMID CONTAINED IN VARIOUS CONCENTRATION OF BACTERIA WITH DIFFERENT COMMERCIALLY AVAILABLE TAQ POLYMERASES IN THEIR RESPECTIVE BUFFER. ............................................................. FIGURE 60. INCREASING MG CONCENTRATION IN A PCR RUN WITH ONETAQ POLYMERASE. ................................................... FIGURE 61. TEST OF BST 3.0 AND VENT(EXO-) IN THE IPA-PCR REACTION AND VARIOUS BUFFER OPTIMIZATION TESTS. .............. FIGURE 62. TEST OF THE BST EVOLVED BY JOHN MILLIGAN FROM THE ELLINGTON LAB IN A TURNOVER AND IPA-PCR REACTION WITH BACTERIA. ..................................................................................................................................................... FIGURE 63. TEST OF TWO NEW POLYMERASE IN THE IPA-PCR REACTION.. .......................................................................... FIGURE 64. INITIAL TRIALS TO FOLLOW IPA-LIKE TURNOVER REACTIONS WITH BACTERIA LYSATE.. ............................................ FIGURE 65. FIRST SIGN OF AN IPA-PCR ALMOST TAKING PLACE IN BACTERIA LYSATE. ............................................................ FIGURE 66. TESTS OF DIFFERENT CONDITIONS TO PERFORM IPA-PCR WITH VARIOUS CONCETRATION OF BACTERIA LYSATE.. ......... FIGURE 67. EFFECT OF MOLECULAR PROGRAM CONCENTRATION ON THE IPA-PCR REACTION IN BACTERIA OR IN VITRO. .............. FIGURE 68. DEGRADATION OF PROTECTED BIOT MOLECULAR TEMPLATES IN BACTERIA LYSATE. ................................................ FIGURE 69. BUFFER OPTIMIZATION IN BACTERIAL LYSATE. ................................................................................................ FIGURE 70. FOLLOWING THE PRODUCTION OF PRIMER P17R3 IN INCREASING CONCENTRATION OF BACTERIA LYSATE.. ................ FIGURE 71. P17R3 PRIMER PRODUCTION BY COMMERCIAL NBI OR COMMERCIAL NBI IN PRESENCE OF BACTERIA LYSATE EXPRESSING AN INACTIVE VERSION OF THE ENZYME.. .............................................................................................................. FIGURE 72. EFFECT OF RLYS AND STREPTAVIDIN ON PRIMER PRODUCTION ACCORDING TO THE BUFFER.. ................................... FIGURE 73. IPA-PCR IN SUPERNATANT OF BACTERIA LYSATE. ........................................................................................... FIGURE 74. NEW SELECTION PRIMERS AND TEMPLATES PCR AND IPA-PCR ASSAY. .............................................................. FIGURE 75. IN VITRO IPA-PCR WITH VARIOUS IPA INCUBATION TIMES FOR VARIOUS COMBINATION OF TEMPLATES. . ................. FIGURE 76. INVESTIGATING NEW TEMPLATES RESISTANCE TO BACTERIA LYSATE. ................................................................... FIGURE 77. IPA-PCR WITH 100 BACT/NL FOR VARIOUS COMBINATION OF PPT AND EITHER 15 OR 30 MIN OF IPA INCUBATION TIME. ................................................................................................................................................................... FIGURE 78. EFFECT OF RLYS ON IPA-PCR USING NEW TEMPLATES. .................................................................................... FIGURE 79. EFFECT OF 1-OR 2-MIN HEAT SHOCKS ON IPA-PCR REACTION IN BACTERIA. ....................................................... FIGURE 80. SUMMARY OF BACTERIA LYSIS FOR IPA-PCR. ................................................................................................

FIGURE 81 .

 81 FIGURE 81. INACTIVE MUTANT AMPLIFICATION IN BACTERIA LYSATE.. ................................................................................. FIGURE 82. EFFECT OF V11 (TRIGGER) OR SSV11 (SCET) ON THE PRIMER PRODUCTION IN VITRO. ............................................ FIGURE 83. COMPARISON OF PRIMER PRODUCTION SPEED USING EITHER V11 (T) OR SSV11 (SCET) AS INITIATING TEMPLATE WITH VARIOUS BACTERIA CONCENTRATION. ................................................................................................................. FIGURE 84. COMPARISON OF IPA-PCR YIELD WHEN USING EITHER 20NM OF V11 (T) OR 2NM OF SSV11 (SCET) IN 100 BACT/NL OF INACTIVE AND ACTIVE BACTERIA WITH VARIOUS IPA INCUBATION TIME. ..................................................................... FIGURE 85. USING THE SOURCE TEMPLATE SSV11 AS INITIATING TEMPLATE FOR IPA-PCR IN ACTIVE AND INACTIVE BACTERIA. ..... FIGURE 86. PRIMER P17R3 PRODUCTION IN INACTIVE BACTERIA LYSATE WITH INCREASING CONCENTRATION OF COMMERCIAL NBI.158 FIGURE 87. YIELD OF IPA-PCR IN INACTIVE BACTERIA LYSATE WITH INCREASING AMOUNT OF ACTIVE COMMERCIAL NBI. ............. FIGURE 88. PCR WITH SELECTION PRIMERS (P17R3 AND P25F2) FOR A RANGE OF LOW PRIMERS CONCENTRATION. ................... FIGURE 89. MEASUREMENTS OF PCR PRODUCT QUANTITY BY QPCR FOR VARIOUS PRIMER CONCENTRATION. ........................... FIGURE 90. REPRESENTATION OF A PUTATIVE FITNESS LANDSCAPE AND THE DIFFERENT PROBLEM ARISING FROM ITS SHAPES. ........ FIGURE 91. THE DIFFERENT TYPES OF MUTATIONS THAT CAN BE OBSERVED. ......................................................................... FIGURE 92. ACCESSIBLE AMINO ACIDS STARTING FROM THE CODON GAA ACCORDING TO THE ALLOWED NUMBER OF MUTATIONS IN THAT CODON. ................................................................................................................................................ FIGURE 93. STATISTICAL ANALYSIS OF AMINO ACID SUBSTITUTION PATTERNS OF P450 BM-3 HEME DOMAIN GENERATED EITHER BY A THEORETICAL NON-BIASED MUTAGENESIS METHOD OR AN EPPCR USING THE TAQ POLYMERASE.. .................................. FIGURE 94. TOOLS FOR LIBRARY CREATION. ................................................................................................................... FIGURE 95. WHAT IS THE BEST LIBRARY GENERATION METHODS ACCORDING TO YOUR GOALS, MEANS, AVAILABLE INFORMATION AND EVOLUTION STAGE ? ........................................................................................................................................ FIGURE 96. SDS PAGE GEL OF PURIFIED NBI WITH (LEFT) OR WITHOUT (RIGHT) CO-EXPRESSION OF PLE1 ............................... FIGURE 97. COMPARISON BETWEEN PURIFIED NBI PROTEIN OBTAINED IN KRX/PLE1 BACTERIA WITH OR WITHOUT INDUCTION OF THE PLE1 METHYLTRANSFERASE. .............................................................................................................................. FIGURE 98. IPA-PCR WITH 50 BACT/NL OF KRX/PLE1/PNBI CONTAINING AN UNEXPECTED INSERT. ...................................... FIGURE 99. EFFECTS OF INDUCER QUANTITY, INDUCTION OD600 AND INDUCTION TIME ON NBI ACTIVITY IN BACTERIA. .............. FIGURE 100. MICROSCOPE OBSERVATION OF T7 EXPRESS/PLE1 BACTERIA EXPRESSING EITHER THE WILD-TYPE OR AN INACTIVATED VERSION OF THE NBI ENZYME. .......................................................................................................................... FIGURE 101. MICROSCOPY OBSERVATION OF T7 EXPRESS/PLE1 BACTERIA EXPRESSING VARIOUS VERSIONS OF NBI WITH OR WITHOUT 0.1% TRITON IN THE CULTURE MEDIUM. ............................................................................................................. FIGURE 102. EXPRESSION LEVEL AND NBI ACTIVITY ACCORDING TO INDUCTION TIME AND CONCENTRATION FOR T7 EXPRESS CELLS.190 FIGURE 103. EFFECT OF THE CULTURE CONDITION AND TEMPERATURE ON THE BACTERIA SHAPE AND NBI ACTIVITY.....................FIGURE 104. SINGLE CELL NBI EXPRESSION LEVEL............................................................................................................ FIGURE 105.NEMGFP-NBI EXPRESSION IN BACTERIA. ................................................................................................... FIGURE 106. THE FOUR MAIN METHODS OF NEXT GENERATION SEQUENCING IN 2018. ........................................................ FIGURE 107. MUTATIONAL BIAS OBSERVED IN THE SEQUENCED LIBRARIES. .......................................................................... FIGURE 108. MUTATION RATED ACCORDING TO INITIAL TEMPLATE QUANTITY. ..................................................................... FIGURE 109. REPARTITION OF THE READS FOR EACH PART OF THE GENE IN THE DIFFERENT EXPERIMENTS ................................... FIGURE 110. ACTIVITY TEST OF THE LIBRARIES COMPARED TO THE WILD-TYPE NBI. ............................................................... FIGURE 111. ACTIVITY ASSAY OF 93 PICKED CLONES.. ..................................................................................................... FIGURE 112. ACTIVITY TEST AND PRIMER PRODUCTION RATE OF 13 PICKED CLONES.. ............................................................ FIGURE 113. AMINO ACIDS SUBSTITUTION PATTERN PREDICTED BY MAP 2.0 3D FOR NBI USING THE MUTAZYME II KIT. ............ FIGURE 114. SOME EXAMPLES OF DROPLETS GENERATION METHODS. ............................................................................... FIGURE 115. MICROFLUIDIC CHIP FABRICATION. FIRST THE DESIGN OF THE MOLD IS PRINTED ON A MASK. ................................. FIGURE 116. SEVERAL WAYS OF PRODUCING DROPLETS IN MICROFLUIDIC CHIPS.. ................................................................. FIGURE 117. DIFFERENT REGIMES OF DROPLET PRODUCTION.. .......................................................................................... FIGURE 118. KIM'S DEVICE ........................................................................................................................................ FIGURE 119. DIFFERENT TYPES OF FILTERS. ................................................................................................................... FIGURE 120 GEOMETRY OF THE DEVICE NOZZLE COMPARED WITH THE INSPIRING DEVICE FROM SHIM AND AL.. .......................... FIGURE 121. EVOLUTION OF DROPLET GENERATION DEVICE DESIGNS. ................................................................................ FIGURE 122. TROUBLED ENCOUNTERED WITH FILTERS DESIGN. ........................................................................................ FIGURE 123. THE FINAL DEVICE DESIGN. ....................................................................................................................... FIGURE 124. TRIDIMENSIONAL REPRESENTATION OF THE FINAL DEVICE.. ............................................................................. FIGURE 125. ANTHONY AND ALEX'S DEVICE. ................................................................................................................ FIGURE 126. ANALYSIS OF DROPLETS GENERATED BY DIFFERENT DEVICES.. .......................................................................... FIGURE 127. MEASUREMENTS OF IN VITRO IPA-PCR GENE YIELD USING A FLUORESCENT PROBE IN DROPLETS. .......................... FIGURE 128. OBSERVATION OF DROPLET SIZE DISTRIBUTION AFTER PCR IN DROPLETS WITH VARIOUS SLOPE FOR TEMPERATURE CHANGE. .......................................................................................................................................................

FIGURE

  FIGURE 129. TESTS REALIZED TO RECOVER DNA FROM DROPLETS. .................................................................................... FIGURE 130. OBSERVATION OF BIOFILM FORMED BY BACTERIA AT THE LEVEL OF THE FILTER. ................................................... FIGURE 131. ENCAPSULATION OF BACTERIA IN DROPLETS.. .............................................................................................. FIGURE 132. PURIFICATION AND ACTIVITY ASSAY OF THE WILD-TYPE AND INACTIVATED TAQ POLYMERASES. .............................. FIGURE 133. GENERAL SCHEME OF CSR. ...................................................................................................................... FIGURE 134. SCREENING OF EXPRESSION CONDITION TO INCREASE TAQ EXPRESSION. ............................................................ FIGURE 135. SELF-REPLICATION OF WILD-TYPE KLENTAQ WITH VARIOUS BACTERIA CONCENTRATION. ...................................... FIGURE 136. DIFFERENT MOCK LIBRARIES ..................................................................................................................... FIGURE 137. ASSAYS TO MEASURE PROPORTION OF ACTIVE AND INACTIVE CLONES. .............................................................. FIGURE 138. ENRICHEMENTS VALUES FROM LITERATURE DEPENDING ON THE INITIAL FRACTION (P). ......................................... FIGURE 139. THEORETICAL PREDICTION OF CHANGE IN FREQUENCY UPON ONE ROUND OF SELECTION IN A VARIETY OF SELECTION DYNAMICS. .................................................................................................................................................... FIGURE 140. SQI SCORES CALCULATED FOR THE DATA IN FIGURE 138. ............................................................................... FIGURE 141. ON/OFF MOCK EXPERIMENT SELECTION SCHEME. ....................................................................................... FIGURE 142. DIFFERENCE BETWEEN ACTIVE AND INACTIVE MUTANT FOR SELF-REPLICATION.. .................................................. FIGURE 143. RHQPCR WORKFLOW USED TO DETERMINE QUANTITY OF ACTIVE AND INACTIVE GENE ......................................... FIGURE 144. SELF-REPLICATION OF KLENTAQ DEPENDING ON BACTERIA CONCENTRATION.. .................................................... FIGURE 145. INCREASING CONCENTRATION OF BACTERIA IN SELF-PCR REACTION. ................................................................ FIGURE 146. COMPARISON OF THEORETICAL OBTAINABLE FINAL ACTIVE FRACTION COMPARED TO THE OBTAINED DATA ACCORDING TO THE MODEL. .................................................................................................................................................. FIGURE 147. RECOVERY OF THE CORRECT WILD-TYPE AND INACTIVE MUTANT SEQUENCE.. ..................................................... FIGURE 148 VERIFICATION OF NEGATIVE MUTANT EXPRESSION AND INACTIVITY. .................................................................. FIGURE 149. RHQPCR ON SOME PICKED CLONE PLASMIDS............................................................................................... FIGURE 150. ACTIVITY ASSESSMENT OF THE CHOSEN AD-1 AND NEB+4 CLONES. S. ............................................................. FIGURE 151. SCHEME OF MOCK EXPERIMENTS WORKFLOW. ............................................................................................. FIGURE 152. EXPECTED OUTCOME OF IPA PCR IN DROPLETS CARRYING THE ACTIVE, INACTIVE OR CO-ENCAPSULATED ACTIVE AND INACTIVE NBI EXPRESSING BACTERIA.. ................................................................................................................ FIGURE 153. SUMMARY OF DROPLETS IPA-PCR ISSUES .................................................................................................. FIGURE 154. MEASURED RELATIVE ACTIVITY OF THE LIBRARY BEFORE AND AFTER SELECTION COMPARED TO THE WILD-TYPE NBI ENZYME AFTER HIS-TAG PURIFICATION. ............................................................................................................... FIGURE 155. SELECTION COEFFICIENTS OF AMINO ACIDS BY POSITION FOR THE NEUTRAL AND HEAT SHOCK SELECTION.. ............... FIGURE 156. MEAN PERMISSIVITY BY POSITION. ............................................................................................................ FIGURE 157. NBI STRUCTURE WITH PERMISSIVITY. ......................................................................................................... FIGURE 158. MUTATION RATE OBSERVED BY MUTATION TYPES. ........................................................................................ FIGURE 159. PERMISSIVITY BY POSITIONS IN THE NEUTRAL SELECTION (NS) VERSUS THE HEAT SHOCK SELECTION (HS).. .............. FIGURE 160. PRIMER PRODUCTION MEASURED BY DIFFERENT REPORTING PROBES. ............................................................... FIGURE 161. TIME DELAY FOR DIFFERENT CONCENTRATION OF PRIMERS DETECTION BY EITHER 50NM OF THE V1 OR V2 OF DOUBLE STRAND PRIMER REPORTING SYSTEM WITH OR WITHOUT BST 2.0 WS POLYMERASE. .................................................... FIGURE 162. LOOPP17 PROBES REACTIVITY ASSAY. ........................................................................................................ FIGURE 163. ISOLATION OF GFP EXPRESSING BACTERIA IN DROPLET. ................................................................................. FIGURE 164. RH-QPCR PRINCIPLE. .............................................................................................................................. FIGURE 165. CALIBRATION OF RNASEH2 QUANTITY_1. ................................................................................................... FIGURE 166. CALIBRATED OF RNASEH2 QUANTITY_2 ..................................................................................................... FIGURE 167. PRINCIPLE OF LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LAMP) METHOD.. ............................................... FIGURE 168. LAMP AMPLIFICATION OF NBI GENE ......................................................................................................... FIGURE 169. GENERAL SCHEME OF THE STRAND DISPLACEMENT ACTIVITY ASSAY. ................................................................. FIGURE 170. RESULTS OF THE EXPERIMENTS. ................................................................................................................

  FIGURE 129. TESTS REALIZED TO RECOVER DNA FROM DROPLETS. .................................................................................... FIGURE 130. OBSERVATION OF BIOFILM FORMED BY BACTERIA AT THE LEVEL OF THE FILTER. ................................................... FIGURE 131. ENCAPSULATION OF BACTERIA IN DROPLETS.. .............................................................................................. FIGURE 132. PURIFICATION AND ACTIVITY ASSAY OF THE WILD-TYPE AND INACTIVATED TAQ POLYMERASES. .............................. FIGURE 133. GENERAL SCHEME OF CSR. ...................................................................................................................... FIGURE 134. SCREENING OF EXPRESSION CONDITION TO INCREASE TAQ EXPRESSION. ............................................................ FIGURE 135. SELF-REPLICATION OF WILD-TYPE KLENTAQ WITH VARIOUS BACTERIA CONCENTRATION. ...................................... FIGURE 136. DIFFERENT MOCK LIBRARIES ..................................................................................................................... FIGURE 137. ASSAYS TO MEASURE PROPORTION OF ACTIVE AND INACTIVE CLONES. .............................................................. FIGURE 138. ENRICHEMENTS VALUES FROM LITERATURE DEPENDING ON THE INITIAL FRACTION (P). ......................................... FIGURE 139. THEORETICAL PREDICTION OF CHANGE IN FREQUENCY UPON ONE ROUND OF SELECTION IN A VARIETY OF SELECTION DYNAMICS. .................................................................................................................................................... FIGURE 140. SQI SCORES CALCULATED FOR THE DATA IN FIGURE 138. ............................................................................... FIGURE 141. ON/OFF MOCK EXPERIMENT SELECTION SCHEME. ....................................................................................... FIGURE 142. DIFFERENCE BETWEEN ACTIVE AND INACTIVE MUTANT FOR SELF-REPLICATION.. .................................................. FIGURE 143. RHQPCR WORKFLOW USED TO DETERMINE QUANTITY OF ACTIVE AND INACTIVE GENE ......................................... FIGURE 144. SELF-REPLICATION OF KLENTAQ DEPENDING ON BACTERIA CONCENTRATION.. .................................................... FIGURE 145. INCREASING CONCENTRATION OF BACTERIA IN SELF-PCR REACTION. ................................................................ FIGURE 146. COMPARISON OF THEORETICAL OBTAINABLE FINAL ACTIVE FRACTION COMPARED TO THE OBTAINED DATA ACCORDING TO THE MODEL. .................................................................................................................................................. FIGURE 147. RECOVERY OF THE CORRECT WILD-TYPE AND INACTIVE MUTANT SEQUENCE.. ..................................................... FIGURE 148 VERIFICATION OF NEGATIVE MUTANT EXPRESSION AND INACTIVITY. .................................................................. FIGURE 149. RHQPCR ON SOME PICKED CLONE PLASMIDS............................................................................................... FIGURE 150. ACTIVITY ASSESSMENT OF THE CHOSEN AD-1 AND NEB+4 CLONES. S. ............................................................. FIGURE 151. SCHEME OF MOCK EXPERIMENTS WORKFLOW. ............................................................................................. FIGURE 152. EXPECTED OUTCOME OF IPA PCR IN DROPLETS CARRYING THE ACTIVE, INACTIVE OR CO-ENCAPSULATED ACTIVE AND INACTIVE NBI EXPRESSING BACTERIA.. ................................................................................................................ FIGURE 153. SUMMARY OF DROPLETS IPA-PCR ISSUES .................................................................................................. FIGURE 154. MEASURED RELATIVE ACTIVITY OF THE LIBRARY BEFORE AND AFTER SELECTION COMPARED TO THE WILD-TYPE NBI ENZYME AFTER HIS-TAG PURIFICATION. ............................................................................................................... FIGURE 155. SELECTION COEFFICIENTS OF AMINO ACIDS BY POSITION FOR THE NEUTRAL AND HEAT SHOCK SELECTION.. ............... FIGURE 156. MEAN PERMISSIVITY BY POSITION. ............................................................................................................ FIGURE 157. NBI STRUCTURE WITH PERMISSIVITY. ......................................................................................................... FIGURE 158. MUTATION RATE OBSERVED BY MUTATION TYPES. ........................................................................................ FIGURE 159. PERMISSIVITY BY POSITIONS IN THE NEUTRAL SELECTION (NS) VERSUS THE HEAT SHOCK SELECTION (HS).. .............. FIGURE 160. PRIMER PRODUCTION MEASURED BY DIFFERENT REPORTING PROBES. ............................................................... FIGURE 161. TIME DELAY FOR DIFFERENT CONCENTRATION OF PRIMERS DETECTION BY EITHER 50NM OF THE V1 OR V2 OF DOUBLE STRAND PRIMER REPORTING SYSTEM WITH OR WITHOUT BST 2.0 WS POLYMERASE. .................................................... FIGURE 162. LOOPP17 PROBES REACTIVITY ASSAY. ........................................................................................................ FIGURE 163. ISOLATION OF GFP EXPRESSING BACTERIA IN DROPLET. ................................................................................. FIGURE 164. RH-QPCR PRINCIPLE. .............................................................................................................................. FIGURE 165. CALIBRATION OF RNASEH2 QUANTITY_1. ................................................................................................... FIGURE 166. CALIBRATED OF RNASEH2 QUANTITY_2 ..................................................................................................... FIGURE 167. PRINCIPLE OF LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LAMP) METHOD.. ............................................... FIGURE 168. LAMP AMPLIFICATION OF NBI GENE ......................................................................................................... FIGURE 169. GENERAL SCHEME OF THE STRAND DISPLACEMENT ACTIVITY ASSAY. ................................................................. FIGURE 170. RESULTS OF THE EXPERIMENTS. ................................................................................................................

  (IN EQUIVALENT PLASMID NG/µL) FOR TRIAL NUMBER 14 ............... TABLE 25. IPA-PCR MOCK EXPERIMENTS PART-I ...........................................................................................................TABLE 26 IPA-PCR MOCK EXPERIMENTS PART-II ........................................................................................................... TABLE 27. QPCR MEASURED NBI GENE AMPLIFICATION VERSUS THE BEFORE SELECTION SAMPLE IN THE DIFFERENT SAMPLE AFTER SELECTION ..................................................................................................................................................... TABLE 28.FOCUSED MUTAGENESIS TECHNIQUES ............................................................................................................ TABLE 29. RECOMBINATION METHODS FOR LIBRARY GENERATION ..................................................................................... TABLE 30. DIFFERENT HAIRPIN LOOP USED TO DETECT NBI ACTIVITY AND SPECIFICITY. ........................................................... TABLE 31. THE DIFFERENT PRIMER CONCENTRATION REPORTING PROBES. ........................................................................... TABLE 32. HOME MADE ELECTROCOMPETENT CELLS TESTS ...............................................................................................

  RNAs have since then proven to be very versatile molecules than sometimes behave like proteins[START_REF] Jijakli | The in vitro selection world[END_REF] , but, because of the limited physico-chemistry of their building blocks, their functional diversity is not as rich as polypeptides based on 20 chemically different amino acids.A few years later, in 1993, Frances Arnold and her colleagues[START_REF] Chen | Tuning the activity of an enzyme for unusual environments: sequential random mutagenesis of subtilisin E for catalysis in dimethylformamide[END_REF] applied the random mutagenesis technique to the subtilisin E protease. After expression, the mutants were tested for activity in organic solvent and the best ones were picked to undergo another cycle of mutagenesis and activity tests. This was the first time that this process, now called directed evolution, was used in protein engineering. Directed Evolution (DE) now refers to a large number of techniques, including methods generating variability and methods to select variants, used in combination to produce molecules whose properties conform to a user-defined goal. DE can also be used to examine the mechanisms of evolution at the molecular level.

a. Mimicking Darwinian evolution

Rational protein design is based on structural information and accumulated knowledge on sequence-structure-function relationship. But as explained above, it is currently difficult to predict exactly the outcome of mutations, especially when it comes to catalytic activity. Despite great achievements in protein engineering rational design, there is not yet a general method or a simple route to new enzymes with novel properties.

Natural proteins and enzymes have been produced by millions of years of evolution.

The general principles of evolution were theorized by Darwin in 1859, based on the natural selection of individuals. Genetic diversity in a population results in various phenotypes.

Individuals carrying theses various phenotypes undergo a selective pressure exerted by the environment. Fittest individuals with characteristics allowing them better to survive and reproduce in a given environment are selected and transmit their genes to the next generation.

Heredity allows to maintain the selected characteristics while mutation and recombination during reproduction generates new diversity among the new generation. Several factors in the physical, chemical and biological environment in which a population evolves induce a selection at each generation cycle whose result is a differential reproductive success.

Natural evolution acts on full organisms, but, with the spread of gene mutagenesis techniques, researchers started thinking about applying evolution principles at the scale of molecules. As proteins are uniquely the products of evolution, they could easily be redesigned by similar approaches. The idea was that, if the secret of protein structure/function relation cannot be unveiled, one may bypass this problem, and rather bias and accelerate the evolution process to obtain new functional biopolymers. In 1990, two teams,

Ellington et al. and Tuerk et al.

used random libraries of short RNA molecules to select aptamers able to bind specifically a target molecule

[START_REF] Tuerk | Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase[END_REF][START_REF] Ellington | In vitro selection of RNA molecules that bind specific ligands[END_REF]

. The process, nicknamed SELEX for Systematic Evolution of Ligands by Exponential Enrichment, is similar to natural selection. The random RNAs are retained, and then artificially replicated, if they can bind the target. No previous knowledge on the sequence is required.

The general scheme is presented in Figure

8

. The first step is to introduce mutations in the target gene sequence, generating a library of variants. Random and/or directed mutations can be introduced. After expression of the corresponding variants (in the case of proteins; this step is not necessary for the selection of functional DNA or RNA molecules), a screening or selection step allows to keep only the mutants matching the desired requirements. The retrieved encoding DNA is then mutated again to explore more neighboring sequences. Iterative cycles of diversification and selection are repeated. Doing so, proteins improve toward a desired activity in the course of the evolution campaign.

As proteins have a long sequence of amino acids (each encoded in the genetic code by 3 DNA bases) the number of possible sequences to be tested in DE experiments is astronomically large. For instance, a protein with 300 amino acids (aa) has 20 300 ~ 10 390 different possible sequences. Considerations about library generation are presented in part II of this thesis and in the annexes. It is impossible to test all the possible sequences during a DE campaign. It is therefore essential for DE to have a good starting point in sequence space, and to test the largest possible number of mutants. Conversely, DE does not require information on the protein sequence or structure. It can find solutions to biological "problems" without requiring any a priori understanding of its mechanism. In that sense, it overcomes the knowledge gap which prevents straightforward rational design.

Note that, while DE is a black box method, the information gathered by DE method coupled to next generation sequencing could be of great value to better understand structure/function relationships, the evolution process itself and ultimately improve rational design experiments.

1 Structure of the DNA molecule

	What in the DNA structure makes it so unique as a computing material? DNA was
	discovered in 1868, but the structural knowledge that we have on the molecule only arrived in
	the 1950s. It is a polymer composed of nucleotide monomers. These building blocks are
	composed of a deoxyribose, with a nitrogenous base attached on the first carbon, and one or
	several phosphates attached on the fifth carbon. In the DNA strand, nucleotides possess only
	one phosphate linked via a phosphodiester bond to the next nucleotide's sugar on the third

  The classic right-handed helix observed by Watson and Crick and abundantly found in the biological world is the called the B-form. But the B-form is not the only shape that DNA

	can have. Still based on hydrogen bonds and stacking effects, other structures have been
	observed in the living world. The A-shape is a bit shorter and has been observed in desiccated
	bacteria spores. It can also be the shape taken by DNA, RNA duplexes. RNA differs from DNA
	by the type of sugar -a ribose-used in its backbone, and by the use of an uracil instead of a
	thymine among the nucleobases. Even if base pairing is happening in the same manner, RNA
	is not able to form the same duplexes as DNA because of the constraints generated by the extra
	hydroxyl group on its sugar. Structures of RNA strand are very diverse, but they can adopt
	tridimensional structures granting them numerous functions, including catalytic ones. Another
	observed DNA shape, the Z-form is left-handed and observed in DNA methylated regions,
	certainly having a role in expression regulation. With a putative role in expression regulation,
	the H-DNA is a triplex involving 3 DNA strands. Hairpin structures observed in RNA but also
	in DNA are present in Holliday junctions formed during DNA recombination. Finally,
	quadruplex or G4-DNA can form in GC rich double stranded regions that self-aggregates during
	meiosis, recombination and also to regulate gene expression.
	Sequence-guided DNA self-assembly properties, and the various shape it can take can
	be used to engineer nanoscale devices. The predictable nature of DNA strands interactions
	enables researchers to program self-assembly. Back in 1982, Seeman proposed to combine

An in vitro modeling and computing tool based on polymerase extension and nicking

	And of course, building nanoscale computation system is still an option. transiently hybridize on a molecular template creating a starting point for the polymerase. The
	Molecular computing systems are still mainly based on DNA molecules, because a lot polymerase uses the template to elongate the trigger thus, building the fully complementary
	of tools exist to manipulate them and interaction between DNA strands are easy to predict. The strand of the template. Various kind of polymerases with or without strand displacement acivity
	field started more as a DNA-computing field and is now turning into molecular computing with can be used like for instance the bst Large Fragment, bst 2.0 or the vent (exo-) polymerases.
	new computation methods involving RNA or enzyme. One can think that if proteins structures The nickase, the second type of enzymes, come into play to restore the molecular template and
	and folding were understood as well as DNA or RNA structures, computation and nanoscale support dynamic production of single stranded DNA. The nickases have specific recognition
	construction involving proteins would be of great interest 112 . Proteins have binding and catalytic sites and cut one of the two DNA strands forming a duplex. Principally two different nickases
	activities and can also self-assemble to create big structures such asviral capsids. Using directed are used in the PEN DNA Toolbox, the Nt.bstNBI and the Nb.BsmI enzymes, but in principle
	evolution to improve enzymes used in the molecular computing field can be of huge interest to any nickase can be used provided buffer and temperature compatibility.. Once a strand has been
	better control the various parameters of the networks like leakage or reaction speed. It could complemented by a polymerase it can be targeted by the nickases leading to an opening in the
	also be used to create protein actuators and exclusively protein-based molecular programs. Such DNA strand. This nick can allow the two top strands to detach and diffuse freely in the reaction
	programs would have the advantage of having a limited half-life and would not risk to because of a reduction of the melting temperature due to the shortening of the strand. If the
	incorporate in the cells' genome. This can be interesting in the case of therapeutic applications. melting temperature is still too high, the nick will allow the polymerase to start producing a
	However, it is In my project, the PEN DNA toolbox was used to create a programmable selection new DNA strand by strand displacing the previously polymerized one preventing by-product
	true that enzymatic idiosyncrasies, such as substrate specificity (e.g. the polymerase can have function. This molecular programing technique is detailed in the next section. inhibition. Figure 19 describes the mechanism of oligonucleotides strand production by these
	different affinities for similar primer-template duplex), side reactions, lifetime limitations, two kinds of enzymes using DNA molecular templates. The last kind of enzymes, the
	purity, and other non-ideal behaviors can create difficulties 111 . On the contrary molecular b. The PEN DNA Toolbox exonucleases, can degrade produced single strand oligonucleotides thus, keeping the systems
	program based exclusively on DNA are relying on pure components, well-mapped DNA out of equilibrium. The exonucleases used in the PEN DNA Toolbox is RecJ, either
	thermodynamics and kinetics and can beneficiate for a lot of tools enabling precise estimation thermophilic or not thermophilic. It is a processive single strand specific 5'-3' exonuclease that
	of the network outcomes. Better understanding of protein structure-function relationship via The PEN DNA Toolbox stands for Polymerase Exonuclease Nickase Dynamic Network cleaves the strands base per base so that no intermediate oligo product is created so that the
	directed evolution could be one way to improve the prediction of hybrid molecular programing Assembly Toolbox and was first presented in 2011 110 . The strategy behind this toolbox consists wastes do not interfere with the program. Such intermediate oligos are one of the big issues in
	systems. of building simplified dynamic systems analog to that employed by gene-regulation networks the genelet molecular programming system. The RNaseH used is such system is not cleaving
	in a bottom-up approach, and within an artificial, well-controlled environment. It aims to all the strand leading to accumulation of RNA transcripts and other nondegradable a.4 Perspectives promote a better appreciation of the molecular origins of biological complexity and may also intermediates, which eventually poisoned the system 109 . Molecular programming is a very recent field, spanning only 2-3 decades with many serve in technological applications. This in vitro molecular programming approach uses a set The typical size of the molecular templates is from 22 to 26 base pairs and is about 11 major advances less that 10-year-old. Many perspectives can be envisioned for this field. At the of three kind of enzymes (P, E, N) combined with various short DNA strands encoded to be base pairs for a trigger strand 115 . They are synthetics strands whose sequence can be completely level of interactions with biology, molecular nanorobots able to interact with living cells, like used as molecular templates. These molecular templates are single-stranded chosen by the user, designed in adequacy with desired purpose and easily ordered to DNA the logic-gated nanocage, could be used to specifically deliver drugs or monitor some blood deoxyoligonucleotides composed of a 3′ input site and a 5′ output site. Interacting with the synthesis companies. The relatively short length of these strands prevents having secondary parameters. Molecular detection systems usable in diagnosis can be envisioned, thanks for the emergence/creation of complex behaviors. The basic reaction used is a classical example to aptamers. The HCR was already used to detect some molecules with a modified templates, the enzymes can dynamically produce and degrade short oligonucleotides enabling structures that could lead to program design issues. With such a short length produced 'input'
	aptamer initiator strand. A lot of researchers are interested in creating minimal cells. This could amplification systems first presented by Walker et al. in 1992 113 and further studied in 2003 by
	allow to test the various hypothesis of life's origin and better disentangle the important Jeffrey Van Ness et al. 114 . The first enzyme type is a polymerase that can complement primed
	parameters of biological networks. On a more physical approach, programmable DNA molecular templates by elongation of a trigger strand, seen as an input. Indeed, by base pair
	nanostructures can be used to create smart materials and, eventually, to drive chemistry process. complementarity the trigger, whose sequence is matching the 3' side of a template strand, can

selection function The Compartmentalized Self Replication methods

	bifurcations in nonlinear biochemical circuits 130 . The nonlinear and continuous nature of the
	PEN DNA Toolbox network yields to a not fully tractable link between structure and function
	with an intricate functional landscape. This can sometimes make the design counter-intuitive
	and the characterization over various range of concentrations of the different involved species
	laborious. In this study microfluidics was used to generate water-in-oil microdroplets
	comprising various amount of several species involved in studied networks. In a relatively short
	time droplest with different combinations of chemicals were generated and studied by
	microscopy.
	Table 3 gives a general overview of the PEN DNA Toolbox and all the advantages and
	disadvantages of the toolbox. More generally the encoding of the programs in short DNA
	strands that are nowadays easily synthesized, chemically modified, resistant to a lot of harsh
	conditions and can be attached to a lot of various support makes the PEN DNA Toolbox very
	convenient for the study of networks in a lot of different environments. Also, the functional
	plasticity of nucleic acids that can be used as ligand, scaffold, information storage tool and even
	catalyst (cf. DNAzyme) allow to envision infinite possibilities of interaction between the PEN
	DNA Toolbox and various in vitro and/or in vivo systems. That is probably why a growing
	number of teams are using the PEN DNA Toolbox all over the world. Some of them contributed
	to the previous works and are now developing their own applications like the team of Tom F.A.
	de Greef in Netherlands that designed a translator module that translates output strands from an
	upstream PENDNA network to unique regulatory DNA strands able to drive enzymatic
	actuators without adding load on the network 131 . This work can be seen as the inverse of the
	detection module, as the ouput of the PEN DNA Toolbox allows to trigger another chemical
	mechanism. We can also cite the work of André Estevez-Torres in Paris who studied the
	reaction-diffusion waves of the predator-prey system in microreactors 132 or the teams of
	. In this Masami Hagiya, Ibuki Kuwamata and Nathanael Aubert-Kato in Tokyo associated to the team
	work, Gines et al. created microscopic agents programmed by DNA circuits by attaching PEN of Nicolas Bredeche in Paris working on swarm robotics systems based on the PEN DNA
	DNA Toolbox template on beads. It was possible to see activation of the beads through Toolbox 133 . Very recently, Ievgen Kurylo in the team of Alexis Vlandas in Lille used DNA-
	propagation of input strands traveling front. Molecular programs were also combined with grafted micro-fabricated gold electrodes to trigger the PEN DNA system. Their microfluidic
	microfluidics to be observed and monitored in water-in-oil emulsions. The first implementation system allows the release at specific times and locations of DNA-based instructions and provide
	was carried on the Predator Prey 126 in picoliter droplets in 2013 and allowed to observe new opportunities of interaction between a bio-electronic system and DNA-based circuits. As presented previously, directed evolution methods are based on screening or selection
	sustained oscillations with slow desynchronization between the compartments and kinematic This project of directed evolution is part of this widening of the perspectives of the usage techniques. Selection methods are very convenient because they do not require time-consuming
	spatial waves. This approach clerified the influence of small volume effects in such networks of PEN DNA Toolbox. We will see now how we can use PEN DNA circuits to link enzyme or throughput-limiting measurement steps. One just links an enzyme activity to an organism and paved the way to technological applications of compartmentalized molecular programs 128 . activity and gene amplification and create a programmable selection function. survival and propagation or its own gene retrieval by binding or replication. Selection methods
	It was pushed forward in 2016 with the realization of a high-resolution mapping of

c. Using artificial network as

Table 3 .

 3 General overview of the PEN DNA Toolbox molecular programing system.

	limit experimental intervention and their unavoidable bias. But all the developed selection
	methods are case specific, and each protein requires ingenuity to design a specific feedback
	loop. This is especially true for the selection of catalytic activities. The Phage Assisted
	Continuous Evolution or other methods based on organismal replication can easily be used to
	evolve enzymes but always in in vivo environment that cannot be fully controlled.
	The Compartmentalized Self Replication (CSR) technique 79 developed by Ghadessy et
	al. in 2001 is one particularly interesting example among selection methods. It was the first
	method to generate in vitro replication of the coding genes by PCR. In the CSR, a Taq
	polymerase is evolved by encapsulating bacteria expressing the mutant enzymes in water-in-oil

Table 4 .

 4 Efficiency in enrichment in good mutants in the population by the different selection methods.

Table 5 .

 5 Advantages and drawbacks of the different compartmentalized selection methods directly linked to oligos production speed Nt.bstNBI has been chosen as a first target to develop this method.

Table 7 .

 7 Different primers sequences and melting temperature

Table 9 .

 9 Random Mutagenesis Techniques

Table 10

 10

	OPTCOMB or OPTOLIGO can help the design of combinatorial libraries. The program
	DNAWorks automate the design of oligos for gene assembly. Backtranslation according to the
	host organism and defined codon preference as well as definition of primers junctions,
	overlapping regions and avoidance of hairpin formation are provided.

) answer the following type of questions. What random mutagenesis method should I use for my target enzyme? What oligos should I use for my gene assembly (see annex i p.316)? What library size should I target? Can I expect to have all my desired amino acids mutation using such techniques? Among them, PEDEL or PEDEL-AA can assess diversity in an epPCR method and the Mutagenesis Assistant Program (MAP) (or its 2.0 version taking the structure into account) can estimate what is the best random mutagenesis method for your enzyme by comparing the bias in various parameters of the different methods improving chances to have functionally efficient and/or stable variants. For saturated mutagenesis experiments, GLUE or GLUE-IT can find degenerate codons (see Box 2 in annex i p.324) that encode user-specified sets of amino acids and estimate the maximum library size according to the throughput of your method.

Table 10 .

 10 Computational tools assisting library creation

	Identification of target regions, hot-spots and target amino-acids
	Protein-ligand interaction	LigPlot+, PDBePISA , LPC
	Analysis of the binding pocket	CASTp, Hotspot wizard, Meta Pocket 2.0
	Analysis of access tunnel	CAVER 3.0, MOLE 2.0, MolAxis
	Thermostability	B-FITTER, Popmusic2.1, FoldX 3.0, consurf, 3DM
	Amino-acids conservation	Hotspot wizard, consurf, Evolutionary Trace, 3DM, REAP
	profile
	Avoid structural disruption	SCHEMA, OPTCOMB
	Prediction of mutations effect
	on:
	Stability	Dmutant, CUPSAT, or I-Mutant2.0, FRESCO, REAP
	Function	SIFT, PROVEAN, REAP
	Learning from previous run	PROsar, ASRA
	Library creation predictive and analytical tools
	Random mutagenesis	PEDEL and PEDEL-AA, Volles and Lansbury, MAP 2.0 3D
	Site saturated mutagenesis	GLUE, GLUE-IT, CASTER, AA-Calculator, Kong, TopLib, DC-Analyzer, OneClick, DNAWorks
	Recombination	DRIVeR, OPTCOMB, OPTOLIGO, SCHEMA (+RASPP)

Table 11 .

 11 Growth of E. coli bacteria containing the pNBI active plasmid

Table 13 .

 13 Difference in techniques, read length, throughput, total number of reads and error rates for the four main NGS techniques.

Table 14 .

 14 The different types of mutations and their frequencies in various sequenced libraries.

Table 15 .

 15 The inactive clones may correspond to empty vector or to inactive enzymatic mutants. 13 clones with various activity were picked, sequenced and tested in more accurate conditions(Figure 111 & Figure

Top: Difference between mutation rates before or after transformation in bacteria. Bottom: Mutation rates obtained for a library generated from 255 ng of template and the wild-type gene in independent experiments 206 is inactive.

Table 16 .

 16 Obtained reads for each experiments and proportion of each libraries for multiple sequencing experiments

Table 17 .

 17

PEDEL_AA predicted parameters of a library of 107 variants with 2, 3 or 4 mean mutations per daughter sequence. (30 PCR cycles, PCR efficiency 0.6, nucleotides mutation matrix and indel parameters from Mutazyme II kit) Figure 113. Amino acids substitution pattern predicted by MAP 2.0 3D for NBI using the Mutazyme II kit.

Table 20 .

 20 Data collected on screening experiments with emulsion generated by shaking or filtersScreening experiments with emulsion generated by shaking or filter Several λ process : gene on beads, beads in IVTT, beads in selection droplets

	#
					initial	final
							†ratio
					1:10	1:0,7	14	0.71
	1				1:100	1:2,1	47	0.41
					1:1000	1:4,6	217	0.23
							† 2 not
							defined
					1:10	~1000:1**
	2
					1:1000 ~100:1**
					1:10 5	~1:1**	10 5
	Encapsulation of cells (bacteria or yeasts)
				¤gating
					1/1000	9/31	290	0.23
					1/100	12/19	63	0.28
	3				1/1000	2/50	40	0.04
					1/100	9/13	69	0.42
					1/1000	0/55	-	?
					1/100	0/19	-	?
							† 2 not
	4	Ultrahigh Throughput Screening System for Directed Glucose Oxidase Evolution in Yeast Cells (2011)	Glucose oxidase GOx gene vs empty pYES2 vector Counting cells with GOx gene	0,01*	1:10 1:100		defined 4,7 13	?	1,8 increase in k cat (1)
					1:1000		33
	Encapsulation of genes by extrusion through filter
			in the 2nd emulsion step β-Galactosidase LacZ vs LacZmut PCR and gel analysis a bit of exchange between drops	? (300)	25% 0,8% 0,8% 0,1% enrich §	50% 45% 14% 16%	2 57 138 20		Turn a protein of unknown function into β-Galactosidase

Table 21 .

 21 Data collected on screening experiments with emulsion generated by microfluidics 250

Table 22 .

 22 Data collected on selection experiments

	initial	final

λ Fraction active:inactive Active genes enrichmen t (n-fold) Bulk final 1st selection experiment SQITable 23 .

 23 Enrichment

		5,0%		96,0%		19,2	0.96	31,7%
	0,1	7,3%		84,0%		11,5	0.87	20,7%
	1	25,0%		80,0%		3,2	1.16	57,9%
	5	33,0%		55,0%		1,7	1.65	24,1%
	2nd selection experiment
	0,01	20,7%	±6,9%	99,9%	±0,1%	4,8	1.00	24,63%	±10%
	0,1	19,3%	±4%	97,7%	±0,7%	5,1	1.02	29,7%	±4,5%
	1	16,8%	±3,5%	83,3%	±2,8%	5,0	1.26	36,6%	±11,4%
	3	24,0%	±6,5%	65,9%	±9,5%	2,7	1.74	37,3%	±7%

and SQI values for KlenTaq self-replication in droplets with various lambda. Green numbers are not trustworthy as they contained more bacteria lysate.

  stabilizer from Raindance was added, as well as rlyzozyme concentration divided by 2 and molecular program templates concentration doubled. These last two changes were guided by bulk experiments results presented in the part I. Droplet stabilizer was used in an attempt to prevent droplets merging. For the first time, a gel electrophoresis of the PCR product of the selection was run and produced surprising results: bulk selection experiments ran either with only active bacteria or a mixture with 10% of active bacteria showed a proper band in the gel; but for droplet experiments a band appeared only in the 10/90 case. Nothing was visible for the active-only droplet experiment. It was first thought that it could be the result of an experimental error, but when looking closely at the gel it was noticed that actually the genomic DNA that we usually recover from bacteria was also not visible in the loading well of the gel. In general, less genomic DNA was seen in the wells for droplets experiments compared to bulk. This discovery lead to the work presented in part II, concerning the capture of DNA by water-oil interfaces and our attempts to recover DNA after emulsion breaking. However, as we can see in the 6 th trial, even in absence of visible band on the gel, the small amount of DNA retrieved is enough to be able to measure DNA quantity and evaluate active/inactive ratios. This trial was the first-time selection in droplet was observed. It suggested that selection efficiency increased with incubation time and was very promising. Unfortunately, an exact duplicate of this experiment, showed a band on the gel, but did not result in any selection. It is still unclear what could explain the difference between these 2 experiments. It seems that a lot of parameters including interfacial DNA and proteins capture, unpredictable

Table 27 .

 27 Qpcr measured NBI gene amplification versus the before selection sample in the different sample after selection

Table 3 -

 3 Microfabrication protocol for the two main thicknesses used in this work

Table 4 -

 4 DNA oligonucleotides necessary for IPA

Loopact test/ Lysate activity assay

  To realize the loopact tests, 250 nM of either the protected, unprotected or unspecific loopact strand were mixed in an IPA (or its derivative) buffer and the sample of interest. Various amount of purified Nt.BstNBI proteins or the commercial Nt.BstNBI (NEB 10 000U/mL) were

	Selec_FwdT7		GCGTATCACGAGGCAGAATTT
	Selec_RevT12		GCCTGCATAACGCGAAGTAAT
	IPA-PCR
	The IPA (Isothermal Primer Amplification)-PCR reactions were performed in various
	used for in vitro assays. In lysate assay, various concentration of rLysozyme (Merck) were used IPA-type buffers (1x) with 200µM of each dNTPs (NEB), 0.1% of Bst 2.0 WarmStart® DNA
	in addition to various concentrations of bacteria expressing either the active or inactive NBI Polymerase (NEB), 1% OneTaq® Hot Start DNA Polymerase (NEB, 5 000U/mL) and various
	enzyme. The 10µL reactions were monitored in CFX96 Touch Real-Time PCR Machine (Bio-amount of the following molecular program templates: cv11r2p (aT), either ssv11 (sceT) or v11
	rad) set up at 45°C and taking a measurement every ~30s. (T), and various combination of mainly the following ppT ensuring production of a reverse and
	a forward primer: v11top17r3 (biot or unbiot), v11top25f2 (biot or unbiot), v11top25r2
	PCRs (unbiot), v11toFwdT7 (v1 or v2 both biot), v11toRevT12 (v1 or v2 both biot). Other templates
	For all classical PCRs, mostly used in cloning steps, the reactions contained the were tested and are listed in the table below.
	commercial polymerase buffer (1x), 200µM of dNTPs (NEB.), 200nM of each forward and When performing the IPA-PCR in bacteria lysate, 6 µg/mL of Streptavidin (NEB 1
	reverse primer, up to few ng of template DNA to be amplified, if necessary up to 4 mM of mg/mL) and various amount of rlysozyme (Merck 27-33 KU/µl) were added to various
	MgSO4 (NEB) and between 0.5% and 2% of DNA polymerase. The mixtures were then concentration of bacteria (10 to 1000 bact/nL).
	incubated in a classical thermocycler (PCR machine) following procedures as summarized
	below:
	Init. Currently optimized conditions are: IPA V0 buffer, 200 µM of each dNTPs (NEB), Number of Denaturati Anneali Elongati Final Storag
	Denaturation 0.1% of Bst 2.0 WarmStart® DNA Polymerase (NEB), 1% OneTaq® Hot Start DNA cycles on ng on Elongation e
	Taq Polymerase (NEB, 5 000U/mL), 40 nM cv11r2p (aT), 2 nM ssv11 (sceT), 20 nM of each ppT, 95°C, 2min ~30 95°C, 30s X°C, 30s 68°C, 1min/kb 68°C, 5min 4°C
	Q5 0.125% of rlysozyme (Merck 27-33 KU/µl), 6µg/mL of Streptavidin (NEB 1mg/mL) for a 98°C, 2min ~30 98°C, 30s X°C, 72°C, 72°C, 2min 4°C
	30s maximal bacteria concentration of ~150 bact/nL with an IPA incubation time of 15 to 30 min. 30s/kb
	Annealing temperatures were determined using NEB Tm Calculator or the thermofisher IPA Init. Number of Denaturati Anneali Elongati Final Storag
	Incubation equivalent. 45°C up to	Denat. 95°C,	cycles 35	on 95°C, 30s	ng 56°C,	on 68°C, 2	Elong. 68°C,	e
	For the PCR linked to the IPA step, reactions were run in IPA buffer or its derivative 2h 2min, 1min min 5min
	5min in with 200µM of dNTPs (NEB.), dilution of the either the active or inactive gene on the pNBI lysate
	plasmid and primers concentration ranging from 0 to 500 nM. Different polymerases were
	tested with PCR program adapted according to the manufacturer instruction. Primers are listed
	here:
		Primers for IPA-linked PCR trials
	Name		Sequence (5'-3')
	p17r3		TGGTGGTGGTGCCATGG
	p17f2		CCTAAGGAGGTGATCTA
	p17r2		CAGATTTCCCTTCACGA
	p25f2		CCTAAGGAGGTGATCTAATGGCTAA
	p25r2		CAGATTTCCCTTCACGAATTTTACC

The mixture was then incubated for various amount of time during the project (up to 2 hours) at 45°C in a standard PCR machine, before the PCR protocol described below started.

  the lib 255, 255 ng of NBI gene (600 ng of plasmid pNBI+) were used in the error prone PCR reaction. For lib 50, 100 and 200, respectively 50, 100 and 200 ng of dam methylated NBI gene PCR product was used. The NBI gene was obtained via a PCR using the Q5® Hot :100 in the first two experiments). In the first experiments, 1µM of dextran alexa 647 was added in the bacteria premix in order to differentiate the two solution in order the microscope and adjust their contribution to the droplets.

	over inactive bacteria was 1:10 (1The premixes were then flowed to the microfluidic device with equal hopefully equal
	contribution to generate droplets of ~26µm of dimeters, with bacteria at indicated λ<1, in
	fluorinated oil (Novec 7500, 2% RAN or EA). The collected droplets were then transferred in
	a PCR tube and incubated for various amount of time (up to 45 min) at 45°C in a standard PCR
	machine, before the PCR protocol described below started.
	During the project, addition in both tubes of droplet stabilizer (Raindance) or 1ng/µL of
	Yeast RNA (Merck) and 0.4% of Pluronic F-127 (Sigma-Aldrich) was tested.
	Currently optimized conditions allows to go from 10% to ~90% of active gene in one
	cycle: IPA V0 buffer, 200µM of each dNTPs (NEB), 0.1% of Bst 2.0 WarmStart® DNA
	Polymerase (NEB), 1% OneTaq® Hot Start DNA Polymerase (NEB, 5 000 U/mL), 40 nM
	cv11r2p (aT), 2nM ssv11 (sceT), 20 nM of each ppT, 0.125% of rlysozyme (Merck 27-33
	Start High-Fidelity DNA Polymerase (NEB), 0.6 ng of pNBI plasmid, 200µM of dNTPs and
	200nM of primers (XbaIprpNBIvide: TAATCTAGATAATACAACTCACTATAGGG and
	SpeIprpNBIvide: TTAATACTAGTCAAAAAACCCCTCAAG). Then ~1µg of purified PCR
	product (PCR clean-up kit Machery & Nagel) was methylated with 2% of the dam
	Methyltransferase (NEB 8000U/mL) in 1x dam Methyltransferase Reaction Buffer
	supplemented with 80 μM S-adenosylmethionine (SAM) at 37°C for 1h. The methylated DNA
	was purified before use.
	For the mutagenic PCR, reactions were run in Mutazyme II reaction buffer with 200 µM
	of dNTPs, 2% of Mutazyme II DNA polymerase (Agilent technologies, 2.5 U/µl), ~290 nM of
	primers	(NBI_LG_#1_SalI:	TATGTCGACAAACCTTACCTCCTTGTC	and
	NBI_LG_BamHI: ATAGGATCCTAAGGAGGTGATCTAATG) and the indicated quantity of
	template DNA.
	Init.		Number	Denaturation Anneali	Elongati	Final	Storage
	Denaturation	of cycles			ng	on	Elongation
	95°C, 2min		30	95°C, 30s	51°C,	72°C,	72°C,	4°C
						30s	2min	10min

KU/µl), 6 µg/mL of Streptavidin (NEB 1mg/mL), 1ng/µL of Yeast RNA (Merck) and 0.4% of Pluronic F-127 (Sigma-Aldrich) for λ = 0.4 with an IPA incubation time of 15 or 30 min.

  To palliate the limitations of random mutagenesis, focused mutagenesis techniques have been designed. They use the information that one can gather on a target protein to reduce the studied sequence space and have a better and more complete view of it. They allow to target some specific positions or regions in the protein sequence. Sometimes, when the crystal structure is known, binding and catalytic residues or active site can be determined. Homologous enzymes sharing the same scaffold but having few different amino acids can catalyze very different reactions. Thus, it implies that targeting a few key residues can be enough to optimize binding capacity or improve catalytic properties of an enzyme while saving experimental time.

	KlenTaq Mutant ON Detection	0.3 mU in 10µL Annexes
	KlenTaq Mutant OFF	0.2 mU in 10µL
	Init. Denaturation Detection 95°C, 3 min Nt.BstNBI Mutant OFF Number of cycles 35 Nt.BstNBI Mutant ON Detection i. Library generation methods complement Denaturatio n Annealing Elongation Final Elongation 98°C, 10s 53°C, 30s 72°C, 1min30 72°C,2min 7.5 mU in 10µL 7.5 mU in 10µL	Storage 8°C
	Detection
	1. Focused mutagenesis
	Library selection			), 1.5mM of extra MgSO4 (New
	England Biolabs, Inc.), 200µM of dNTPs (New England Biolabs, Inc.), 1% Evagreen (Biotium, Selection of the Lib 50 library was performed in IPA V0 buffer, 200µM of each dNTPs
	inc.), 200nM of the two adapted rhPrimers (IDT DNA technologies), 0.5% of DreamTaq DNA (NEB), 0.1% of Bst 2.0 WarmStart® DNA Polymerase (NEB), 1% OneTaq® Hot Start DNA
	polymerase (Thermofisher) and a specific amount of RNase H2 (IDT DNA technologies) -cf Polymerase (NEB, 5 000U/mL), 40 nM cv11r2p (aT), 2 nM ssv11 (sceT), 20 nM of each ppT
	Table. The 10 µL reactions were monitored in CFX96 Touch Real-Time PCR Machine (Bio-(v11toFwdT7, v11toRevT12), 0.125% of rlysozyme (Merck 27-33 KU/µl), 6µg/mL of
	rad) following 40 standard qPCR cycles (see below). Standard were run with plasmid of either Streptavidin (NEB 1mg/mL), 1ng/µL of Yeast RNA (Merck) and 0.4% of Pluronic F-127
	active or inactive version of the gene starting from ~1 ng in 10 µL and diluted sequentially by
	5 for 5 dilutions.
	To determine the proper amount of RNase H2 giving the best assay dynamic range,
	ranges of [RNase H2] were realized for each reaction and each primer range. The results were
	compared to positive and negative control reactions performed with the corresponding classical
	Denaturation PCR primers. Init.	cycles Number of	on Denaturati	ng Anneali	Elongation	Elongation Final	Storage
	95°C, 5min	35	95°C, 10s	56°C,	68°C, 2	68°C, 5	8°C
	Target	Init.	Number of	Denaturation 1min	Annealing min	min	Elongation
	Denaturation	cycles
	NBI Taq MinIon Sequencing 95°C, 3min 95°C, 3min	40 40	95°C, 10s 95°C, 10s		52°C, 20s 60°C, 40s	68°C, 20s None
	rhPCR primers sequences MinIon protocol "1D PCR barcoding genomic DNA (SQK-LSK108)" and "Low input
	Name rhprimf_nbiON genomic DNA by PCR Barcoding (SQK-LWB001)" were mixed and modified. The library Sequence CCATTCCACGTAAGCCATrUCAAAC_3SpC3
	rhprimf_nbiOFF Lib50 after heat shock selection (HSS), neutral selection (NS) or before selection (bFS) as well CCATTCCACGTAAGCCATrGCAAAC_3SpC3
	rhprimf_nbiON primF_qNBI as 12 clones picked from the initial library were sequenced simultaneously using the SQK-ACAAGCAGATTTTAATGACCCrACGTCC_3SpC3 CCATTCCACGTAAGCCAT primR_qNBI ACAAGCAGATTTTAATGACCC LSK108 1D 2 R9 version kit. DNA was barcoded using PCR amplification with barcoded
	rhprimf_KTON_v2 TTGGCTGCTGGTTGCACTGGrATTATC_3SpC3 primers. For two of the samples, it was not possible to amplify them with barcoded primers, so rhprimf_KTOFF_v2 TTGGCTGCTGGTTGCACTGGrGTTATC_3SpC3 rhprimr_KT_v2 GCTTCACGCGGAACACCAAACrATCCAC_3SpC3 we used the minion kit. Q5® Hot Start High-Fidelity DNA Polymerase (NEB) was used for all
	primf_qKT_v2	TTGGCTGCTGGTTGCACTGG
	primr_qKT_v2	GCTTCACGCGGAACACCAAAC
	RNase H2 Concentrations in rhPCR
	Experiment		[RNase H2]

(Sigma-Aldrich), for λ = 0.7 with an IPA incubation time 30 min in 26µm droplets in fluorinated oil (Novec 7500, 2%EA). Bacteria and rlysozyme were introduced in separate solution mixed at the last moment in the nozzle. A 3 min heat shock at 60°C was added before the IPA for the heat shock selection. the amplification outside of the MinION protocol. HSS and NS were respectively barcoded with the 26 and 29 barcodes from MinION barcode list by PCR amplification. A selection of the barcodes with the largest divergence in sequence was done before ordering the primers. bFS

  Target residues are identified in a knowledge-guided manner but are then generally mutated randomly in terms of amino acids. It has already been used successfully to improve thermostability, substrate specificity or enantioselectivity205 . For a simple screen of one position, one can easily order 20 different versions of a synthetic gene or use site-directed mutagenesis or mutagenic plasmid amplification (Stratagene, NEB) to introduce the different codon with a randomized primer. Some genes/oligos synthesis company like Twist can also provide saturation mutagenesis libraries for several unique positions for 50$ per position with same constraints on relative positions. But for more complicated screening involving several positions and their permutations, more sophisticated methods are needed as efficiency decreases when you try to apply saturation mutagenesis to distant locations. Oligosynthesis companies can also provide combinatorial saturation mutagenesis libraries but this time at relatively prohibitive costs. However, this can be envisioned for small proteins with few targeted positions. Available methods to build combinatorial libraries in the laboratory use cassette insertion, mutagenic oligonucleotide PCR amplification, splicing by overlap extension (SOE-ing) of DNA fragments or gene 323 . Since the number of mutants that have to be screened to allow full coverage of the reduced sequence space increased exponentially with the number of saturated positions, in addition to the means developed to insert the mutations at specific position, work has been done on the codon usage to minimize the number of sequence coding for an amino acid. (cf Box 2 p.324) In the Combinatorial Active-site Saturation Test 324 (CAST) large cassettes with degenerate codons (see Box 2 p.324) allow to mutate pairs of positions exploring all 20 amino acids with only 32 codons for each position reducing drastically the size of the library that has to be screened. One-Pot Simple Methodology for Cassette Randomization and

Table 28 .

 28 Focused mutagenesis techniques

  C Stemmer in 1994 332 . It relies on the random fragmentation of the genes by the DNaseI and reassembly of sized selected fragments through what is now sometimes called a sexual PCR despite the non-exponential nature of the process. High sequence identity regions between parents allow annealing of strands from different parents and therefore recombination by strand extension. After the reassembly reaction a PCR using external primers is needed to generate and amplify full-length chimeras usable for cloning. In the random-priming in vitro recombination 333 (RPR) random hexanucleotide primers are used to generate a pool of different sized random extension products. Like the DNA shuffling technique, a PCR-like process is then used to reassemble the Methods based on Golden Gate xvii Shuffling 338 and enabling recombination of fragments based on site-selective ligation can also be used. Mixing gene shuffling and oligonucleotides cassettes like in the previously presented OSCARR focused mutagenesis technique can also allow to create recombined libraries. Several techniques based on the assembly of oligonucleotides were developed to increase recombination resolution while reducing the bias of generated by highly homologous sequences. These methods appeared to many researchers as a promising option, probably because of the decreasing prices of oligosynthesis. Synthetic Shuffling 339 (SS), Degenerate Homologous Recombination 340 (DHR) and Assembly of Designed Oligonucleotides 341 (ADO) were all developed in 2002-2003 and are very similar to the Gene Assembly Mutagenesis presented if the focused mutagenesis methods. They typically use a reduced amino acid alphabets for saturation mutagenesis encoded through degenerate codons at some positions of the synthetic oligonucleotides, but the classical 20 amino acids can also be used. While DHR is using hybridization and ligation of modified oligos followed by PCR, SS is using Polymerase Overlap Extension (POE) PCR also named Polymerase Cycling

Table 31 .

 31 The different primer concentration reporting probes.

Table 32 .

 32 Home made electrocompetent cells tests

:

  FIGURE 1. THE VARIOUS ROLES OF PROTEINS IN BOLD WITH SOME EXAMPLES TO ILLUSTRATE THEM. ........................................... FIGURE 2. THE 20 COMMON AMINO ACIDS AND THEIR PROPERTIES. ..................................................................................... FIGURE 3. THE DIFFERENT LEVELS OF STRUCTURE OF PROTEINS. ........................................................................................... FIGURE 4. PROTEIN-FOLDING ENERGY LANDSCAPE. .......................................................................................................... FIGURE 5. ENZYMATIC CATALYSIS. ................................................................................................................................. FIGURE 6. BEGINNING OF PROTEIN ENGINEERING TIMELINE. ............................................................................................... FIGURE 7. DE NOVO DESIGN OF LIGAND BIDING PROTEIN....................................................................................................

  ..... FIGURE 49. OBSERVATION OF GFP CARRYING BACTERIA LYSIS IN DROPLETS UNDER THE FLUORESCENT MICROSCOPE.. .................. FIGURE 50. SURVIVING (INTACT) BACTERIA AS A FUNCTION OF TIME IN ONE MICROSCOPIC FRAME, WITH (TOP) OR WITHOUT (BOTTOM) THE LYSING AGENT BUGBUSTER AT 10%.R. ......................................................................................................... FIGURE 51. DEGRADATION OF PROTECTED SPECIFIC OR UNSPECIFIC LOOPACT PROBE BY BACTERIA LYSATE WITH OR WITHOUT LYSING AGENTS. ....................................................................................................................................................... FIGURE 52. EFFECT OF RLYSOZYME AND BUGBUSTER ON THE IN VITRO IPA-PCR REACTION WITH 1% COMMERCIAL NBI.. ........... FIGURE 53. EFFECT OF RLYSOZYME AND BUGBUSTER IN NBI ACTIVITY RELEASE (MEASURED BY LOOPACT DIGESTION RATE) ...........

  129. TESTS REALIZED TO RECOVER DNA FROM DROPLETS. .................................................................................... FIGURE 130. OBSERVATION OF BIOFILM FORMED BY BACTERIA AT THE LEVEL OF THE FILTER. ................................................... FIGURE 131. ENCAPSULATION OF BACTERIA IN DROPLETS.. .............................................................................................. FIGURE 132. PURIFICATION AND ACTIVITY ASSAY OF THE WILD-TYPE AND INACTIVATED TAQ POLYMERASES. .............................. FIGURE 133. GENERAL SCHEME OF CSR. ...................................................................................................................... FIGURE 134. SCREENING OF EXPRESSION CONDITION TO INCREASE TAQ EXPRESSION. ............................................................ FIGURE 135. SELF-REPLICATION OF WILD-TYPE KLENTAQ WITH VARIOUS BACTERIA CONCENTRATION. ...................................... FIGURE 136. DIFFERENT MOCK LIBRARIES ..................................................................................................................... FIGURE 137. ASSAYS TO MEASURE PROPORTION OF ACTIVE AND INACTIVE CLONES. .............................................................. FIGURE 138. ENRICHEMENTS VALUES FROM LITERATURE DEPENDING ON THE INITIAL FRACTION (P). ......................................... FIGURE 139. THEORETICAL PREDICTION OF CHANGE IN FREQUENCY UPON ONE ROUND OF SELECTION IN A VARIETY OF SELECTION DYNAMICS.

TABLE 3 .

 3 GENERAL OVERVIEW OF THE PEN DNA TOOLBOX MOLECULAR PROGRAMING SYSTEM. ............................................... TABLE 4. EFFICIENCY IN ENRICHMENT IN GOOD MUTANTS IN THE POPULATION BY THE DIFFERENT SELECTION METHODS. ................. TABLE 5. ADVANTAGES AND DRAWBACKS OF THE DIFFERENT COMPARTMENTALIZED SELECTION METHODS ................................... TABLE 6. SOME SEQUENCES OF THE USED TEMPLATES AND TRIGGER. .................................................................................. TABLE 7. DIFFERENT PRIMERS SEQUENCES AND MELTING TEMPERATURE ............................................................................. TABLE 8. RESISTANCE TO BACTERIA LYSATE FOR DIFFERENT COMMERCIALLY AVAILABLE PCR POLYMERASE ................................. TABLE 9. RANDOM MUTAGENESIS TECHNIQUES ............................................................................................................ TABLE 10. COMPUTATIONAL TOOLS ASSISTING LIBRARY CREATION ..................................................................................... TABLE 11. GROWTH OF E. COLI BACTERIA CONTAINING THE PNBI ACTIVE PLASMID ............................................................... TABLE 12. DIFFERENT CLONING EXPERIMENTS AND THEIR RESULTING LIBRARY SIZES .............................................................. TABLE 13. DIFFERENCE IN TECHNIQUES, READ LENGTH, THROUGHPUT, TOTAL NUMBER OF READS AND ERROR RATES FOR THE FOUR MAIN NGS TECHNIQUES. ................................................................................................................................. TABLE 14. THE DIFFERENT TYPES OF MUTATIONS AND THEIR FREQUENCIES IN VARIOUS SEQUENCED LIBRARIES. ........................... TABLE 15. TOP: DIFFERENCE BETWEEN MUTATION RATES BEFORE OR AFTER TRANSFORMATION IN BACTERIA. ............................ TABLE 16. OBTAINED READS FOR EACH EXPERIMENTS AND PROPORTION OF EACH LIBRARIES FOR MULTIPLE SEQUENCING EXPERIMENTS ................................................................................................................................................................... TABLE 17. PEDEL_AA PREDICTED PARAMETERS OF A LIBRARY OF 107 VARIANTS WITH 2, 3 OR 4 MEAN MUTATIONS PER DAUGHTER SEQUENCE. .................................................................................................................................................... TABLE 18. VARIATIONS OF PARAMETERS IN TRIED MICROFLUIDIC DEVICE. ............................................................................ TABLE 19. RESULTS OF DROPLET SIZE ANALYSIS BEFORE AND AFTER PCR WITH VARIOUS TEMPERATURE CHANGE SLOPE ................ TABLE 20. DATA COLLECTED ON SCREENING EXPERIMENTS WITH EMULSION GENERATED BY SHAKING OR FILTERS ........................ TABLE 21. DATA COLLECTED ON SCREENING EXPERIMENTS WITH EMULSION GENERATED BY MICROFLUIDICS ............................... TABLE 22. DATA COLLECTED ON SELECTION EXPERIMENTS ................................................................................................ TABLE 23. ENRICHMENT AND SQI VALUES FOR KLENTAQ SELF-REPLICATION IN DROPLETS WITH VARIOUS LAMBDA.. ................... TABLE 24. MEASURED NEB GENE QUANTITY WITH RHQPCR

température à 45°C, et après un certain temps d'incubation, une concentration d'amorces dépendante de l'activité de l'enzyme est atteinte. On lance alors une PCR dont le rendement dépendra de la concentration d'amorces. Après cassage de l'émulsion on retrouve une banque de mutants enrichies avec les meilleurs gènes qui sera utilisée pour créer une nouvelle banque et répéter les cycles d'évolution. L'enzyme étudiée comme premier modèle de cette méthode d'auto-sélection est une des nickases utilisées dans les programmes moléculaires de la PEN DNA Toolbox : Nt.Bst.NBI. Les étapes de développement de cette nouvelle méthode ont permis d'optimiser les conditions de cultures et de co-encapsulation des bactéries avec les réactifs nécessaires à l'étape de programmation moléculaire et de PCR. Elles ont rendu possible la sélection du gène wild-type de Nt.Bst.NBI parmi une large majorité de mutants inactifs passant d'une fraction initiale de 10% à une fraction finale de 90% en un seul cycle démontrant ainsi la forte capacité de sélection de la méthode. Les cycles de sélection sur une banque de mutant de cette même enzyme sont en cours et l'analyse des résultats de séquençage NGS des mutants sélectionnés dans un tout premier cycle montre déjà les signes d'une sélection efficace.(1) F. J. Ghadessy and al. Proc. Natl. Acad. Sci. U.S.A., vol. 98, Apr. 2001. (2)K.Montagne and al. Mol. Syst Biol., vol. 7, Feb. 2011.

i Hidden Markov Model (HMM) is a statistical model based on anlysis of a system external observable outcomes to infer the rules governing the observed phenomenon.

Table 26 IPA-PCR mock experiments part-II

the price for the most impressive screening capacity is awarded to the Phage-Assisted Continuous Evolution experiment (PACE). Taking advantage of the high mutation frequency in the phage whose titer can reach 10 12 pfu/ml, although it is not explicitly estimated, with 200 rounds in 8 days the number of mutants that can be screened in a PACE experiment in greatly exceeds 10 [START_REF] Arnold | Evolution: Bringing New Chemistry to Life[END_REF] .

The Figure 94 summarize the different tools that can be used for library creation.

Mutations rate and library metrics

We sequenced the different epPCR libraries with the IonTorrent PGM sequencer. This sequencer was acquired by the laboratory because of its relatively cheap price and the possibility to use it in other experiments. It's relatively high read accuracy of 99% would allow us to determine the mutation rate and frequencies in our produced libraries. Because of the read size limitation of the machine (~300-400bp), the gene of NBI was split in six parts ~300 bp long, by PCR amplification. The last fragment was 366 bp. Unfortunately, the not deeply thought primers design led to some unsequenced parts in the gene (because of primer overlapping). The sequencing run led to ~200 000 reads, which were almost all of sufficient quality for further analysis (Table 16). Libraries with various initial template during the epPCR were generated and we sequenced either after the mutazyme PCR or after transformation of the library in bacteria. For the later, two possible IonTorrent protocols were used, OT2 based on PCR amplification for the clonal amplification of DNA on beads or the RPA, an isothermal amplification protocol. The RPA protocol led to less reads than the OT2 one. Barcodes were used to combine several libraries in a single sequencing run.

The observed mutation rates were of 3.4, 2.6 and 1.9 mutations per kb for libraries produced from respectively 50, 100 or 200 ng of template DNA (Table 14) without taking insertion and deletion into account. A mutation rate of 1.3 mutations per kb was observed for 255 ng of template in an independent experiment. The wild-type gene, sequenced together with the three 50, 100 and 200 libraries exhibit a mutation rate of 0.5 mutation per kb in the same conditions, 0.25 in a test run realized with only the wild-type. Attributing this mutation rate to sequencing errors and the different amplification steps necessary to prepare the libraries for sequencing, we can therefore count an average mutation rate of 3, 2, 1.5 and 1 mutations per kb for the 50, 100, 200, and 255 ng based libraries. Regarding the insertions and deletion, we can see in the Table 14 that they are more frequent in the sequenced libraries than what is announced in the Mutazyme II kit protocol. An increase can also be noticed for the wild-type gene, suggesting that most of these insertions and deletions are due to the sequencer. Transitions and deletions are similar to Mutazyme II kit values except for the wild-type, here again pointing on mutations introduced by manipulations and sequencing for this sample. Comparing the mutational bias presented on Figure 107 we can see that they are not exactly the expected ones. Some values are similar and other a bit different, but the general tendency is the same.

One important problem with this sequencing method when using barcodes is to try to have the same amount of each sample so that the same or desired throughput is obtained for each sample. DNA concentration of our samples were measured and mixed accordingly to get

Conclusion and Discussion

I reported here the first example of the Programmable External Network Compartmentalized Self-Replication (PEN CSR). This directed evolution method is based on the use of molecular programs encoded by the PEN DNA Toolbox. The program encodes a selection function able to link an enzyme activity to the yield of its own gene replication via the production of primers, later used in a PCR amplification. The combination of the molecular program isothermal primers amplification and the PCR is called the IPA-PCR step and was introduced as an intermediary step in the classical CSR scheme. Bacteria expressing the target protein library are encapsulated in microfluidic droplets with the program, thus providing a high capacity for parallel selection. In this first proof of principle, the PEN CSR was applied to the selection of a nicking enzyme nt.bstNBI. To do so, we linked the catalytic activity directly to the oligonucleotides production in the molecular program. While a priori, many MP design can implement this linkage, we selected a fail-safe design where nicking activity is necessary at the initiation (source template), amplification (autocatalytic template) and actuation (primerproducing template) stages. The ability of the method to select genes based on the NBI activity of their protein product has been demonstrated with a mock experiment in which a selection cycle allowed to go from an initial fraction of 10% of active gene among inactive variants to ~90% in the final fraction. A selection cycle was also performed on a library and NGS sequencing results tend to attest for an efficient selection, for example by looking at the disappearance of stop codon inducing mutations. Unfortunately, due to cloning issues, we did not yet confirm the activity enrichment in the selected libraries. Indeed, cloning of the selected libraries yield to an important quantity of empty vectors. Bacteria carrying these empty vectors affect the global activity of the tested library. Their presence decreases the global activity of the library, first because less bacteria are active but also potentially because their growth is faster due to the absence of toxic expression.

The development of the project was the occasion to combine the PEN DNA Toolbox for the first time with cellular content, bacterial lysate in particular, and revealed the important modifications that need to be done in this context. After adaptation of the Taq polymerase necessary to perform a PCR in the lysate, it was discovered that protection of the templates with chemical modifications was also necessary. We introduced phosphorothioate bonds as well as biotin on both template extremities to protect them from exonuclease degradation. With such modifications, it was possible to obtain NBI-dependent primer production in low bacteria Note: * represent phosphorothiates bonds in the nucleotide sequence.

Material & Methods

Buffer's composition

Microfluidics

Device design

The masks used for the microfabrication were first designed using AutoCAD in 2DWire mode, from simple shapes (lines, circles…). Depending on the physical support and technology used to print the masks (film mask or chrome mask), two rules were observed: 1) Create closed lines so that the printer can infer the color (full or empty) by changing each time it crosses a line, 2) Always separate patterns by a distance greater than the printer resolution (6µm for the film mask, 2µm for the chrome mask). For the step designs, two masks were prepared. Special attention was payed to the alignment between the two masks. During the project T7 express cells (NEB, #C2566) carrying the ple1 plasmid were made electrocompetent (T7E/ple1 bacteria).

Transformation

• Chemocompetent cells

The cells (50 µL aliquot) were taken from the -80°C fridge and thawed on ice for 10min.

From 1 pg up to 100 ng in less than 5 µL of vector were added and the mixture was incubated on ice for 30min. The cells were then heat shocked at 42°C for 10 to 30 s (depending on the strain used) and put back on ice for 5 min before 950 µL of SOC medium (Sigma-Aldrich) was added and the mixture was incubated at 37°C for ~1h in an orbital shaker. Dilutions were then spread on LB (LB miller Sigma-Aldrich) agar plate containing the proper antibiotic (100 µg/mL of ampicillin and/or 30 µg/mL of kanamycin). The plate was then incubated at 30°C or 37°C in an adapted incubator overnight.

• Electrocompetent cells

The cells (25 µL or later 200 µL aliquot) were taken from the -80°C fridge and thawed on ice for 10 min. 1 µL (up to 2 µL) of vector were added and the cells were transferred to a ice-chilled electroporation cuvette (resp. 1 mm or 2 mm large). The cuvette was then placed inside an electroporator (resp. 1800 V, 5 ms or 2500 V, 5 ms). 975 µL of SOC medium prewarmed at 37°C were added immediately before cells were incubated at 37°C for ~1h in an orbital shaker. Dilutions were then spread on LB Agar plate containing the proper antibiotic (10 µg/mL of ampicillin and/or 30 µg/mL of kanamycin) and the plate was incubated at 30°C or 37°C in an adapted incubator overnight.

Plasmids

The pGFPS1 plasmid used to obtain fluorescent bacteria was given by the Kiga Lab in Japan, it contains the emGFP plasmid.

Cloning & production (miniprep)

• pUCIDT-PleI

The PleI_M gene was ordered directly cloned in the high-copy pUCIDT vector from IDT DNA Technologies.

• Supreme Run 20µL of sequencing primer at 10µM and 20µL of plasmid at 30-100ng/µL or PCR product at 10-50ng/µL were put in two different tubes and brought to a GATC collection box.

The reaction was then detailed on the GATC website. In first experiments, the culture was done at 37°C (overnight and day culture) then an optimal temperature of 30°C was determined for bacteria expressing active NBI in T7E/ple1 cells with 40µM of IPTG.

Purification

KRX bacteria containing the plasmids coding for the proteins were cultured in 3 mL of LB broth (Sigma-Aldrich) with 100 µg/mL of ampicillin and/or 30 µg/mL of kanamycin at

Cloning

• Restriction-Ligation:

For the restriction-ligation protocol, several protocols were tried. The vector pNBI_vide For the Gibson assembly, NEBuilder® HiFi DNA Assembly Cloning Kit (NEB) was used according to manufacturer protocol. The vector was amplified either from pNBI+, pNBIor pNBIvide using 200 nM of GA.Vf, GA.Vf.2 and/or GA.Vr primers with the Q5 polymerase (NEB). The library or test NBI-gene was amplified with 200 nM of GA.NBI.f, GA.NBI.r and/or GA.NBI.r2 primers also with the Q5 polymerase. After PCR, the vector was digested with dpNI 1 h at 37°C in the cutsmart buffer (NEB). After purification of both vector and insert with PCR clean-up kit (Machery and Nagel) and quantification using Clariostar, Gibson assembly was done with a 1:2 molar ratio (vector:insert). After Hi-fi assembly, the product was concentrated in 8 µL in a zymoresearch column and 1 or 2 µL was used to transform electrocompetent cells.

Primers

Selection

ON/OFF selection experiments

NBI:

For mock selection experiments, two premix solutions were prepared containing both

Other

Gels

Agarose gels were prepared with 0.8% of agarose in TBE buffer (1x) (Sigma Aldrich).

TapeStation

TapeStation experiments were run using the TapeStation 4200 protocol for the 5000D screentape and reagents (Agilent technologies).

Primers for MinION sequencing Primers

Sequence ( 5 To overcome the issue due to the preferential self-recombination of parental genes, methods using either top or bottom strand of each parent genes proved to be very efficient. The Random Chimeragenesis on Transient Templates 337 (RACHITT) is addressing this issue by using a single stranded template. It can be used to create libraries averaging 12 or even 19 crossovers per gene in a single round of gene family shuffling. To do so, parental gene "donor" fragments are annealed on a full-length uracilated template at relatively high ionic strength.

Flap trimming by endo and exonuclease activities removes unannealed parts, before ligation of all the fragments along the strand. Finally, an uracil-DNA glycosylase treatment is preventing amplification of the initial template strand so that only the chimeric strand can be amplified and cloned. This ensures a low frequency of the parental gene in the generated library. The technique provides an important control on the crossover fragments size.

ii. Bacteria counting

To calibrate bacteria concentration determination with the OD600 cuv measurements, Anton Zadorin counted the bacteria directly by microscopy observations. The cells were treated with the LIVE/DEAD BacLight kit to increase the visibility. In parallel, I counted the colony forming units after an overnight growth on agar plates for a given OD600 cuv.

From the literature, we could find several estimations giving similar conversion values between OD600 cuv and bacteria concentration. For example, in the work of Kintses et al. 166 , the

Probes for detection of primer production rates

Two distinct kinds of probes have been designed to detect the quantity of primer produced overtime; hairpin loops or stable double strand DNA duplexes. On the Table 31

Material & Methods

• Primers design.

Primers were design according to TwistDX former recommendation and had to be of length 30-35 pb. They were chosen at various position on pGFPS1 to try the amplification of different sizes of amplicon by using always the same forward primer (See Table S1). The T7 promoter was also amplified to be able to try expression from RPA products later (See Figure S1).

• RPA.

A solution containing PFRB (Primer Free Resuspension Buffer) 1X, 200nM of each primer and 10000 copies of pGFPS1 plasmid was used to resuspend freeze dried RPA pellets of either S type or T type reaction. Half of the premix was added with EvaGreen 0.8 X in a separate tube. Then 11.2mM of MgAc was added to start the reaction in each tubes. The 4 sets of primers were used giving different expected amplicon sizes. Reaction was then run at 37°C

for up to about 3 hours.

Discussion

TwistDX propose the type S reaction for amplicon size between 500 and 1000 pb and T reaction for 1000 to 2000 pb. Yet the S reaction didn't amplify nicely even the shortest length.

Generally, even if the correct bands are produced by RPA a lot of parasites products are present therefore some optimization should be done. Maybe the new formula fron TwistDX could give cleaner results, primers could be changed as theey now claim that short pcr primers can be used.

Also, higher temperatures could be tried as TwistDX said they could use up to 40°C. But T reactions should be preferred over S reactions. For this experiment 6 strands were designed containing for each of them a v11 (11bp) sequence with NBI restriction site and Ba12 (12bp) sequence that can bind specifically a random coiled single strand reporter containing on one side a quencher and a fluorophore (Cy5)

Supplementary data

Name

on the other side. The difference between each strand is the number of repeats of a 10 bp sequence between Ba12 and V11. This experiment was designed to estimate the effect of the length of the output template on the reaction speed.

We observed a rapid decrease of the production (or reporting) of Ba12 with the introduction of the repeats. It decreased by about 5 with just one repeat.

The reaction was run either in newIPA or PP buffer, with 3mM DTT, 1% BSA9000S, 0,025% Bst 2.0 w.s., 2% Nt.bstNBI, 100nM V11, 800nM rBa12BQCy (reporter), 100nM

V11toriBa12, 1% SybrGold and either 10 or 100µM dNTPs.