And little Frida, as I write these lines, you are looking into the world for the first time. I can't wait to meet you and I am looking forward to all the adventures, big and small, that we will live together in the future.

Throughout my time as a PhD student, I experienced the support and friendship of numerous people, to whom I am most grateful.

First, I would like to thank Prof. Andrew Gri ths for welcoming me in his laboratory. Andrew, you can be counted on, no matter what. I am grateful for your trust and positive input all throughout. I am greatly indebted to Dr. Marco Ribezzi for the supervision of my doctoral studies. Marco, without you, I would not be where I am now. You gave me, a mechanical engineer, the chance to enter the world of molecular biology which allowed me to grow both, scientifically and personally. Therefore, I will be forever grateful to you. During this journey you always stood by me with your ideas, valuable discussions and scientific support. This PhD was a challenge, but with your patience and enthusiasm, you encouraged me to take it on. It is a great honor for me that the jury members Prof. Anne-Marie Gué, Prof. Patrice Soumillion, Dr. Clément Nizak and Dr. Maria Mañosas have agreed to participate in the evaluation of this PhD work. I acknowledge thankfully to have received funding from the European Union's Horizon 2020 research and innovation programme as a Marie Sk≥odowska-Curie Actions fellow. I would also like to take this opportunity to thank the Europe department team, in particular Alexandra, Lucie, Virginie and Lior who made the international doctoral programme "UP-toPARIS" a platform for inspiring trainings and encounters. I would like to thank Dr. Clément Nizak, Dr. Olivier Rivoire and Dr. Yannick Rondelez for stimulating discussions during the Data Analysis / Modelling meetings. Isa and Hélène, you were always there to rescue me from the tentacles of the French bureaucracy. Thank you so much for your unfailing support. Thank you to Maria Mañosas for showing me her lab after a wonderful biophysics conference in Barcelona. ¡Me encantaron nuestras conversaciones en español! I am grateful to my previous supervisor Dr. Lorenzo Bordonali and to Prof. Anne-Kristin Kaster, both from the Karlsruhe Institute of Technology. You advised me on the choice of my PhD position and I would like to thank you for your guidance. Your advice was invaluable. A big shoutout goes to all current and past members of the LBC. It is you who make this lab such a warm and welcoming place. I have always felt very comfortable as part of this team. You are amazing people, incredibly helpful and always supportive of one another. Thank you, Adeline, for introducing me to my project with a lot of heart and soul (starting with the correct use of a pipette!). Tatyana, we made our first steps in Paris together and I have always enjoyed working next to you at the bench. Antoine, thank you so much for the great time we had toi Chapter 0. Acknowledgements gether -in the lab but especially at your wedding in Bidart! Many thanks to Pablo, my seatmate in the o ce, you were always there with good advice, practical help and an unlimited supply of chocolate cookies. Jonathan, we had fantastic conversations, whether about our life with polymerases or about what is to follow afterwards. Jérémy, your sense of humour is wonderful and only topped by your baking skills. Fortunately, you share both with the LBC. Satyam, I will never forget the fun times we had at Café Oz and at the beach of Biarritz. Adrien, you always have a good line on your lips, which makes it a real pleasure to work with you. Sebastian, thanks for sparkling good advice with british humor. Andrea, thank you for your great organisation of the lab events and the little encounters at the co ee machine. Diab, Jean-Baptiste and Shiraz, with your management you keep the place running. I am grateful for your tireless support and practical help. Zoran, late in the evening you were the light in the gel electrophoresis room. I always enjoyed our nightly talks in the lab. Gillian and Darja, I am very happy that we continue to see each other beyond LBC, for hikes and festivities. Here, I would also like to address a word to the new PhD generation: Ellyn and Ahmed, you'll swing it! I wish you much success and "bon courage"! My dear LBCees, especially our lunch breaks and conversations -sometimes crazy, sometimes serious, sometimes scientific but always funny and relaxed -will stay in my mind.

A very special thanks goes to Karolis. You were my rock in the surf of science (and in the bouldering hall!). But just as much in the bars and on the Velibs of this city. My friendship with you has made the three years at LBC very special. My life in Paris is unimaginable without a very dear group of people: It fills me with pride to be a member of the "Party Animals". Anastasia, Bruno, Claudia, Larissa, Magda, Mariam, Anna, Charlie, Chiara, Darius, Diego, Elena, Estel, Federico, Gabriel, Henrique, Ibo, Maria, Mathieu, Olivia, Pascal, Simon and Valeria, you are my second family in the urban jungle of Paris and I am so grateful to each and every one of you. You have been the source of happiness, courage and hope. In the last three years, throughout dark days of the pandemic and lockdowns, we have created a strong bond of love and community which will always unite us.

Yannick, I cannot express how much you enrich my life. You show me the French "savoirvivre" in Paris, Bordeaux, Marseille, Lyon, and a lot of places to come! Your smile and your embrace make me forget my worries; your strength and your boundless love make me invincible. Thank you for taking this journey with me. I love you.

The greatest thanks goes to my mother Margret and my sisters Franziska and Teresa. Thank you for the phone calls and the walks in the forest. Thank you for the motivational care packages with cake and "Lyonerwurst" and for just always being there for me. Through your support and motivation, your strength and joy, I have never given up. I am grateful that you stand behind me and always believe in me. Teresa, your experience and your help in the form of books and personal advice have been incredibly helpful to me in mastering this project.

List of figures

1.1 The central dogma based on Francis Crick's revised version of 1970 [Crick, 1970].

The solid arrows represent so called "general transfers", while dotted arrows represent so called "special transfers". The absence of an arrow means that no information flow is possible ("unidentified transfers"). Error rates of the E. coli system are from Bebenek et al., Ninio et al., and Kramer et al. [Bebenek, 1990], [Ninio, 1991], [Kramer, 2007]. Created with Biorender.com ............. 1.2 DNA replication: The leading strand is synthesised continuously, while the lagging strand is assembled by the same replication machinery in a discontinuous fashion by connection of Okazaki fragments. Figure from [Bell, 2006] Primer is coloured in red, template DNA in yellow, incoming dNTP space-filling purple B) Schematic representation of the enzyme in complex with DNA. In the foreground: Outlines of the exonuclease domain in red, TPR2 subdomain in cyan, thumb subdomain in green. The rest of the enzyme is in the background and is marked in grey. The polymerase active site is indicated by an asterisk. Figure altered, from [Kamtekar, 2004] Side chains of residues T15, N62, F65, Y148, P129 (yellow), and L567 (green sticks) contact the single stranded template DNA. The polymerase domain and two 5' bases are omitted. A segment of the thumb backbone is shown in green (positions 560-575). DNA residues are labeled T3-T5 and metal ion A is represented by a blue dot. B) Di erence electron denisty map of cocrystallised ssDNA and native protein. Figure from [Kamtekar, 2004]. . . . . . . . . . . . . . . . . . 12 1.7 Multiple sequence alignment on AA level covering the region between exonuclease and polymerisation domains of protein-priming DNAPs. The ExoIII motif, putative limit of the exonuclease domain, and the "YxGG/A" motif, putative limit of the polymerisation domain, are shown. The "YxGG/A" motif and R/S/K/W/H at position 223 are suggested to be a specific feature of protein-priming DNAPs.

The numbers denote the position of the first aligned amino acid with respect to the N-terminus of the corresponding DNA polymerase. Invariant residues (white over black background) and conserved residues (boldface over grey background) are shown. Figure altered, from [Truniger, 2005]. . . . . . . . . . . . . . . . . . . 15 1.8 Working principle of the MagNIFi assay. A) The extension template consists of three bases and one NT position (i.e. the Error-Enriched Site) where the fourth base occurs (i.e. the base complementary to the rare base). In an imbalanced dNTP pool DNAPs are forced to introduce misincorporations at the EES. In this illustration base titration is illustrated using the example of dATP. At a threshold rare base consentration a low fidelity (LF) DNAP is introduces errors at the EES, while a high fidelity (HF) DNAP is able to copy the template still faithfully. B) The extension template. It consists of a primer binding site (blue), a conserved region (yellow) and two combinatorial sequence context library regions (gray), which flank the EES (pink). The conserved region and the library regions are made of three di erent bases, while the EES consists of the fourth base and is only one base long. Figure from [Paz, 2018]. . . . . . . . . . . . . . . . . . . . . . 16 1.9 Error rates of Φ29 DNAP WT in imbalanced dNTP concentrations. Figures a) to d) show the four di erent template contexts. The preferred nucleotide substitutions (and single nucleotide deletions) are quantified for each context.

The sum across all error types is the so-called "total error". Figure altered, from [Paz, 2018]. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 1.10 The FC 50 value is derived from error rates measured in imbalanced dNTP concentration conditions. FC 50 is then correlated to reported error rates from the literature. Figure altered, from [Paz, 2018]. . . . . . . . . . . . . . . . . . . . . . 18 1.11 heat map representation of Φ29 DNAP WT error preferences at the rare base concentration of 10 -7 µM. Results from "T", "A", "C" and "G" template contexts are averaged. Figure altered, from [Paz, 2018]. . . . . . . . . . . . . . . . . . . . 19 1.12 E ect of sequence context on the FC 50 value of Φ29 DNAP WT for a BBBABBB template library. The influence of template bases neighboring the "A" EES is displayed for each base identity in figures a) to c), respectively. In this example, base "A" occurs only in the EES but not in the 6 base pairs flanking the EES. For each base identity, influence on FC 50 value is displayed in position-wise resolution. Fidelity is measured as "logF C 50 = logF C 50Average -logF C 50Fixed . Figure altered, from [Paz, 2018] .13 E ect of sequence context on the total error of Φ29 DNAP at a rare base concentration of 10 -7 µM. The influence on the total error of template bases neighboring the "T", "A", "C" and "G" EESs is displayed in figures a) to d), respectively. The figures resolve influence of base identity and position of the 6 base pairs flanking the EES. The grey dotted line denotes the average total error of Φ29 in a given template context. Figure altered, from [Paz, 2018]. . . . . . . . . . . . . . . . . 1.14 E ect of sequence context on Φ29 error preferences at a rare base concentration of 10 -7 µM, using the example of BBBABBB (left) and DDDCDDD (right) template libraries. Identity and position of template bases flanking "T", "A", "C" and "G" EESs impact the distribution of nucleotide substitutions (and single nucleotide deletions). This Error preference was determined by normalizing the frequency of error sub-types to the total error rate. Figure altered, from [Paz, 2018]. . . . . 1.15 One cycle of directed evolution. Starting point is a gene library from which variants are expressed and then screened. Subsequently genes are selected based on the phenotype which requires genotype-phenotype linkage throughout the process. Selected genes can serve as starting point for the generation of a new library for the next directed evolution cycle. Figure inspired by Gargiulo et al. [START_REF] Gargiulo | Directed evolution for enzyme development in biocatalysis[END_REF], created with Biorender.com. . . . . . . . . . . . . . . . . . . . . . . . . . 1.16 (Ultra-) High-throughput screening strategies for directed evolution assays. The threshold from high-to ultra-high-throughput is reached at 10 5 analyses par day. Each of the assays can be cell-based, cell lysate-based, cell-free, or all of them (versatile). Compartments containing the target phenotype are coloured in red whereas compartments without target phenotype are coloured in blue. Autour et al. [Autour, 2017] distinguish two formats of the emulsion-based approach (right panel): They consider classic IVC to manipulate in polydisperse emulsions and set microfluidic-assisted IVC (µIVC) apart from it, as an approach that manipulates highly monodisperse emulsions. This schematic representation is not to scale.

Figure from [Autour, 2017]. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.17 An aqueous droplet is part of a water-in-oil emulsion. An IVTT system is encapsulated in the aqueous droplet, together with a single gene from which the enzyme is expressed in vitro. A selection step can follow (right). E.g. the enzyme converts a non-fluorescent substrate into a fluorescent product and compartments can be sorted based on fluorescence. This schematic representation is not to scale. Figure redrawn from [Gri ths, 2006], created with Biorender.com. . . . . . . . . 1.18 In vivo CSR scheme: 1) A library of DNAP variants is cloned and expressed in E. coli. 2) Cells are isolated in an aqueous solution containing reaction bu er, dNTPs and primers by compartmentalisation. 3) Cells are lysed by heat shock in their respective compartments releasing polymerase enzyme and encoding genes.

The double stranded plasmid is denatured by heat and primers are allowed to anneal. Active proteins (green and blue) are able to replicate their encoding gene, while non-active polymerases (red) do not. Eventually, the reaction is stopped, emulsion is broken and synthesised DNA is re-cloned for another round of CSR. Denaturation and hybridisation of primers limit this assay to application to thermostable DNAPs like the Taq polymerase. Transformation imposes a bottleneck on the throughput of this approach. This schematic representation is not to scale. Figure redrawn from [Ghadessy, 2001], created with Biorender.com. . . . . . . . . xiv List of figures 1.19 Geometries typically used for droplet generation in microfluidic chips. a) In the co-flow geometry an aqueous phase ("w", blue) arrives from a capillary that is immersed in a stream of oil ("o",yellow). b) to d) are planar geometries. e) In the step geometry the downstream channel is of increased height with respect to the channel of the incoming aqueous phase. Arrows indicate flow directions, figure from [Autour, 2017]. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 1.20 Schematics of the two types of ME. With direct emulsification (left) the dispersed phase is forced through a membrane into the continuous phase. In contrast, with premix emulsification (right), a premixed emulsion is forced through the membrane, where shear forces align the particle size. Figure altered and redrawn from [Berendsen, 2014], [Nazir, 2021] and [Technology Co, 2021]. . . . . . . . . . 30 1.21 a) Dependency of average emulsion droplet size and average membrane pore size is directly proportional. Black dots denote measurements of oil-in-water emulsions and white dots water-in-oil emulsions. In this example soybean oil (containing 0.5% Phosphatidylcholine and Polyglycerol esters of polycondensed ricinoleic acid) and an aqueous phase containing 5.0% glucose were used in direct emulsification with a Al 2 O 3 -SiO 2 type microporous glass membrane. Figure from [Mine, 1996]. b) The pore size distribution of an SPG membrane with an average pore size of 1 µm. external pressure system (SPG membrane, direct ME), c) internal pressure system (SPG membrane, direct ME), d) stirred cell (SPG membrane, direct ME), e) external pressure system (SPG membrane, premix ME). f) in the study of Vladisavljevic et al. [VladisavljeviÊ, 2019] a nanoemulsion was produced with a syringe filter by i) premix formation by pushing back and forth pure oil and pure aqueous phase several times between two syringes and ii) premix homogenisation by pushing it back and forth several times through a syringe filter mounted between two syringes. Dispersed phase is coloured in red and continuous phase in blue.

Figure from [VladisavljeviÊ, 2019]. . . . . . . . . . . . . . . . . . . . . . . . . . . 34 1.24 Schematic of an aqueous droplet (central dotted white sphere), stabilised in an oil continuous phase by a surfactant. 1) Surfactant molecules are present in the oil phase and adsorb to the oil-water interface The surfactant molecules thus form an interfacial surfactant layer, which stabilises the emulsion. 2) Biomolecules (DNA, RNA, protein) or 3) cells are present in the aqueous phase and adsorb to the oil-water interface if no surfactant layer is present. In this schematics, the surfactant is a a non-ionic PEG-PFPE 2 fluorosurfactant that does not interact with biomolecules or cells. This schematic representation is not to scale. Figure from [Holtze, 2008] A library of DB shaped mutant genes is encapsulated (1) in a transcription, translation and expression system (2). Expressed active variants are able to replicate their own gene in an RCA (3). After incubation the emulsion is broken (4) and the library recovered. The methylated template is selectively digested by DpnI (5) and the remaining RCA product is reformatted by PCR (6a) to be read in NGS (7b) and to serve as input (in DB shape, (7a)) for another round of iviCSR. A quality control can be carried out by qPCR (6b). Created with Biorender.com. . . . . . . . . . . . . . . . . . . . . . . 3.2 Workflow of library recovery and reformatting. A) RCA product is loaded on an agarose gel after it has been retrieved from the emulsion and undergone DpnI digestion. B) Primers (yellow) flank the mutated region on the gene and amplify it in a recPCR reaction using the RCA product (dark blue) as a template. In this example the primers flank a region that consists of two NNK scanned regions that are coloured in red and light blue, while the conserved regions of the gene are coloured in grey. C) The PCR product (yellow) is circularised together with a backbone fragment (dark blue) in a Gibson Assembly (a more detailed representation of the circularised product can be found in SuM 5). The template is amplified in an outPCR reaction. For each round a di erent primer set (green)(targeting di erent regions on the plasmid) can be used to minimise contamination throughout rounds. D) The outPCR product is linear and contains the whole DNAP gene (green). E) Comparison of backbone fragment, recPCR product and outPCR product on an agarose gel. . . . . . . . . . . . . . . . . . . 3.3 Standard curve of qPCR primers targeting Q1WT and Q1DD templates. The two templates can easily be discriminated in an iviCSR reaction. . . . . . . . . . 3.4 Characteristics of Q1WT and Q1DD genes. a) Sequence alignment of Q1WT and Q1DD genes. Numbers at the 3' end of the aligned sequences denote sequence position. The Q1DD gene codes for an inactive variant due to NT substitutions at positions 747 to 756, with respect to WT (coloured in green). The two genes can be discriminated in qPCR due to specific primers that target the mutated site (blue FW primer). The RV primer (red) targets both, Q1WT and Q1DD gene. b) Protein model of Φ29 DNAP. Green arrow indicate inactivating mutations in Q1DD on an alpha helix in the PolA motif. Mutations in this region interfere with metal B coordination and render polymerisation activity impossible [Pérez-Arnaiz, 2010], [Blasco, 1993a]. A) The biotinylated DNAP forms a complex with the template-primer dimer. The resulting ternary complex is tethered at the bottom of a ZMW by biotin-streptavidin binding. Addition of fluorescently labeled dNTPs allows the DNAP to resume DNA synthesis. The ZMW is illuminated from the bottom by a laser. Its geometry provides excitation confinement which allows for phospholinked dNTP incorporation detection against against the bulk solution background. B) Schematic representation of the phospholinked dNTP incorporation cycle (top panel) and the corresponding time traces (bottom panel). 1 and 2) Cognate phospholinked dNTP -template associatio in the DNAP acive site commences the fluorescence pulse on the time trace. 3) Formation of the phosphodiester bond dissociates the dye-linker-pyrophosphate product which diffuses out of the ZMW and ends the fluorescence pulse. 4) After translocation of the DNAP, the subsequent phospholinked dNTP can be incorporated (5). This schematic representation is not to scale. Figure from [Eid, 2009]. . . . . . . . . . 48 3.7 Illumina NGS workflow. Top panel: The to-be-sequenced DNA templates are hybridised to the flow-cell and a polymerase creates a complement strand. The original template is then washed away. Left middle panel: A complementary strand is synthesised through bridge amplification. The double stranded bridge is denatured, leaving two single stranded copies of each template immobilised on the flow cell. Parallelisation of the bridge amplification generates clusters. Right middle panel: Reverse copy strands are cleaved and washed away. Sequencingby-synthesis is carried out on the remaining forward strands as shown in the bottom panel. This picture does not show the generation of the second read: After completion of the first read, the read product strand is dehibridised and washed o . A complementary strand is synthesised through bridge amplification.

The bridge is dehibridised and the already read forward strand is cleaved and washed o , leaving the reverse strand which can then be sequenced to give read 2. This schematic representation is not to scale. Figure from [Weinstock, 1752] . 49 3.8 The preprocessing analysis pipeline of Illumina and PacBio reads. Left panel:

Illumina reads have two read senses (Read 1 and Read 2). They are merged with the program Flash and aligned with the program Bowtie2 which outputs a machine-readable alignment file in .bam format. This Bam file is then further processed in R. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 3.9 A) Gene counts as determined by NGS. B) CL-Frequency and CL-Enrichment calculation. C) Graphical representation of the di erent genes in the NGS dataset before (left panel, R=0) and after selection (right panel, R>0). Genes are coloured in blue. Mutations on the genes are marked with a red "x". Genes f, b' and f' have no mutations and represent the WT (occurrence n=1 before and n=2 after selection), genes b' and d' code for the same variant, they also have the occurrence n=2. Created with Biorender.com. . . . . . . . . . . . . . . . . . . . . . . . . . . 52 3.10 A) Counts along the DNA fragment as determined in NGS are stored in a onehot encoded data representation. B) PW-Frequency and PW-Enrichment calculation as well as the relation of WT PW-frequency at AA position x f PW,WT,x to f PW,Var,x , the PW-frequency of all variants at position x. C) Graphical representation of the di erent genes in the NGS dataset before (left panel, R=0) and after selection (right panel, R>0). Genes are coloured in blue. Mutations on the genes are marked with a red "x". Created with Biorender.com. . . . . . . . . . . 54 xvii List of figures 3.11 A) Counts along the gene on AA level as determined in NGS. The data of the standard equimolar condition of Titration Assay R2 is taken for this example. B) At each given position x the frequency f SpS,x of each AA substitution is calculated. C) Graphical representation of the di erent genes in the NGS dataset before (left panel, R=0) and after selection (right panel, R>0). Genes are coloured in blue.

Mutations on the genes are marked with a red "x". Created with Biorender.com.

4.1 The QuIK setup for emulsification. An SPGE is installed between two syringes and an emulsion is conveniently created by pushing two phases (aqueous and oil phase) multiple times back and forth through the extruder. . . . . . . . . . . . . 4.2 Emulsification of de-ionised water and 35% IVTT in water mixture. Test of di erent membrane types with 50 pushes through the SPGE. SLS measurements suggest that droplet size can be decreased by smaller pore sizes, emulsification of viscous sample and membrane passivation. Sample size is 1000 droplets, mean droplet radius of QuIK 10 pushes is r = 0.9 µm (V = 3 fL), mean droplet radius on-chip is r = 1.9 µm (V = 30 fL), standard deviation on the radius is SD = 0.28 µm (QuIK) vs. 0.1 µm (on-chip). 4.8 iviCSR kinetics scheme illustrating di erential DNA increase between a highly active variant (blue) and a low activity clone (ocher). a) The replication plateaus in the observed time range. At t = t 1 the two variants can be clearly discriminated. However, at t = t 2 , the reaction has reached a maximum DNA concentration and discrimination is not possible anymore. . . . . . . . . . . . . . . . . . . . . . . . . 4.9 The Time Assay for a non-digested sample. Both, x-and y-axis are in log scale.

A iviCSR reaction is incubated for di erent times (1h, 1.5h, 2h, 2.5h, 3h, 5h, 6h, 8h, 16h). Input concentration of the model system is set to 1 pM (for Q1WT and Q1DD, respectively). DNA concentration is derived by multiplication of initial DNA concentration with fold increase (fold increase is calculated by ∆Cq =C q incubated -Cq (1h, 1.5h, 2h, 2.5h, 3h, 5h, 6h, 8h, 16h). Input concentration of the model system is set to 1 pM in emulsion (for Q1WT (blue) and Q1DD (red), respectively) and set to 10 pM, 20 pM and 100 pM in bulk (for Q1WT (light green, yellow, light blue) and Q1DD (dark green, brown, violet), respectively). DNA concentration is derived by multiplication of initial DNA concentration with fold increase (fold increase is calculated by ∆Cq =C q incubated -Cq non-incubated ). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 4.11 The Time Assay for a non-digested sample. Both, x-and y-axis are in log scale.

A iviCSR reaction is incubated for di erent times (1h, 1.5h, 2h, 2.5h, 3h, 5h, 6h, 8h, 16h). Input concentration of the model system is set to 1 pM in emulsion (for Q1WT (blue) and Q1DD (red), respectively) and set to 10 pM, 20 pM and 100 pM in bulk (for Q1WT (light green, yellow, light blue) and Q1DD (dark green, brown, violet), respectively the standard equimolar dNTP concentration. One RCA copy of the 2.5 kb long DB template requires 5000 dNTPs as both, the template and its complementary strand are copied. Assuming an equal distribution of ∼25% of each base, this results in a requirement of 1250 dTTPs per copy. In the standard condition 320 copies marks the theoretical ceiling of amplification. This value drops by a factor 10 for each 10-fold dTTP titration. As the Titration Assay comprises of a one-pot expression and replication reaction, the maximal copy numbers should be considered as theoretical maximal values. Inhibitory factors (e.g. T7-RNA polymerase, rNTPs and bu ering conditions present to enable expression) could reduce the copy number. De Paz et al. [Paz, 2018] maps the gene ‡ i , captured in NGS, to the frequency f r ( ‡ i ).T h e h e a v i s i d e function ‰(s, x) makes sure that only frequencies f r ( ‡ i ) of genes ‡ i are taken into account, that possess the AA substitution s at position x along the gene. . . . . . 4.47 Slope of the linear fits of enrichment correlations in the three mutated regions(R1, nt level, in black I would like to begin this thesis with a closer look at its quite broad title "Information Transmission Across Generations -Thermodynamics and Evolutionary Implications". If we place this title into the realm of biology, we see that the genome is the data carrier through which information is passed on across generations. The genome comprises of the information needed for an organism to develop and function. It is stored in each cell and consists of nucleotide (NT) sequences, the deoxyribonucleic acid (DNA). Molecular biology studies biomolecules like DNA, ribonucleic acid (RNA) and proteins, that carry out the biological processes essential for the function of cells and for the organism those cells are part of. Life has not only sustained but evolved over millions of years as the information in the genome can be altered -often when when being copied -which paves the way for organisms to adapt throughout generations. This process is known as the evolution of species. Reading and writing the genetic code are thus two of the most fundamental processes in present day life. They are performed by polymerases, enzymes that catalyze DNA replication by the consecutive incorporation of nucleotides in the enzyme's "polymerisation domain". They replicate the genetic information before cell division [Benkovic, 2001] and their enzymatic activity is believed to have been one of the earliest enzymatic activities in the history of evolution, as the accurate replication of genomes is a prerequisite for evolution itself [Steitz, 1998] Many polymerases also have an "exonuclease domain" that performs an additional function: The excision of misincorporated nucleotides. While the polymerization domain is responsible for the elongation of the copy DNA strand, the exonuclease domain accounts for error correction. Polymerases are core components of many state-of-the-art laboratory practices, from DNA amplification to sequencing. Polymerases with strand-displacement activity have the ability to replicate DNA alone in isothermal conditions. Such polymerases are especially useful for sequencing, diagnostics and genomic applications. However, in nature and in the laboratory DNA is not the only biomolecule that can store information. Moreover, DNA to DNA is not the only way information can be passed on. Thus, it comes handy that Watson and Crick not only described the double helix structure of DNA but also found the time to propose descriptions for all possible information flows between biopolymers [Watson, 1953], [Crick, 1970]. I would like to mention here, that the description of the double helix structure of DNA is also based on an early fiber di raction image of DNA fiber, "Photograph 51", for which Rosalind Franklin as a female researcher in the 1950s (and her graduate student Raymond Gosling) have so far received little recognition in science [Ferry, 2019].

1 The central dogma of molecular biology DNA, RNA and protein are biopolymers that store information in their sequence. The nucleic acids are made up of chains of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), while proteins are made up of chains of amino acids (AA) (polypeptides). The "central dogma" proposes a set of rules for the transfer of information between mentioned

General Special Unknown DNA → RNA RNA → RNA protein → DNA RNA → protein RNA → DNA protein → RNA DNA → DNA DNA → protein protein → protein
Table 1.1: The nine theoretically conceivable residue-by-residue transfers of sequential information as described by the central dogma of Crick [Crick, 1970].

2 Polymerases: The central component of the DNA replication machinery I would like to focus on one specific part of the central dogma: The information transmission from DNA to DNA. As mentioned before, Watson and Crick are known not only for their work on the central dogma, but even more so for their groundbreaking description of the double helix structure of DNA in 1953 [Watson, 1953], based on research of Rosalind Franklin [Ferry, 2019]. Only 5 years later Maurice J. Bessman and I. Robert Lehman discovered the first enzyme responsible for DNA synthesis in Arthur Kornbergs laboratory [Bessman, 1958][Lehman, 1958]. They named this enzyme "DNA polymerase" (DNAP) and Arthur Kornberg was later granted the Nobel Price together with Severo Ochoa for the discovery of the mechanism in the biological synthesis of ribonucleic and deoxyribonucleic acid.

The "replisome" is the multiprotein molecular machinery that replicates DNA. The molecular motors of this machinery are proteins that couple chemical energy to a mechanical task. Polymerases are the pivoting protein in this system of enzymes and helping processivity factors that reads, writes, moves and edits megabase-pair long genomes [Bocanegra, 2021].

In more detail, Bocanegra et al. describe the "[...] beautiful sophistication of DNA replication and its regulation[.]" [Bocanegra, 2021] as follows: Each of the two complementary DNA strands serves as a template for a new DNA strand. After separation of the two template strands, the so called "leading strand" is processed by the polymerase in 3' to 5' direction. Thus, synthesis of the copy strand occurrs continuously in 5' to 3' direction by a stepwise addition of complementary deoxynucleoside triphosphates (dNTP) to the terminal 3' end of the primer or nascent DNA strand [Snedeker, 2017].

Due to the antiparallel nature of DNA, the other strand (lagging strand) is synthesised discontinuously by formation of short Okazaki fragments, that are later joined together (figure 1.2). DNA polymerases that are mainly in charge of copying entire genomes are called "replicative" [Maloy, 2013]. To enable dNTP binding, the fingers switch between an opened and a closed state and there is evidence, that this conformational change contributes directly to translocation of the polymerase along the gene towards the next template base [Berman, 2007]. In fact, Morin et al. [Morin, 2015] have conducted single molecule studies to investigate conformational changes and intermediate states that are involved in coupling the chemical reaction of nucleotide incorporation to the mechanical DNAP translocation along the DNA. Φ29 DNA polymerase (DNAP) was used as a model system and the findings are presented in more detail in section 1.3. Polymerases strongly select for correct Watson-Crick base pairing before covalently incorporating nucleotides, distinguishing each of the 12 possible mismatches [Echols, 1991]. In this way, fidelity is already given without additional proofreading activity. Polymerases of families A, B and C having an an additional conserved exonuclease domain can further increase fidelity with an additional function: Proofreading is the ability to correct incorporated errors by nucleotide hydrolysis. This function is catalyzed by two metal ions which are both bound to the enzyme via four carboxylate groups [Freemont, 1988][ Beese, 1991]. The Φ29 DNAP from polymerase family B has an extraordinary processivity and fidelity. The enzyme serves as a model system for the work in this thesis and is presented in more detail in the following section 1.3.

The Φ29 DNA polymerase

Spanish scientist Margarita Salas started working on bacteriophage Φ29 of Bacillus Subtilis in 1967 [START_REF] Salas | Margarita Salas. "My life with bacteriophage ϕ29[END_REF] and for the following 52 years dedicated her academic life to it. Bacteriophage Phi29 which targets Bacillus Subtilis has a linear, genome length of only 19,285 base pairs. Its DNA polymerase consists of 575 amino acids and has a mass of 66,714 Da. The DNAP of the Φ29 bacteriophage (in the following: "Φ29 DNAP") was discovered by Blanco et al. in Salas' group in 1984[Blanco, 1984]. It is mainly thanks to Salas' extensive work that Φ29 DNAP is known to great detail today. The Φ29 DNAP is a highly processive enzyme that possesses both, isothermal 5' → 3' DNA synthesis activity, preferrably at 30°C, and 3' → 5' exonuclease activity [Blanco, 1989]. It is part of the polymerase B family and its architecture fits well with the architecture of other polymerases of that family which was first described for the structure of RB69 DNA polymerase [Kamtekar, 2004][ Wang, 1997].

DNA replication by Φ29 DNAP can be protein or DNA primed

Unlike in the case of RNA, DNA replication cannot be initiated de novo. Φ29 DNAP tackles this circumstance by use of a protein side chain as a primer to provide the hydroxyl group to which the initial 5' nucleotide of the strand to be synthesised is attached. Similar solutions are observed in other bacteriophages but also linear plasmids and viruses, such as poliovirus, adenovirus, and hepatitis B, as well as in bacteria of genus Streptomyces [START_REF] Ito | Bacteriophage phi29 terminal protein: its association with the 5'termini of the phi29 genome[END_REF]][Salas, 1991][Salas, 1996][Chen, 1996]. The linear Φ29 gene is covalently linked to a protein at both 5'-ends. In the case of Φ29 this "Terminal Protein" (TP) is 266 amino acids long and attaches the first nucleotide of the phage genome by a phosphoester bond between the hydroxyl (OH) group of 5'-dAMP and Ser-232 [Hermoso, 1985][Kamtekar, 2004]. The ability to bind to the terminal protein is associated with the terminal protein regions 1 and 2 (TPR1 and TPR2) which are inserted in the polymerisation domain of Φ29 DNAP [Blasco, 1990][Dufour, 2000]. TPR1 is an insertion between the palm and the fingers subdomains. It can directly contact TP and DNA, comprises of residues 261 to 358 and has no homology with palm subdomains of other B family polymerases. TPR2 on the other hand has structural similarities to the specificity loop of T7 RNA polymerase, a member of polymerase family A [Kamtekar, 2004]. In the absence of DNA the TP and the polymerase form a tightly associated heterodimer [Blanco, 1987]. When dAMP is covalently linked to a free molecule of the TP, DNA replication takes place by continuous, strand displacing synthesis [Blanco, 1989]. The second base from the 3' end of each strand of the double-stranded genome serves as the template for the addition of the first nucleotide. It is located within a six base pair inverted repeat that serves as an origin of replication from either end of the linear DNA molecule [START_REF] Escarmís | Nucleotide sequence at the termini of the DNA of Bacillus subtilis phage phi 29[END_REF][START_REF] Salas | Margarita Salas. "My life with bacteriophage ϕ29[END_REF] [ Salas, 1978].

Φ29 DNAP driven DNA replication does not include any lagging strand synthesis. Instead, the polymerase synthesises from one origin of replication a long ssDNA stretch. There appears to be no coordination of initiation between the twin origins of replication. Thus, the ssDNA stretch is protected from decomposition by a single strand binding protein until it serves as a template for a Φ29 DNAP which now synthesises the complementary strand from the opposite origin of replication [Inciarte, 1980][Gutiérrez, 1991].Under laboratory conditions Φ29 DNAP is also able to use the 3'OH group of a nuceleotide to initiate DNA replication.

Chapter 1. Introduction

The first incorporated nucleotide has to "slide back" before elongation can be resumed [Méndez, 1992] and it is only after 10 nucleotides that the polymerase dissociates from the TP to resume synthesis of the phage genome [Méndez, 1997].

Φ29 DNAP topology contributes to its high processivity and intrinsic helicase activity

The polymerase lacks inherent helicase activity, but performs an equivalent role by its strong binding a nity to ssDNA which facilitates the debranching of double stranded DNA. A structural model of the enzyme reveals orifices giving access to the active site and o ering topological mechanisms as an explanation for Φ29 DNAP's intrinsic denaturation function and high processivity [Kamtekar, 2004]. Figure 1.4 shows the three orifices:

1. Downstream template tunnel: Debranching of template and non-template strand happens at the tunnel entrance. Residues that form this tunnel are part of the TPR2, exonuclease domain, fingers and palm (sub-) domains.

2. Upstream duplex tunnel: Template strand and the newly synthesised copy strand leave this larger tunnel in double helix configuration. Residues of the following domains participate in the formation of this tunnel: Exonuclease domain, palm, thumb, TPR1, and TPR2 subdomains.

3. Incoming dNTP entrance: Gives access to dNTPs to enter the active site and allows pyrophosphate to leave. This is the largest of all three pores.

Homology modeling from the ternary primer:template:polymerase complex of RB69 polymerase suggests that downstream template DNA passes through tunnel 1 prior to entering the Φ29 DNAP active site. This tunnel seems to be a structural basis for the polymerase's intrinsic strand displacement and processivity: With a length of less than and a diameter of approximately 10 Å it is only large enough to allow for the passage of single stranded DNA, necessitating the separation of template and non-template strand. In addition, the exonuclease domain, together with thumb, TPR1, TPR2 and palm subdomains appear to enclose upstream double stranded product DNA, forming tunnel 2. This clamp ensures maximal DNA-binding stability which is required for the enzymes intrinsic processivity [Franklin, 2001], [Salas, 2008]. Due to this structural configuration the polymerase is not dependent on additional helicases or clamps as replication accessory factors. Kamtekar [Kamtekar, 2004] suggests that the tunnel must be able to open. Otherwise ssDNA circular template could not be "threaded" into the tunnel. In vivo, the tunnel appears to be too small for the terminal protein to reach the active site, and also dissociation from the replicated genome would require the polymerase to open the tunnel. A proposed mechanism for tunnel opening involves peeling away of the extended --turn -structure of the TPR2 domain from exonuclease domain and thumb subdomain.

Chapter 1. Introduction strand displacement activities of the protein and study combined e ects of load (20 to -50 pN) and nucleotide concentration (seven dNTPs concentrations, from 2 to 500 µM) on real time kinetics. Optical tweezers are a single molecule approach (magnetic tweezers, a similar technique, are described in chapter 6.3.5) that allows to apply opposing or aiding loads on the DNA-DNAP duplex. With this single molecule assay, Morin et al. aimed to find insights into the coupling mechanism between the chemical polymerisation reaction and the mechanical translocation of the DNAP along the gene [Morin, 2015]. Their model proposed that translocation is only loosely coupled to the chemical catalysis and occurs after PPi release and before nucleotide binding. The latter might unilaterally direct thermal di usion of the polymerase between pre-and post translocational states.

According to Morin et al. the dNTP / PPi-free DNAP-DNA complex can shift between pro-and post-translocated states. Once a nucleotide comes in, the post translocated state is stabilised.

In this state the polymerase can generate force and work against large opposing loads of up to ∼ 50 pN. This force is an indirect e ect of dNTP binding. The polymerase spends a considerable amount at the dNTP / PPi-free conformation, which would suggests that the translocation step could be modulating the balance towards a switch to the exonuclease function of the protein. This intramolecular balance between polymerisation and exonuclease activity is further discussed below. Replication rates for Φ29 DNAP are reported to be v ∼ 80 nt/s, associated average free energy change for translocation ∆G trans = -0.46k B T , e ective translocation step size " =0 .4nm and Michaelis-Menten constant

K M = 31 ± 3uM (previously, Saturno et al. reported K M =0.5 ± 0.2
µM [Saturno, 1995]). Further kinetic rates, equilibrium constants and force dependencies of Φ29 DNAP can be found in table 1.2. dNTP concentration modulations showed that detachment of polymerase from the DNA template is facilitated at lower nucleotide concentrations. However, for all concentrations the average replication velocity reaches v ∼ 7 nt/s before detachment occurs.

k on (s -1 µM -1 ) 5 k cat (s -1 ) 120 k T (s -1 ) 670 k -T (s -1 ) 420 K " =k T /k -T 1.59 d T (nm) 0.35 d -T (nm) 0.05 " (nm) 0.40
Table 1.2: Kinetic rates, equilibrium constants and force dependencies proposed by Morin et al. [Morin, 2015] for the nucleotide incorporation cycle based on Brownian ratched model. k on denotes the rates at which the enzyme binds the nucleotide, k cat the catalytic rate constant, k T and k -T the forward and backward translocation rates, d T and d -T their respective associated characteristic distances, K " the equilibrium constant of the translocation step and " the distance between the pre-and posttranslocated states.

Chapter 1. Introduction

An AA sequence comparison of prokaryotic and eukariotic DNA-dependent DNA polymerases revealed three conserved N-terminal amino acid motifs in the exonuclease domain: ExoI, ExoII and ExoIII [Blanco, 1991], [Bernad, 1989], suggesting evolutionary conservation of the 3' → 5' exonuclease active site. In the case of Φ29 DNAP the three conserved motifs are found at the following AA positions: 8,9,12,14,15,19,20 (ExoI);58,59,61,62,65,66,69,70 (ExoII);165,168,169,172 (ExoIII) (figure 1.5). In PolI polymerase the aspartase and glutamate residues of ExoI (D12 and E14 in Φ29 DNAP) correspond to positions D355 and E357 and are directly involved in metal binding at the exonuclease active site. The metal ion A (Zn 2+ , Mg 2+ or Mn 2+ ) is interacting with the phosphodiester bond to be hydrolyzed and is therefore important for substrate binding. Position D20 of ExoI in Φ29 DNAP corresponds to PolI position D361, where the residue plays a role in dTMP binding and stabilisation of ssDNA [Bernad, 1989]. Figure from [Kamtekar, 2004].

Residue D424 in the ExoII motif of PolI is linked to binding of metal B (Zn 2+ , Mg 2+ or Mn 2+ ), which is essential for the exonucleolytic catalysis. The corresponding position in the gene of Φ29 DNAP is D66 [Derbyshire, 1988]. The ExoIII segment of PolI includes residues Tyr497 and Asp501 that are involved in exonucleolytic catalysis and metal A binding, respectively [Derbyshire, 1988] [ Freemont, 1988]. These residues correspond to Φ29 DNAP residues F199 and D201. Three variants with mutations in the ExoIII motif (Y165F, Y165C and D169A) were shown to decrease Φ29 DNAPs ability to replicate DNA 80-to 220-fold [Soengas, 1992]. Φ29 DNAP WT has an error frequency of 10 -5 to 10 -6 [Paz, 2018]. Exonuclease deficient variant of Φ29 DNAP have been reported to have an error frequency in the range of 10 -4 to 10 -6 bases [Esteban, 1993] and 2 * 10 -1 to 6 * 10 -3 bases [Saturno, 1995]. Deletions of residues 563-575 in the thumb domain, however, were shown to reduce exonuclease activity below 1% of wild-type (WT)lev el [Truniger, 2004]. Positions D12, E14, D66 and D169 in the three conserved motifs of the exonuclease domain have shown to be catalytic carboxylates. Mutations to alanine at any of these positions lower exonuclease activity by 10 5 [Kamtekar, 2004]. In general proofreading enhances DNAP fidelity by a factor of 10 2 to 10 3 [BÍbenek, 2018]. Figure 1.6 shows the structure of Φ29 DNAP bound to dT 5 . Residues T15, N62, and F65 of the exonuclease active site directly contact ssDNA [De Vega, 1996][ Vega, 1998]. The metal ion A coordinates the two aa residues E14 and D169 as well as the nonbridging oxigen of the phosphate that is linked to the base to be cleaved [Kamtekar, 2004].

Balancing Φ29 DNAPs polymerisation and exonuclease domain

If the dimer consisting of template and primer is perturbed during replication, the thermodynamic equilibrium may shift away from the polymerisation domain towards the exonuclease domain. There are numerous factors which can cause such perturbations: DNA lesions, mechanical tension, protein binding or unbinding events and ionic conditions [Soengas, 1995][Ibarra, 2009]. Most important, however, are incorporated mismatching nucleotides. These perturbations cause the polymerase to stall as the balance between extension and excision is changed. Johnson et al. [Johnson, 2004] investigated the e ects of all 12 possible base-pair mismatches with a thermostable bacterial DNA polymerase (the Bst DNA polymerase I, from Bacillus stearothermophilus) as model system and observed a "short-term memory" of the polymerase responding to mismaches up to four base pairs in from the primer terminus. The transfer reaction between the polymerase and exonuclease domain is performed intramolecularly with a bidirectional communication between the two active sites [Donlin, 1991][ Vega, 1999]. In order to perform this transfer reaction and to stabilise the primer at the exonuclease active site, the polymerase opens the template-primer duplex by three to four base-pairs (bp) [Ibarra, 2009]. Ibarra et al. point out how the coordination between polymerisation and exonuclease activity should be finely balanced in order to guarantee e cient yet faithful replication. The dynamics of this interplay are still little understood [Ibarra, 2009]. To deepen the understanding of DNA replication fidelity Ibarra et al. conducted an analysis of the structural mechanisms leading to stalling with the Φ29 DNAP as a model [Ibarra, 2009]. Using optical tweezers, they are able to apply mechanical tension on a single enzyme-DNA complex. This approach shows to trigger the transfer of the primer to the exonuclease domain in a similar way as the incorporation of a mismatched nucleotide. However, this transfer is not direct, but connected via at least two intermediate stages. One of them is a tension-sensitive and functional "slow" polymerization state in which interaction with the template is relaxed. The other one is a non-active state in which the polymerase is assumed to check the integrity of the DNA before a possible switch to the proofreading mode. This behaviour could be regarded as a "fidelity check point": It favours the selective degradation of misincorporations, triggered by the instability of a mismatched template-primer structure and the high a nity of the exonuclease domain for ssDNA. At the same time, this non-active intermediate state helps to avoid Chapter 1. Introduction unnecessary proofreading of the primer; namely in case of innocuous events that are also known to modify the template elastic properties such as protein binding or unbinding events or ionic conditions. These events, however, are not expected to destabilise the template-primer structure su ciently to trigger the final transition from the inactive intermediate to the exonuclease state. Ibarra et al. conclude that the approach could provide a novel take not only on the study of proofreading dynamics and the fine interplay between polymerase and exonuclease activity but also on dNTP concentration e ects. The interplay between the two opposing catalytic domains is assumed to be coordinated by the linker region that resides in between the putative limits of polymerisation and exonuclease domains [Truniger, 2005]. The YxGG/A motif in between the linker region and the polymerisation domain is highly conserved in protein priming DNAPs belonging to the B family and is proposed to be important for the linker's coordinating function [Truniger, 1996], [Truniger, 1999]. For Φ29 DNAP, the motif is located at AA positions 226, 228 and 229. A preceeding conserved Arginine at AA position 223 seems to be a feature specific to protein priming DNA polymerases and cellular DNA polymerases α. Truninger et al. suggest that the linker's balancing function relies on its involvement in substrate binding including both, TP and DNA. For example, some mutants with AA substitutions in the linker region showed besides reduced DNA binding capacity either reduced exonuclease or reduced polymerisation activity. Joyce et al. [Joyce, 1989] previously described how factors that destabilise the template-primer structure (e.g., high temperatures, low ionic strength or a mismatched primer terminus) can a ect the equilibrium between the two active sites that coordinate DNA polymerisation and excision. Truninger et al. was able to relate a large polymerase to exonuclease balance to poor stability at the exonuclease active site and a small polymerase to exonuclease balance (and also null polymersiation) to a poor stability at the polymerisation active site [Truniger, 1999]. They suggested that the YxGG/A motif is involved in the stabilisation of the template-primer structure at the polymerisation active site and also plays an important role in balancing between the two active sites. In their work, AA substitution R223I eliminated the positive charge of Arginine. Clones with this substitution showed five-fold higher K M for dATP in comparison to that of the WT [Truniger, 1999]. Further, the mutant favoured exonucleolysis and needed higher dNTP concentrations to favour polymerisation. It is suggested that the removal of the positive charge (by R → I substitution) destabilises binding of DNA at the active site and in turn favours stabilisation at the exonuclease active site, which leads to a low polymerisation-exonuclease ratio [Truniger, 1996], [Truniger, 1999]. De Vega et al. [Vega, 1999] highlighted that switching of the primer strand to the exonuclease domain and back to the polymerisation domain is an intramolecular process that does not require dissociation from the DNA which facilitates productive replication. They suggested that the YxGG/A motif and other non conserved residues could play an important role in DNA transfer between the active sites of the two catalytic domains.

Figure 1.7: Multiple sequence alignment on AA level covering the region between exonuclease and polymerisation domains of protein-priming DNAPs. The ExoIII motif, putative limit of the exonuclease domain, and the "YxGG/A" motif, putative limit of the polymerisation domain, are shown. The "YxGG/A" motif and R/S/K/W/H at position 223 are suggested to be a specific feature of protein-priming DNAPs. The numbers denote the position of the first aligned amino acid with respect to the N-terminus of the corresponding DNA polymerase. Invariant residues (white over black background) and conserved residues (boldface over grey background) are shown. Figure altered, from [Truniger, 2005].

It is due to the proofreading function of the exonuclease domain that Φ29 DNAP has such high fidelity. However, the low rate of error incorporation makes it di cult to study the mechanism with direct methods. As previously described, exonuclease deficient variants were studied, as they incorporate errors more frequently. However, these mutants do not capture the behaviour of the WT enzyme. In addition, these variants cannot give insight into the coordination between the synthesising and decomposing catalytic activities of the proofreading DNAP. De Paz et al. [Paz, 2018] developped the MagNIFi assay that allows for the indirect measurement of DNAP WT and mutant error rates. It forces the incorporation of errors by withholding one of the four nucleotides. They claim that a stable fidelity assessment can be achieved by mapping back measured error rates under biased conditions to standard conditions. The Titration Assay is an assay I have developed and will present in the following chapters. It adapts the idea of titrating each one of the four bases individually from the MagNIFi of de Paz et al. This is why I will present the MagNIFi assay in more detail in the following section.

The MagNIFi assay

The rare occurrence of mismatched base incorporations or base deletions of high fidelity polymerases make error rate measurements di cult. However, error rates increase proportionally with biases in dNTP concentrations. The Magnification via Nucleotide Imbalance Fidelity (Mag-NIFi) [Paz, 2018] assay takes advantage of this observation and allows for robust error rate measurement in vitro under the condition of reduced nucleotide concentrations.

Figure 1.11: heat map representation of Φ29 DNAP WT error preferences at the rare base concentration of 10 -7 µM. Results from "T", "A", "C" and "G" template contexts are averaged. Figure altered, from [Paz, 2018].

At "T" and "G" template sites Φ29 revealed a preference for T:dGTP and G:dTTP substitutions in the MagNIFi assay, respectively [Paz, 2018]. At "C" and "A" template the respective substitutions C:dTTP and A:dATP were dominant. Impact of sequence context on DNAP fidelity is well established [Petruska, 1985][ Mendelman, 1989]. De Paz et al. were able to substantiate the findings and aimed to gain a better understanding of the specific influences of sequence context. They looked at sequence context influence in three di erent ways: At first, sequence context specific FC 50 values were compared with average FC 50 of a given template library (i.e. {logFC 50 average} -{logFC 50 fixed template base for a given template base at each position surrounding the EES}). They observed that, for example, on a BBBABBB template library (see figure 1.12) a C template base could increase or decrease fidelity depending on its proximity to the EES, while a G template base would consistently decreased fidelity (except for G template base at position +2 downstream the EES). The template base T would consistently increased fidelity (except for T template base at position -2 upstream the EES). The BBBABBB template library is particularly interesting in the scope of this thesis, as a similar template will be part of a presented assay.

Directed evolution

Summary Φ29 DNA polymerase

Φ29 DNAP has been investigated for five decades and found numerous commercial applications, both due to its high fidelity and outstanding processivity. Nevertheless, the coordination of the two main catalytic functions of the enzyme is still not fully understood. The MagNIFi assay forces DNAPs to introduce errors during replication by base titration. The assay shows how sequence context can influence error rate and substitution preferences [Paz, 2018]. In the following chapters of this PhD thesis I will show how ideas from the MagNIFi assay can be combined with approaches to challenge Φ29 DNAP variants in an evolutionary context. Before showing the results of the developped assays in chapter 4,Iw i l lfi r s ti n t r o d u c et h e concepts of so-called "Directed Evolution" (section 1.4), "Droplet-Based Microfluidics" (section 1.4.1) and "Compartmentalised Self Replication" (section 1.4.1.2) in this chapter. These concepts are combined to develop massively parallelised, ultra-high-throughput, evolutionary assays in vitro to investigate activity-fidelity trade-o s based on large (10 6 ) library screenings with next generation sequencing (NGS), using Φ29 DNAP and a homolog as a model system.

Directed evolution

Enzymes evolved over thousands of years and generations by natural selection. By undergoing the natural selection process catalytic functions were refined to enable and accelerate reactions with specificity and e ciency. The adaptation of nature's selection strategies in laboratory practices allows to guide further evolution of existing enzymes towards medical, agricultural and industrial needs. These so-called "directed evolution" approaches improve natural systems but can also introduce new-to-nature reactions. They are faster than natural evolution and o er better control over the traits to select for, making engineered enzymes an alternative to traditional chemical toolboxes [Stucki, 2021]. The impact of protein engineering as a key biotechnological tool has been recognised by the 2018 Nobel Prize in Chemistry, awarded to Frances H. Arnold for the directed evolution of enzymes and George P. Smith and Sir Gregory P. Winter for phage display of peptides and antibodies. Directed evolution is an iterative process which typically consists of three steps: 1) Generation of genetic diversity by the introduction of mutations, 2) the screening for a desired property, and 3) selection of the best performing variant. A typical directed evolution cycle is depicted in figure 1.15.

Chapter 1. Introduction A further stage of miniaturisation was achieved by adapting compartmentalisation as practised in nature: The compartmentalisation in cells that -in the case of bacterial cells -typically have volumes in the range of femtolitres. This 10 9 -fold miniaturisation of the reaction volume marks the step from high-throughput to ultra-high-throughput screens. The femtolitre-sized cell-like compartments utilised in the presented thesis are organised in water-oil emulsions. Already a 30µM reaction volume dispersed in 3 femtolitre droplets -typical volumes in the presented experiments in chapter 4) -equals the amount of approximately 6 * 10 7 microtitre plates with 1536 wells.

In vitro compartmentalisation

The idea of cellular compartmentalisation seen in nature has been transferred into the laboratory to perform In vitro compartmentalisation (IVC) in volumes of similar size [Gri ths, 2006]. Depending on the emulsification technique the reaction volumes can be adjusted from 1µM to 100µM diameter. All reagents of a chemical or biological reaction are combined to form an aqueous phase. The dispersion in an oil phase ensures that each droplet is a separate microreactor in which an independent reaction can be carried out [Tawfik, 1998]. This makes IVC a useful tool for the directed evolution of proteins: In this application genes that code for variants of a protein are encapsulated together with an in vitro transcription translation system (IVTT). The latter allows for the in vitro expression of the variant and its subsequent study in specific conditions [Agresti, 2005], [Gri ths, 2003], [Miller, 2006], [START_REF] Mastrobattista | High-throughput screening of enzyme libraries: in vitro evolution of a β-galactosidase by fluorescence-activated sorting of double emulsions[END_REF], circumventing the need for transformations and thus increasing the throughput (see figure 1.17). The linkage between genotype and phenotype is crucial in the evolutionary process. In nature it is ensured by the compartmentalisation of genes in cells. In IVC it is likewise maintained: First, the encapsulation of only approximately one gene every 10 droplets in IVC ensures that only few compartments are co-encapsulating two or more genes. Second, the reaction volumes are not in exchange with one another after completion of encapsulation. Gene encapsulation follows the Poisson distribution just like single-cell compartmentalisation [Joensson, 2012]. Originally, IVC was limited to DNA modifying enzymes. However, with advancements, including the combination of IVC with FACS and with droplet-based microfluidic systems, the activity of many enzymes was improved [Gupta, 2011], [Ma, 2014], [Ostafe, 2014], [Fallah-Araghi, 2012]. ash deposit), calcium carbonate (CaCO 3 ), and boric acid (B(OH) 3 ). Spinodal phase separation at high temperature and subsequent washing out of the separated CaCO 3 /B 2 O 3 -rich phase (calcium carbonate and boric anhydride) with acid. Adjusting the parameters of phase separation enables manufacturing of membranes with di erent pore sizes [Kukizaki, 2004][ Kukizaki, 2010] as shown in figure 1.22 a) and b). The highly interconnected non-circular pores have a density of 10 5 -10 9 cm -2 . Pore size can be adjusted precisely and measurement of pore size distribution in the case of an exemplary 1 µm sized membrane is shown in figure 1.21 b). The negatively charged hydrophilic membrane is 700-900 µm thick and can be turned hydrophobic by coating with polyvinylchlorid (PVC), Polymethyl methacrylate (PMMA) or silicone resin, as shown in figure 1.22 c) [VladisavljeviÊ, 2007], [Kukizaki, 2009], [Technology Co, 2021]. external pressure system (SPG membrane, direct ME), c) internal pressure system (SPG membrane, direct ME), d) stirred cell (SPG membrane, direct ME), e) external pressure system (SPG membrane, premix ME). f) in the study of Vladisavljevic et al. [VladisavljeviÊ, 2019] a nanoemulsion was produced with a syringe filter by i) premix formation by pushing back and forth pure oil and pure aqueous phase several times between two syringes and ii) premix homogenisation by pushing it back and forth several times through a syringe filter mounted between two syringes. Dispersed phase is coloured in red and continuous phase in blue. Figure from [VladisavljeviÊ, 2019].

Emulsification

In-emulsion biological assays depend on inert compartments that isolate the independent reactions taking place in each individual microvessel. Fluorocarbon oils like HFE 7500 are both hydrophobic and lipophobic which results in poor solubility of organic molecules like the ones present in a typical IVC reaction. In this way, fluorinated carrier oils limit droplet-droplet molecular di usion and thus cross-contamination between aqueous droplets. It is known that the interface of dispersed aqueous droplets in oil is under high surface tension. Droplet merging reduces this tension by minimizing oil/water surface area. To prevent coalescence, hence to stabilise the emulsion, surfactants can be added to the continuous phase. These Chapter 1. Introduction [START_REF] Brouzes | [END_REF]. Fluorosurfactant-stabilised microfluidic droplets are stable in temperatures below 80°C. In a later study, Chowdhury et al. [Chowdhury, 2019] investigated emulsion stability during PCR cycling that includes temperatures above 80°C. The PEG-PFPE 2 surfactant showed substantial droplet merging after PCR cycling. Another tested surfactant was made up of a tri-glycerolbased dendritic polar head (dTG), with four hydroxy (-OH) groups, and PFPE polymer chains of low molecular weight fluorine chains (L-dTG). The use of the latter surfactant resulted in significantly less droplet coalescence during PCR, while preserving droplet stability at room temperature over 30 days.

Membrane fouling

An obstacle in the use of ME technology is the deposition of particles or solutions on the membrane surface or in the pores. Membrane fouling can lead to membrane degradation and performance decrease. In this context, Nakashima et al. [Kobayashi, 2003] studied the e ect of surfactants on hydrophilic (untreated) SPG membranes in direct emulsification to prodce oil-in-water emulsions (refined soybean oil with 2.0 wt.% Tri-n-octylmethylammonium chloride (TOMAC) and demineralised water with SDS, polyoxyethylene (20) sorbitan monolaurate (Tween 20) or cetyltrimethylammonium bromide (CTAB) in varying ratios). They concluded that the functional groups of surfactants must not carry the charge opposite to the one of the membrane surface. Otherwise electrostatic attractions occur between emulsifier and membrane surface which can lead to increased deposition and pore plugging. Vladisavljevic et al. [VladisavljeviÊ, 2006] produced premix oil-in-water emulsions (40 wt % corn oil with 0.06-2 wt % sodium dodecyl sulfate (SDS) or 0.1-2 wt % Tween 20 at pH 3 or 0.5-2 wt % β-lactoglobulin at pH 7) with an SPG membrane of 8.0 µm, transmembrane pressure of 100 kPa and five consecutive passes. They reported changes in transmembrane flux due to protein adsorption and assumed that membrane fouling occurred due to electrostatic attractions between positively charged groups on β-lactoglobulin and the negatively charged untreated membrane. To my knowledge, no study has investigated fouling of hydrophobic (silicone resin coated) SPG membranes in the premix production of water-in-oil emulsions. No study on fouling has been conducted with block co-polymer surfactant PEG-PFPE 2 in fluorinated oil and no study looked at the fouling e ects if the aqueous phase contained IVTT or other biomolecules like DNA or mRNA.

Summary

As was shown in the previous chapters, numerous studies have been conducted over the last decades to understand the sophisticated machinery of DNA replication. However, some aspects of the Φ29 DNAPs catalytic functions, e.g., the balancing of synthesis and proofreading, remain poorly understood [Ibarra, 2009]. The fine coordination between exonuclease-and polymerisation domains is crucial to ensure high processivity, while faithfully copying the DNA template.

Summary

The linker region located between the putative limits of the two synthetic and decomposing catalytic functions is suspected to have a major influence on this balance. Emulsions present a convenient tool to upscale in vitro directed evolution studies into the realm of ultra-highthroughput screenings of large mutant libraries. Membrane emulsification is most commonly used in the food, cosmetics and pharmaceutical industries [Joscelyne, 2000]. To my knowledge, it has never been applied to compartmentalisation in IVC, CSR and the directed evolution of enzymes. Yet, possible advantages of membranebased emulsification technology for the aforementioned applications are manifold. Membrane emulsification could be particularly suited for biological assays as aqueous phases of microlitre range volumes can be partitioned into droplets in the low micrometer size range with pumping connectors. Current on-chip emulsifications in microfluidic set-ups take time, specifically when the emulsification process relies only on one single nozzle. In this case the emulsification of typical reaction volumes of V = 10µL -30µL takes one to three hours. Yet, they provide fine control over droplet parameters and uniformity that cannot be reached with ME. In the case of premix emulsification, ME provides better control over droplet dispersity than other bulk emulsification techniques.

In vitro isothermal Compartmentalised Self Replication is a tool to study polymerases in vitro. However, the kinetics of the iviCSR reaction are not yet well understood. Membrane emulsification o ers the possibility to emulsify an aqueous phase quasi instantaneously. This does not only save time but can o er new possibilities for CSR kinetics studies and directed evolution assays, which will be shown with the QuIK Assay in chapter 4.2. Many studies have been done to quantify di erent parameter that influence the properties of ME-emulsions produced with SPG membranes. They are covered, for example, in reviews from Joscelyne et al. and Vladisavljevic et al. [Joscelyne, 2000], [VladisavljeviÊ, 2019]. However, these studies do not align with the needs of directed evolution. Usually, oil phases like soybean oil, coconut oil, toluene, mineral oil, kerosene, or corn oil are used. Surfactants are usually Span 80, Tween 80, Tween 20, SDS, Pol, Tyl or SL. To my knowledge, none of the reported studies used an aqueous phase that contained IVTT or any other reagents used in IVC (except for H2O) [Joscelyne, 2000], [VladisavljeviÊ, 2019]. In the previous IVC and CSR studies at the Laboratoire de Biochimie at ESPCI Paris Tech, HFE7500 and fluorosurfactant have been used due to their stabilizing properties and biocompatibility. Additionally, PEG-PFPE 2 fluorosurfactant is available commercially (009A-FS from RAN Biotechnologies), while L-dTG fluorosurfactant is not. The aqueous phase is an optimised IVTT system in purified H20. As the used materials have proven to be a good selection in previous experiments, I decided not to change these parameters but to investigate suited parameters for membrane emulsification with an SPG pumping connector tailored to directed evolution applications. In this work I will follow three main goals: 1) Application of ME to in vitro expression and replication assays to study iviCSR kinetics. 2) Application of ME to in vitro directed evolution studies. 3) Adaptation of the idea to force Φ29 DNAP mutants to incorporate errors -inspired by the MagNIFi assay [Paz, 2018] -to an evolutionary setting and derive insights into exonuclease and polymerisation balance by NGS screening of large mutant libraries.

Chapter 2

Scope of the Thesis Φ29 DNAP derives from bacteriophage Φ29 and replicates DNA under isothermal conditions by rolling circle amplification. It is a particularly interesting enzyme due to its outstanding polymerisation activity, processivity and low error rates in the range of 10 -5 to 10 -6 nucleotides. This high fidelity is due to an additional catalytic function: The ability to correct for base misincorporations by degradation of single stranded nucleotides. In the case of Φ29 DNAP this proofreading function is carried out without dissociation from the template. Since its discovery in 1984, extensive research has been conducted on the polymerase but the coordination between its main catalytic functions, DNA synthesis and proofreading, is not fully understood. In order to switch between the opposing functions, the physical confirmation of the nascent DNA strand is transferred from the active site of one domain, to the active site of the other domain. DNA synthesis and excision of nucleotides during proofreading are opposing catalytic functions and require to be finely balanced to ensure high synthetic rate, while retaining faithful DNA replication. A region located between the coding regions of polymerisation and exonuclease domain on the Φ29 DNAP gene is the so-called "linker". This region is involved in the coordination between DNA synthesis and nucleotide excision. However, questions about its influence on the balance of activity-fidelity trade-o s remain open. For instance, to my knowledge no mutation in the linker region has yet been found, that could circumvent transfer of the nascent DNA strand from polymerisation to exonuclease domain. Many laboratory techniques are available for the study of protein function. I will combine ME, directed evolution, iviCSR and NGS to study the interplay between DNA synthesis and proofreading, as follows: Directed evolution is a laboratory technique that mimics the natural selection process and guides selection towards a defined goal (i.e., a phenotype trait) by application of a selection pressure. By iviCSR, DNAP genotype and phenotype are isolated in a compartment that mimics cellular encapsulation. The process links genotype and phenotype and involves both, expression of the protein from the gene and replication of the gene by the enzyme. These properties make iviCSR a suited tool for the directed evolution of DNAPs in vitro.

Chapter 2. Scope of the Thesis

To accelerate the compartmentalisation process and to enable the study of iviCSR kinetics, a membrane emulsification technique will be adapted to compartmentalise iviCSR reactions virtually instantaneously. This emulsification method will then be used to develop and conduct assays for both, anin vitro directed evolution assay (screening for DNAP activity) and studies of the iviCSR kinetics. DNAP variants with increased activity -as determined by NGS screening -will be studied in follow-up bulk assays to validate the anticipated activity increase.

To study the influence of the linker region on the balance of activity and fidelity trade-o s in Φ29 DNAP, an additional assay is developed: The starvation of variants by titration of individual dNTPs will increase selection pressure to challenge both, the polymerase activity and fidelity. Variants, screened in the assay, will hence be synthesised and subjected to further investigations. The findings can provide information about the contribution that certain AA positions have on the balance of DNA synthesis and proofreading. In conclusion, the goals of my PhD research are threefold: 1) Development of a method to investigate iviCSR kinetics, 2) gain of insight into the interplay between polymerase activity and fidelity, using Phi29 DNAP and a homolog as a model system, and 3) development of massively parallelised, ultra-high-throughput, iviCSR-based evolutionary assays to investigate mentioned trade-o s based on large (10 6 ) gene libraries.

In chapter 3 I will present the Materials and Methods that were applied to obtain the result presented in chapter 4. In chapter 5 the results will be discussed and connected to the literature, presented in chapter 1, before an outlook is given in chapter 6. Supplementary material can be found at the end of the thesis.

2. In vitro isothermal compartmentalised self replication SuM 7) in order to allow the selective digestion of template DNA later on in the workflow (using the DpnI restriction enzyme). The library in DB-format is compartmentalised in water-in-oil droplets of ∼1-3 fL volume, depending on the applied emulsification technique. The continuous phase is here provided by an inert fluorinated carrier oil (protocol in SuM 9). The aqueous phase contains an optimised IVTT system as well as dNTPs and primers that target the single stranded adapters. The complete reaction mix of the aqueous phase is presented in SuM 1. Each droplet acts as an independent compartment, i.e. the occupancy of a specific droplet with one gene does not change the probability of occupation of another droplet. Dumbbell template concentration is in the range of 12.5 pM to 57.5 pM in reaction volumes between 30 µL and 210 µL total volume. This ratio ensures that on average only one in 10 or more droplets will contain a gene (⁄ ≤ 0.1, see also table 3.1). The gene occupation in the droplets follows a Poisson distribution in which only ∼0.5% of the droplets will be occupied by several genes. Droplet Radius (µm) Droplet Volume (fL) DNA Input concentration (pM) ⁄ 0.6 1 12.5 0.01 57.5 0.03 0.9 3 12.5 0.02 57.5 0.10 Table 3.1: ⁄ values for di erent droplet sizes and DNA input concentrations.

The optimised reaction mix leads to transcription and translation of the gene and results in a feedback loop in which, in each compartment, polymerase enzymes interact (and, if active, replicate) their own gene. The template's DB format allows an active variant to make multiple copies of its own gene in an RCA replication. The IVTT system is stable at 30 °C for ∼4h. Before this time, expression and replication are coupled. Afterwards, only replication is occurring. Usually the emulsion is incubated for 16 h before the reaction is heat inactivated and the emulsion is broken (protocol in SuM 9.5). As mentioned above, the DB-shaped DNA template is methylated in vitro, whereas the RCA product, synthesised during incubation, is not. This allows for the selective digestion of template DNA by DpnI after selection (protocol in SuM 11). The recovery of the digested output library of the coupled assay consists of a recovery PCR (recPCR), a Gibson Assembly to form a circular dsDNA product, and a second PCR (hereafter: "outPCR"). The workflow of library recovery is shown in figure 3.2 and the corresponding protocols are found in SuM 12 and 15. A sample (sim0.1 pmol) of the PCR product can be prepared for NGS sequencing and the rest of the product (after outPCR usually in the range of sim1-2 pmol) is reformatted into DB shape to serve as input for the next round of selection (see also section 3.2.2).

2. In vitro isothermal compartmentalised self replication carried on using a "mock" library composed of only two genes: A Φ29 DNAP WT gene (Q1WT) and an inactivated mutant of Φ29 DNAP (Q1DD) with mutations leading to an inactive phenotype (figure 3.4). In a "model selection", the library comprises only of these two genes (in this case, the concentration of both genes is set to 12.5 pM), which allows me to quantify selection pressure via the relative enrichment of Q1WT over Q1DD after one round of selection. With just two variants, such enrichment is easily and quickly measured via qPCR (figure 3.3, see also SuM 12.2). Crucially, the two genes used in the model selection are cloned in a di erent backbone from the one used for libraries. In this way, the two genes can be mixed with a more complex library (in this case, the concentration of both genes is set to 8.2 pM). Q1WT and Q1DD act here as an internal quality control for the emulsification, e.g. to check for co-encapsulation (excessively high λ). After selection, the library can be selectively recovered by PCR without contaminating it with the quality control genes.

Illumina short read sequencing

The Illumina platform takes short linear fragments, typically in the range of 25 to 300 bp, as templates. The workflow is illustrated in figure 3.7 and proceeds as follows: First, specific adapters are added to the template that allow hybridisation on a flow cell. This picture does not show the generation of the second read: After completion of the first read, the read product strand is dehibridised and washed o . A complementary strand is synthesised through bridge amplification. The bridge is dehibridised and the already read forward strand is cleaved and washed o , leaving the reverse strand which can then be sequenced to give read 2. This schematic representation is not to scale. Figure from [Weinstock, 1752] Afterwards, the hybridised template strands are pre-amplified by parallelised bridge amplification (clustering) in a pre-processing step to enhance the signal in the following sequencingby-synthesis step. In the following step, the first read of the clustered templates is obtained with sequencing-by-synthesis. The read product is then cleaved and washed o and a complementary strand is synthesised by bridge amplification. In the following step the second read is obtained. This time the complementary strand serves as template for sequencing-by-synthesis. The fluorophore-label nucleotides serve as a terminator for polymerisation, allowing for simultaneous detection of base incorporation by laser excitement. Subsequent enzymatic cleavage of the nucleotide label allows for incorporation of the next nucleotide.

Comparison of long and short read Sequencing

A comparison of short (PacBio system) and long (Illumina system) read NGS systems is given in table 3.2. The main advantage of the Illumina platform is its high read depth in the range of several hundred thousands to millions of reads. PacBio read depth is in the range of approximately one hundred thousand reads and was in the concrete example of the H0-25 Activity Assay (presented in chapter 4.6) in the range of 10.000 reads. The in-and output libraries of this assay were, in fact, sequenced on both platforms, Illumina and PacBio. The PacBio system, however, is able to read an entire gene of a theoretical maximal length of 25 kb in one read. This advantage made it particularly useful for a random mutagenesis study, presented in chapter 4.5, in which the e ects of mutations across an entire Φ29 DNAP gene were studied.

Read

Next generation sequencing data Analysis

The NGS data analysis workflow is depicted schematically in figure 3.8. Illumina NGS data was stored in .fastq format and scanned with FastQC version 0.11.9 before merging with Flash, version 1.2, and alignment with Bowtie2, version 2.4.5. PacBio NGS data was stored in .fasta format. As it is provided as a single read, no merging was required and sequences could be aligned directly with bowtie2. The output file was in an indexed .bam file which was further processed in R (version 1.2.5001) on a personal computer (Dell Inc. Precision 3630 Tower with an Intel(R) Core(TM) i7-8700 CPU 3.20GHz, 6 Cores and 12 Logical Processors). For the Illumina data counts were in the range of 1.5 Mio for the H0-25 DNAP library and 900.000 reads for the Φ29 DNAP library. Bowtie2 alignment rates were usually in the range of ∼ 60to90%.

Next generation sequencing

In general, an enrichment of E>1 in a round >=1denotes a mutation under positive selection. E<1 denotes a negative selection and and a mutation with E =1is neither under negative nor under positive selection. The "Clone-Level-Enrichment" (CL-Enrichment) between round R0 and any other round R is then calculated by

E CL = f Cl,R f CL,R=0
.

Clone-level analysis is intuitive but it can face important sampling problems. When libraries are large (> 10 7 ), as it is the case, for example, in mutagenic libraries, and enrichments are low, individual clones have just a few counts even at the maximum throughput of Illumina sequencing, leading to a large sampling error. Alternative analysis methods are presented in the following sections.

Position-wise analysis

'Position-wise analysis' measures conservation at a every sequence position, by quantifying enrichment/depletion of the wild-type amino-acid. For each position, all reads are piled-up to measure the counts of every substitution. Typically, i.e. in cases in which mutagenesis is not extreme, the AA with highest count at a given position x (n max,x ) corresponds to the WT (coloured in blue in figure 3.10 A)). The "Position-Wise-Frequency" (PW-Frequency) of the WT is thus:

f PW,WT,x = n max,x q x n x .
The PW-Frequency of mutants at position x is

f PW,Var,x =1-f PW,WT,x ,
The "Position-Wise-Enrichment" (PW-Enrichment) of the entity of variants at position x between round R0 and any other round R is then calculated by

E PW,Var,x = f PW,Var,x,R f PW,Var,x,R=0

Summary

In vitro isothermal compartmentalised self replication is the basis for the assays developed during this PhD project. Alongside the wet laboratory work I developed an NGS analysis pipeline to screen gene libraries of developed iviCSR-based assays according to CL-,PW-and SpS-enrichment. The results will be presented in the following chapter.

Chapter 4

Results

This chapter presents the development of the "QuIK" emulsification assay and four iviCSR-based assays: With the "Time Assay" I developed a better understanding of the iviCSR kinetics. With a random mutagenesis study I gained insight into the e ect of mutations along the Φ29 DNAP gene on the enzyme's activity. With this understanding I was able to find variants of a Φ29 DNAP homolog with improved activity in the "Activity Assay". In the following "Titration Assay" I challenge Φ29 DNAP variants in altered environmental conditions. The behaviour was then modelled and found variants subjected to further orthogonal assays.

Introduction

The results I present in this chapter are all related to iviCSR assays, a method to tailor enzymes in the lab via directed evolution.

The core part of my PhD is the application of membrane emulsification to iviCSR. This, to my knowledge, has never been utilised before in CSR or in direct evolution assays. I named this approach "Quasi-Instantaneous Emulsification for directed Evolution and Kinetics Studies" or "QuIK". Its development is presented in section 4.2. An essential di erence between on-chip emulsification and QuIK is that in the latter I can prepare the whole emulsion at once. In on-chip emulsification, on the other hand, an hour elapses between the encapsulation of the first and the last DNA molecule, so that the reaction starts at di erent times in di erent droplets.

While it is possible to slow down the reaction during the emulsification process by keeping samples at low temperature this is imperfect and practically challenging. The synchronization of QuIK reactions allows me to precisely characterise the kinetics of iviCSR by measuring the accumulation of DNA and RNA products. Also, QuIK is several orders of magnitude faster than chip emulsification with a single nozzle (seconds compared to hours) this becomes very important when juggling multiple selections in parallel as in the case described in section 4.8. Moreover, QuIK can be used for the selection with random mutagenesis libraries, such as the one presented in section 4.5. These large scale mutagenesis studies have motivated the development of the so-called "Activity Assay" (section 4.6) in which variants of the Φ29 DNAP homolog H025, carrying more localised mutations on and close to the TPR2 subdomain, have been subjected to rounds of QuIK-based selection.

For comparison, competing directed evolution assays in vitro require more than 20 rounds to achieve a similar increase in activity [Sakatani, 2018]. The Activity Assay is a one-dimensional directed evolution assay that characterises variant phenotypes based on a single aggregate parameter: "activity". The parameter is measured via the variant's overall DNA synthesis in the iviCSR reaction, which depends on multiple, di erent, microscopic parameters such as protein expression levels, a nity for the template, kinetic rates and processivity. To overcome the limitation of a one-dimensional screening, I developed the "Titration Assay" (section 4.8), which is a two-dimensional screening method, developed to select DNAPs based on two parameters -activity and fidelity. In this assay Φ29 DNAP variants are challenged in altered environments and mutants are selected that thrive better in di erent challenging conditions. Here, the main advantage of QuIK, i.e. the ability to create emulsions instantaneously, facilitates the testing of four conditions across two rounds of selection. NGS data obtained from the Titration Assay libraries enabled me to model average mutant behaviour and better understand the parameters of the assay. Two modelling approaches are presented in section 4.10 that aim to rationalise the findings.

With the results of this study, I can show that it is possible to identify several promising Φ29 DNAP variants using the Titration Assay. Further characterisations with di erent orthogonal assays are presented in section 4.12.

QuIK ME relies on an extruder that can be installed between two syringes (picture 4.1). The company SPG Technology CO., LTD. commercialises an extruder with an SPG membrane (SPGE,S u M9.1). The SPGE comes as a single use product (a syringe pumping connector) available with membranes of di erent pore sizes and was chosen because it is suited for microemulsification. SPGE has previously been applied to many applications in the food and pharmaceutical sectors since the 1990s. An SPGE is installed between two syringes and an emulsion is conveniently created by pushing two phases (aqueous and oil phase) multiple times back and forth through the extruder.

The maximal volume of the emulsion (dispersion phase and continuous phase), however, can be up to 2.5 mL, depending on the syringes used [Technology Co, 2021]. The QuIK protocol is described in the Supplementary Material (SuM 9) and can be carried out in less than one minute. Before being able to apply QuIK to iviCSR assays I needed to gain a clear picture of the characteristics of QuIK made emulsions. First, to develop a protocol that gives emulsions with droplet size and -dispersity suited for iviCSR, I examined the e ects of the following six parameters:

• pore size of the extruder membrane

• type of aqueous phase

• number of pushes through the extruder

• emulsion stability

• extrusion speed

• fouling e ects on the membrane Characterisation of the droplets' physical parameters (size and dispersity) was carried out using Static Light Scattering (SLS), in Supplementary Material 9.4. Emulsion characterisation in terms of ivi-CSR-based model selection, on the other hand, was done by qPCR (SuM 12.1, and SuM 12.2).

Physical characterisation of the emulsion: Droplet volume and degree of polydispersity

The key parameter in IVC is droplet size. To achieve e ective concentration in the nM range (i.e., the molar gene concentration in an occupied droplet), as required by many reactions, I need to produce micrometric droplets. The first investigation was to confirm that QuIK ME can produce droplets in this range. I started out performing emulsification using de-ionised water for the dispersed phase and fluorinated oil and sufactant for the continuous phase. I observed that membranes with smaller pore sizes produce emulsions with smaller droplets (figure 4.2), as was expected due to previously reported proportionality between droplet-and pore-size (chapter 1.5). Furthermore, smaller pores in the membrane also give a more uniform droplet distribution. This latter observation is crucial, as ⁄ values can only be ensured in emulsions with a high degree of monodipersity.

In the case of the 3 µm and 1 µm pore diameter membranes, the force necessary to push the water-oil-mixture through the membrane increased to an extent which rendered extrusion virtually impossible. This was also confirmed by the manufacturer and is the reason why the membrane with 5 µm pores represents the lower end of the manufacturer's product range (the lower pore size membranes were custom-made). For this reason, I selected the 5 µm pore sized membranes (hereafter called 5 µm membrane) to conduct all further tests. To better approximate the iviCSR reactions, the dispersed phase was then changed from water to the iviCSR reaction (SuP 1, however, now DNA template was added, yet). Emulsification of the iviCSR reaction mixture lead to 9.2-fold decreased mean droplet radius and 6.3-fold decreased standard deviation (sd) of the size distribution with respect to the emulsification of an aqueous phase containing only water with a similar membrane. I rationalise this finding based on the di erence in viscosity between the two types of the aqueous phase.

Having already investigated the e ects of membrane pore diameter and aqueous phase, I continued with the investigation of droplet size and polydispersity based on number of pushes through the membrane. The polydispersity decreases with an increase of pushes. Standard deviation is 340 nm for five pushes. After 20 pushes the standard deviation decreased to 170 nm. Again, the negative slope of the curve increases with increasing number of pushes: An additional five pushes leads to a 1.5-fold decrease of polydispersity when starting with five pushes. However, after 15 pushes have been carried out an additional five pushes will decrease polydispersity only marginally by 1.08-fold. At 10 pushes the mean droplet radius is r =0.6µm with a standard deviation of sd =0.2µm. I envisioned the upcoming selection assays to have a maximum incubation time of 16 hours at an incubation temperature of 30 °C. There is a potential for droplets to either shrink or coalesce during this time. Comparison of fresh and incubated emulsions, however, reveals only a small increase in droplet radius (i.e., below the experimental resolution for 5 and 10 pushes, "r =0.06 µm and "r =0.04 µm for 15 and 20 pushes, respectively). For the radius' standard deviation I observe a very similar behaviour (figure 4.3 b)). Here, almost no di erence is observed between the fresh and incubated triplicate emulsions. After 16

The emulsification procedure constitutes of consecutive pushes of premix through the extruder. The first forward push corresponds to push #1, then the syringe is flipped and the following backward push (manipulating the opposite syringe) corresponds to push #2, the syringe is flipped again and the process starts over, now with pushes #3 and #4 etc. To quantify the extrusion speed, emulsification with syringe pumps was tested. The first trial was carried out with the "Harvard Apparatus PHD 2000 Infuse/Withdraw" syringe pump. However, the maximal speed of s = 220.82mm/min is still slower than what is acheived via hand-driven emulsification. The Cetoni Nemesys Base 120 syringe pump system was also tried. The system reaches higher speeds but does not provide an infusion/withdrawal mode. In addition syringe fixation was inadequate and resulted in the syringe falling out of the system. Several SPGE broke during the trials with the Nemesys system each time leading to the loss of the entire sample. Di erent operators of the QuIK protocol will manipulate the syringes with di erent speeds. However, I expect that di erent emulsification profiles will not a ect emulsion quality. For this reason, the hand driven syringe operation was kept. Emulsions made with fast handdriven pushes (approximately one push per 0.5 seconds) gave smaller droplet sizes (and higher degree of polydispersity) with respect to slow hand-driven pushes (approximately one push per two seconds, see SuM 9.3, results not shown). This is consistent with the manufacturer's advice to apply fast speeds for SPG-ME. Unless otherwise stated, fast hand driven pushes were carried out in all following investigations.

As mentioned in chapter 1.5.0.2, fouling generally a ects performance of membranes. Hence, it was important to understand the e ect of fouling on the SPGE membrane in the specific case of iviCSR reactions dispersed in a fluorinated oil-surfactant phase. I wanted to know if the membrane could be reused. Emulsions produced with a new extruder (containing a new membrane) were thus compared to emulsions produced with an extruder that had previously been used 15 times with a IVTT in water mixture. Both membranes with an average pore size of 5 µm (dispersed phase was pure water, figure 4.2). Surprisingly, the emulsion produced with an already used membrane showed 3.6-fold smaller mean droplet radius and 2.9-fold decreased standard deviation of the size distribution compared to the emulsion produced with a similar but new membrane. Storing the membrane in between usages does not alter this e ect (data not shown) and an increase in the force required for extrusion was not observed with reused membranes. I decided that I could reuse membranes (but only in assays that use the same iviCSR template to avoid sample contamination) and that new membranes should be passivated prior to their first application.

Emulsion characterisation in terms of iviCSR-based model selection

As it can be seen from figure 4.3, droplet size and droplet polydispersity decrease with an increasing number of pushes through the SPGE. However, the emulsification technique has to be tailored to the iviCSR reaction stipulated to take place in the droplets. DB-shaped Q1WT and Q1DD templates were added to the iviCSR reaction mix 2). This allowed me to assess iviCSR e ciency by qPCR in terms of di erential increase between Q1WT and Q1DD (figure 4.6, see also SuM 12.2). I observed a drop in e ciency when increasing the number of pushes to 15. An additional test was carried out with a slow hand driven emulsification and no significant di erence could be observed in the qPCR analysis, with respect to the fast emulsification of same number of pushes. Hence, the manufacturer's advice of conducting fast pushes was followed in all further experiments, to maintain consistency. There seems to be a trade-o between droplet radius, and the ability of QuIK-based iviCSR to amplify genes when the number of pushes is increased. I explain the drop of e ciency by an increased impact of shear forces on the IVTT mix with increased membrane passes. I expect sensitive components of the mix, like T7-RNA polymerases and ribosomes, to gradually deteriorate with every pass through the extruder. A 10 passes procedure appears to be a good trade-o as it gives good results in both, droplet size and gene amplification capacity of iviCSR. This parameter is selected for all following experiments.

QuIK compared to on-chip emulsification

The comparison shown in figure 4.7 reveals that the extruder-based application has several advantages with respect to the commonly applied on-chip emulsification. In terms of emulsification speed QuIK allows the process to be completed two orders of magnitude faster than on-chip, requiring only seconds rather than hours. Ultra-high throughput of 10 1 1 compartments allows for unprecedented quasi-instantaneous library emulsification of up to 10 1 0 variants. Furthermore, with QuIK I am now able to produce droplets with average diameters of V = 3 fL instead of 30 fL, i.e. a 10-fold reduction in average droplet volume. These benefits come with a moderate increase of polydispersity which is now at SD/mean = 30% vs. 5% on the radius. A six-fold increase of polydisperisty whose impact is moderate due to the greatly reduced average droplet radius. Finally, the convenient manual operation of QuIK is another very important advantage of the newly developed emulsification assay. This comparison marks the completion of the development of QuIK. In the following, the technology will be used to study the kinetics of iviCSR and to instantaneously emulsify large gene libraries for the direct evolution and investigation of DNAPs.

5. A random mutagenesis study shows that exonuclease domain and TPR2 subdomain changes a ect activity in iviCSR.

a change at a sequence position will result in a gene that codes for a variant with higher polymerisation activity. More likely, however, the mutations will have a negative impact on activity, as nature has already optimised the enzyme over time through the natural evolution process. I am interested in the regions that have the highest impact on activity of the enzyme. This parameter can be measured in terms of the enrichment of variant genes in the autocatalytic iviCSR reaction. I expected that mainly the areas of the gene that code for the polymerisation domain will have an impact on its activity, as this domain is directly linked to the enzyme's ability to synthesise the copy strand. Figure 4.13 maps the conservation patterns found in figure 4.12 to the three-dimensional structure of Φ29 DNAP. Comparison with literature [Blanco, 1989][ Bernad, 1990a][ Blasco, 1990][ Bernad, 1990b] reveals that the red areas correspond with the following regions: In the exonuclease domain there is the ExoI motif (E1) [Bernad, 1989], the ExoI motif (E2) [Esteban, 1992], and the ExoIII motif (E3) [Esteban, 1992] (see also chapter 1.3.4). The latter comes o just above the previously mentioned 10% threshold marked in red in figure 4.12.T h el i n k e r region (LK) [Truniger, 2005] balances exonuclease and polymerisation domain (see also chapters 1.3.5 and 1.3.2). The PolA motif [Delarue, 1990][ Blasco, 1993a] (PA) coordinates one catalytic magnesium and the PolB (PB) motif interacts with the incoming nucleotide [Delarue, 1990][ Blasco, 1993b] [Saturno, 1997], while the PolC (PC) motif coordinates the second catalytic magnesium [Delarue, 1990]. Also showing up in the screening are the key thumb subdomain residues (see also chapter 1.3.2), that stabilise the primer-terminus at the polymerisation active site [Bonnin, 1999], a loop in the TPR1 subdomain (292-365) that has only been recently characterised experimentally [Del Prado, 2019] and the loop at the top of the TPR2 subdomain (398-420) that is involved in strand 6. iviCSR improves the activity of H025 DNAP Due to the correlation of obtained PacBio and Illumina NGS data set and the higher depth of Illumina data I will focus on the analysis of the short read NGS dataset in the following (figure 4.17). At first, I identified the top seven variants with the highest Clone-Level-Enrichment (CLE, see chapter 3.3.4.1), averaged over four rounds. Next, I inspected CL-enrichment across rounds. For this analysis haplotypes with the highest average CL-enrichment throughout rounds R1 to R4 were plotted. For each haplotype the corresponding coding AA variant was determined and average CL-enrichment of the AA variant across all its haplotypes present in the sequencing data was also obtained. The presented 21 most enriched nucleotide sequences each code for a di erent AA variant.

The mutated region at positions 227-229 does not form part of the TPR2 region. In this library variant LTS is not performing as well as its six companions throughout round two and three. In round four, however, it outperforms the other variants, reaching a maximal CL-enrichment of ECL = 925. It is the only variant of the inspected pack, that shows consistent CL-enrichment across rounds. Most variants stagnate or decrease CL-enrichment accumulation in round three and further loose CL-enrichment in round four.

Variant DKR shows a high CL-enrichment of ECL = 817 in round 2 but plummets in the course of rounds three and four to an CL-enrichment of ECL = 215. Variants LRH, RKP and VQT show CL-enrichments of ECL = 403, ECL = 520 and ECL = 587 in round four, respectively. For the mutated region at positions 413-415 the highest CL-enrichment levels are observed. Variant RKC reaches the level of ECL = 1634. VNR is consistently enriched until ECL = 1232.I n round three a drop in CL-enrichment is seen for four of the seven inspected variants. For the library with AA substitutions at positions 430-432 the lowest CL-enrichment values are observed. Ten-fold less. The seven most enriched variants of this library show approximately 10to 15-fold lower CL-enrichments than the variants of the previously mentioned library. However, the seven variants all show consistent CL-enrichment throughout each of the four rounds. Here, mutant LKG is performing best with an CL-enrichment of ECL = 104. The CL-enrichment values of all plotted variants are shown in SuM 2 tables 7.3, 7.4 and 7.5. The highest enriched haplotype of the respective WT is shown at the bottom of each table. Due to the library composition of three sublibraries, each WT also comprises of the pooled other two libraries. For this reason, the WT value is not very meaningful. Also in the PacBio NGS dataset, library 413-415 showed highest and library 430-432 lowest CL-enrichment. 10 promising variants were selected for each mutation region to be synthesised (SuM 10) and tested in a bulk replication reaction on a dumbbell reporter template (SuM 4). This selection was based on both, PacBio and Illumina data, randomly sampling 10 of the 50 most enriched variants after the final round of selection (R4). With this approach, not only top enriched variants (obtained from NGS) were picked, but also variants with lower CL-enrichments. In total, qPCR analysis of the synthesised variants confirmed activity increase over WT for 19 of the 30 variants in bulk (figure 4.18). Individual bulk reactions were carried out for each of the 30 synthesised clones: The clones I expected the random mutagenesis study, presented in section 4.5 to reveal some sites that would have a negative impact on polymerase activity. I furthermore expected these sites to be more pronounced in the polymerization domain, since this is the dominant domain in the synthesis of new DNA. Surprisingly, motifs ExoI-III, if mutated, also seem to have a strong negative influence on enrichment, even though these motifs are located in the exonuclease domain.

The exonuclease domain is responsible for the proofreading activity of Φ29 DNAP as was mentioned in chapter 1.3.4. As a result, I expected alterations in this domain to result in variants with lower fidelity. But how can strong e ects in synthesis be explained?

In the following, I present a hypothesis that might provide an answer to this question. In section 4.8 I will use QuIK to collect evidence for the hypothesis with an iviCSR assay and in section 4.12 I will test the hypothesis using the evidence I found.

The main catalytic domains of Φ29 DNAP are the polymerisation domain and the exonuclease domain. While the first is responsible for strand elongation (figure 4.19 A), the latter fulfills the exact opposite function -the excision of nucleotides. This ensures high fidelity by cutting out errors incorporated during synthesis. The linker region is a mediator between the two domains: As the two catalytic functions are opposing each other, they need to be very finely balanced to find the right trade o between the polymerases activity and its fidelity. In figure 4.19 B) the nucleotide coloured in red does not correspond to the upcoming base in the template. In the case that this nucleotide is nonetheless incorporated into the template, the linker region shifts catalytic function towards the exonuclease domain with predominant probability due to free energy di erences. This involves the actual physical shifting of the nascent strand into the exonuclease domain where the misincorporation is excised (figure 4.19 C). Only then can the physical conformation of the nascent strand is shifted back into the polymerisation domain, where synthesis is resumed. As a first part of the hypothesis, I assume that mutations can cause structural changes in the exonuclease domain, physically hindering proofreading activity and ultimately stalling the polymerase with the nascent strand stuck in the exonuclease domain. To further explore this hypothesis in more detail, I decided to synthesise and screen a gene library with mutations in both exonuclease domain and linker region. Based on the findings of the random mutagenesis study presented in section 4.5, I expect genes with targeted mutations in the exonuclease domain to code notably for variants with low activity. The second part of the hypothesis involves the linker region: If the nascent strand can be prevented from shifting into the exonuclease domain, mutations in the exonuclease domain should lose their ability to stall the polymerase and thus forfeit their negative e ect on the variant's polymerisation activity.

8. Titration Assay: The combination of iviCSR and QuIK enables 2D screenings for activity and fidelity

By introducing mutations also in a second part of the library, namely the linker region, I wanted to discover variants that can impair the linker's mediating abilities and prevent switching of the nascent strand into the exonuclease domain. In this way, the second part of the hypothesis seeks to o er a proof for part one: If the low activity of a variant with exonuclease modification can be restored to WT levels by the introduction of another mutation in the linker region, the hypothesis could prove to be true. Due to the high fidelity of Φ29 DNAP WT error incorporations are a rare event. For Φ29 DNAP variants error rates might be higher but can not be estimated in advance.

To challenge polymerase variants in the evolutionary setting of my assay and to obtain a more robust measurement of errors I altered the environmental conditions during selection in section 4.8. By individual base titration I am depriving the polymerase variants of the building blocks needed for faithful synthesis. In this way, I am starving the polymerase variants of dNTPs and force them to incorporate errors.

Titration Assay: The combination of iviCSR and QuIK enables 2D screenings for activity and fidelity

In the previous Activity Assays on Φ29 and H0-25 DNAPs, iviCSR has only been applied to screen for and identify polymerases with high polymerisation activity. This is what I call a one-dimensional (1D) screening. The QuIK Assay has proven to be a quick and e cient tool for kinetics studies, and has already been successfully applied with two rounds of iviCSR directed evolution of H025 variants as shown in section 4.6. Combination of QuIK and iviCSR will allow to extend 1D activity screenings to a 2D fidelity and activity screening. Therefore, two modifications are introduced:

1. A Φ29 DNAP library disposing of mutations in both, the polymerisation and the exonuclease domain.

2. Similarly to the MagNIFi assay, presented in chapter 1.3.6, one of the four dNTPs is titrated to force polymerase variants to incorporate errors.

The aim of these two modifications is to investigate the trade-o s between polymerase activityand fidelity-based on the screening of large gene libraries (protocol in SuM 3). I call this assay the "Titration Assay" (figure 4.20).

During RCA, error introduction is expected to happen as follows: One RCA copy (i.e., the polymerase makes one circle around the DB-shaped template) consists of 1) a strand equaling the template's coding stand and 2) a reverse complement to the template's coding strand (figure 4.23, top panel). The e ect of titrating dTTP thus has an e ect on sites corresponding to the template's coding strand "A" and "T" sites, corresponding "T" sites lead to "T" misincorporations in the coding part of the RCA product (RCA Product Part 1 in figure 4.23) and corresponding "A" sites lead to "T" misincorporations in the reverse complementary part of the RCA product (RCA Product Part 2 in figure 4.23). The PCR primers target both parts of the RCA product (figure 4.23, bottom panel). If mutants are expressed from RCA product, only part 2 of the RCA product would be the template for transcription, as RNA polymerase reads the non-coding strand.

Short read NGS analysis of the Titration Assay

With the "Titration Assay" fidelity of expressed Φ29 DNAP variants is challenged in an evolutionary setting. The synthesised library with mutations in both, exonuclease domain and linker region serves as input for round one. The recovered genes after selection are reformatted into dumbbells and serve as input for a second round. In both rounds the respective titration of one of the four dNTPs is carried out to shift the selection pressure towards an environment that is supposed to favour ine ective proofreading and/or altered intramolecular coordination of polymerisation and proofreading activity. The synthesised library (i.e. round zero or R0), the recovered genes after the first round (R1) and recovered genes after the second round (R2) of selection are analyzed by short read NGS on the Illumina system and then compared. The average counts after alignment for Rounds R0, R1 and R2 of the Titration Assay are presented in figure 7.18,S u M19. The results of the accompanying quality control by qPCR are shown in SuM 3.1, figures 7.9, 7.10 and 7.11.

UMI count analysis

The short read NGS analysis presented in the previous section 4.9 does not consider consensus sequences, based on identical UMIs. Instead, the whole data set (filtered by MAPQ and quality scores in Bowtie2) is taken into account. After rec-/outPCR and bottlenecking (see SuM 19.1) a UMI is ligated to the genes that are subsequently prepared for NGS. The UMI serves to capture bias of the PCR during NGS library preparation. The analysis of UMI counts reveals that 0.71% of UMIs are detected only once (figure 4.24). 4% of UMIs are detected two times. UMIs with three to 40 counts make almost 25% of the reads. There are manifold possible reasons for UMI sequences with only one appearance in the data set. Bias of the PCR step during NGS library preparation can leave some sequences simply non-amplified. Also, it is possible that errors change one or more bases in the UMI sequence: 9. Short read NGS analysis of the Titration Assay

The average per base and per sequence quality scored for all sequences with a UMI count # < 3 and # > 4 are displayed in figure . The comparison shows that there are no major di erences, however, between the quality of the sequences of the two subsets. For this reason, I continued the data analysis with the untrimmed dataset to begin with.

Frequency scanning

An early stage integrity test of the NGS data can be done on the level of sequence counts to compare frequencies in mutated and non-mutated regions. The NT position-wise average frequency of mutations is calculated as described in chapter 3.3.4.2 and shown in figures 4.26 for round R0 and the standard condition in R2. (a) (c) Figure 4.26: Average position-wise frequencies on NT level for a) the entire sequenced region of the library prior to selection (R0), b) the entire sequenced region after two rounds of selection in standard conditions, c) positions 0 to 300 and 700 to 925 of the library prior to selection (R0) and d) positions 0 to 300 and 700 to 925 after two rounds of selection in standard conditions. Positions are related to bases along the sequenced DNA fragment and do not correspond to positions along the Φ29 gene. The high frequencies observed in between positions 300 to 450 and 475 to 925 (top panel) are due to a decrease in sequencing quality. Those regions are cut out in the bottom panel to highlight frequency changes between NNK-scanned and conserved positions. Vertical grey lines represent the positions at which the data was clipped.

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The three regions subjected to NNK scanning (one region in the exonuclease and two regions in the linker region) can be distinguished from the rest of the points due to their comparatively high frequency (figures 4.26, bottom panel). Figures 4.27 plot again the position-wise frequency on NT level, but this time comparing R0 and all titration conditions of R1 and R2. For each condition, the observed frequencies are in a similar range.

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400 600 800 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Index Frequency Per NT Position

• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •
Round 0 Round 1, 0 uM dTTP Round 1, 7 uM dTTP Round 1, 67 uM dTTP Round 1, 667 uM dTTP (a)

• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 0 200
400 600 800 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Index Frequency Per NT Position I assume that error prone variants thrive in rare base environments better than their high-fidelity counterparts. Error prone variants could be likely to discriminate less between bases and thus integrate mismatching nucleotides more readily if the correctly matching base is not at once available. My first attempt to verify my assumption was to check for unbalanced nucleotide distributions in the copied genes. The enrichment distribution for the individual bases in the four dTTP concentrations of R2 is plotted (figure 4.28): For all three challenging conditions enrichment of the four bases is almost equally distributed and corresponds to the standard condition. No significant base imbalance could be found in the data.

• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 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• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • Round 0 Round 2,
(b) • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 0.
• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 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• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • Round 0 Round 1, 0 uM dTTP Round 1, 7 uM dTTP Round 1, 67 uM dTTP Round 1, 667 uM dTTP (c) • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 0.
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Enrichment comparison

Comparison of PW-enrichment on clipped and unclipped data sets

During NGS data preprocessing a set was created where sequences with a UMI count < 4 were clipped o . The PW-enrichment matrices presented in section 4.9.5 o er a direct way to compare results between trimmed and untrimmed and investigate impact of low UMI count sequences on the presented data. To make this "rescaling" clearly visible and identify possible deviations, in figure 4.39 the PW-enrichment on nucleotide level across all dTTP concentrations in R2 is plotted against the PW-enrichment on nucleotide level in the R2 standard condition (thus, the standard condition in black lies exactly on the diagonal). Each point in this plot compares PW-enrichment in R2 standard condition (x-axis) to PW-enrichment in challenging conditions (y-axis). Clearly, positions that have a positive (negative) PW-enrichment in standard conditions are still enriched (depleted), but less. The slope of the di erent clouds can be interpreted as the overall activity of the library (relative to the standard condition). A linear fit is applied to the challenging conditions. The slope of this fit gradually decreases for lowered dTTP concentrations, as starvation of dTTPs is lowering the reaction e ciency. The decrease in dTTP concentration ultimately results in a virtually horizontal fit with a slope of m ≈ 0. The 10-fold and 100-fold titration of R2 can be found in between the diagonal fit of R2 at 700µM and the horizontal fit of R2 at 0µM . Just as observed before, deleterious and favourable e ects of PW-enrichment are generally preserved throughout the titration conditions. However, there are again outliers from the general pattern.

In the case of the 0 dTTP titration I assume that virtually no replication is happening in the reaction as mentioned in section 4.9.5. Thus, the deviation of the data points from the linear fit results only from the sum of sampling noise occurring during reformatting, NGS library preparation and sequencing errors. I will refer to this aggregate error as "ReSe Error". The residual standard error (RSE) is a measure for the deviation as it describes how well the linear regression fits the data. Therefore, I use the RSE of the 0 dTTP condition as a quantification of errors that derive from ReSe. Figure 4. 40 (b) shows the RSE for the three challenging conditions (no RSE can be obtained for the standard conditions, as it is the dependent and independent variable of the linear fit). The errors of the c =0µM dTTP and c =7µM dTTP conditions are very similar (RSE =0.01 and RSE =0.064). This is a consequence of the fact that also in the c =7µM dT T P sample not much replication is happening. Otherwise it could be expected that replication error would accumulate on top of the ReSe error. However, it is also possible that replication error in the 7 µM dTTP condition on average is simply zero. In the c = 70µM dT T P condition it is RSE =0.11. If ReSe error was zero, all dots of the 0 µM dTTP condition (figure 4.39 blue dots) would be distributed along a horizontal line at y =1 . As a consequence, the dispersion of the points of the 0 µM dTTP condition (figure 4.39 blue dots), gives an idea of the noise in this experimental dataset which can be used to benchmark the measurements. Leveraging this estimate gives confidence in the error measurement. RSE =0.01 of the c =0µM dT T P condition is therefore plotted on the slopes of figure 4.40. Already the slope of the 7 µM condition in figure 4.40 a) can be distinguished from the 0 µM condition as it is significant within the experimental error. After two rounds of selection the first 10-fold dTTP titration step already has a high e ect on the reaction e ciency. In this first step the fitted slope drops by 0.69% from m 1 =1to m 10 =0.31. In the following two steps the slope only drops to m 100 =0.22 and m 0 =0.01 ≈ 0. The drop in slope at 10-fold reduction of dTTP is not as severe in R1 as it is in R2 (figure 4.41 (b)). It drops by 52% from m 1 =1to m 10 =0.48 after the first titration. Overall, the enrichment correlation of dTTP titration after R1 and also after R2 suggests that already a 10-fold reduction of dNTPs in the assay strongly impacts the ability of Φ29 DNAP variants to polymerise, even though this condition is still far from the K D of the WT.

Enrichment correlations of dATP-, dCTP-and dGTP titrations

Enrichment correlations have been carried out with the remaining dNTPs similarly to the correlations presented in section 9.

A Michaelis-Menten fit to the enrichment slope

On the one hand, the rate of a reaction is described by the Michaelis-Menten equation. On the other hand, decreasing dTTP concentration slows down the iviCSR reaction. I interprete this decrease of reaction speed with a fit based on Michaelis-Menten theory. Starting from the Michaelis-Menten equation we write:

‹ = [E] 0 * [S] * k cat K M +[S] . (4.1)
The dimensionless part of this equation is described by

m = [S] [S]+K M . (4.2)
From [Morin, 2015] we obtain K M = 31µM ± 3µM . Thus, we can write equation 4.2 for the di erent dTTP concentrations as follows:

• for S T = 700µM : 700µM 700µM + 31µM ± 3µM ≈ 1 (4.3) • for S T = 70µM : 70µM 70µM + 31µM ± 3µM ≈ 3 4 (4.4) • for S T =7µM : 7µM 7µM + 31µM ± 3µM ≈ 1 6 (4.5)
We can normalise the dimensionless quantity m to the quotient of enrichment in challenging and typical conditions E * E 0 and superimpose the resulting model to the data points from NGS (figure 4.39). The fit is shown in section 10.0.4 of this chapter for two di erent Michaelis-Menten constants found in the literature.

The selection dynamics model

With NGS sequencing we are only able to sample a subset of the sequences of the library. But we assume that this subset is a representative sample of the library population (figure 4.45). Let n r ( ‡ i ) be the number of occurrences of a sequence ‡ i in the population subset that has been sampled for sequencing after round r. Let d be the diversity of the library sampled, thus the number of di erent sequences present in the subset. We can quantify the number of occurrences of each sequence sigma i ,i =1 ...d in the library sampled at round r. And we can do the same for the library sampled at round r+1. I.e. we can measure n r ( ‡ i ) and n r+1 ( ‡ i ).

Chapter 4. Results

ry i e l d st h ef r e q u e n c yf, of sequences ‡ i , in the sampled population of round r:

f r ( ‡ i )= n r ( ‡ i ) q ‡ i n r ( ‡ i ) . (4.8)
The relation of frequencies across rounds is thus:

f r+1 ( ‡ i )= n r+1 ( ‡ i ) q ‡ i n r+1 ( ‡ i ) = -( ‡ i ) * n r ( ‡ i ) q ‡ i -( ‡ i ) * n r ( ‡ i )
.

(4.9)

A more readily accessible representation of equation 4.9 can be obtained by expansion with a factor that equals to one:

f r+1 ( ‡ i )= -( ‡ i ) * n r ( ‡ i ) q ‡ i -( ‡ i ) * n r ( ‡ i ) * q ‡ i n r ( ‡ i ) q ‡ i n r ( ‡ i )
.

(4.10) and

f r+1 ( ‡ i )= -( ‡ i ) * nr( ‡ i ) q σ i nr( ‡ i ) q ‡ i -( ‡ i ) * nr( ‡ i ) q σ i nr( ‡ i ) = -( ‡ i ) * f r ( ‡ i ) q ‡ i -( ‡ i ) * f r ( ‡ i ) . (4.11)
leading to the simple expression

f r+1 ( ‡ i )= -( ‡ i ) * f r ( ‡ i ) <-> r (4.12)
in which <> denotes population averages. As this sequence representation in terms of frequencies is normalised to the populations averages it is

ÿ ‡ i f r+1 ( ‡ i )= ÿ ‡ i -( ‡ i ) * f r ( ‡ i ) <-> r =1. (4.13) Furthermore, it is -( ‡ i ) <-> r ≥ 0. (4.14)
With 4.13 and 4.14 equation 4.12 fulfills the criteria of a probability distribution over the sequence space with the shape

P (x)= ÿ x Q(x) * f (x), (4.15)
where

x = ‡ i , Q(x)= -( ‡ i ) <->r and f (x)=f r ( ‡ i ).
For now, only the o spring has been considered in the calculation. However, the original gene ‡ i is also transferred to round r+1 even if no o spring are produced (-( ‡ i )=0. This can be accounted for by summing -( ‡ i ) with one in equation 4.7 110 10. Modelling the reaction dynamics of the Titration Assay

g(n r ( ‡ i )) = (-( ‡ i ) + 1) * n r ( ‡ i ).
(4.16)

We have now found an equation that allows us to describe the frequency f r+1 ( ‡ i ) of a gene ‡ i in round r+1, based on its frequency f r ( ‡ i ) in round r:

f r+1 ( ‡ i )= (1 + -( ‡ i )) * f r ( ‡ i ) 1+ <-> r , ( 4.17)
where the denominator has been derived in the following way, following the associative property of the sum:

ÿ ‡ i (1 + -( ‡ i )) * f r ( ‡ i )= ÿ ‡ i 1 * f r ( ‡ i )+ ÿ ‡ i -r ( ‡ i ) * f r ( ‡ i ).
(4.18)

The first part of the sum in equation 4.18 equals to one by definition. The second part denotes the population averages again. Hence the denominator in equation 4.17 is:

ÿ ‡ i (1 + -( ‡ i )) * f r ( ‡ i ) = 1+ <-> r .
(4.19)

We can now write

f r+1 ( ‡ i ) f r ( ‡ i ) = 1+-( ‡ i ) 1+ <-> r . ( 4.20)
This fraction gives us the CL-enrichment of any variant gene i across rounds r to r +1.

During the selection assay, I do try to eliminate the library of generation r by digestion. However, enzymatic reactions are virtually never e cient to 100%. Also, there is a probability that during PCR of the library prep, residues of the original generation r library will be amplified to some extent. Therefore, I do not neglect the generation r library, but assign a probability ' to its influence. Equation 4.20 becomes thus

f r+1 ( ‡ i ) f r ( ‡ i ) = ' + -( ‡ i ) '+ <-> r . (4.21)
So far, this approach applies only to equimolar nucleotide conditions. To take titrations into account we will rewrite equation 4.21 such that we explicitly take nucleotide concentration into account:

f r+1 ( ‡ i ,c) f r ( ‡ i ,c) = ' + -( ‡ i ,c) '+ <-(c) > r (4.22)
where c denotes the nucleotide concentration. In the same way equation 4.23 accounts for a challenging concentration c * .

f r+1 ( ‡ i ,c * ) f r ( ‡ i ,c * ) = ' + -( ‡ i ,c * ) '+ <-(c * ) > r . (4.23)
This equation immediately points us to an interesting interplay: The e ect of nucleotide concentration on one mutation ‡ i=x will be di erent from the e ect of the same titration on a di erent mutation ‡ i=y . The dependence of -( ‡ i ,c * ) on nucleotide concentration and sequence information is thus a non-trivial problem. The behaviour seen in figure 4.41 suggests that a first approximation of the overall behaviour can be performed by a factorisation that separates the dependencies:

' + -( ‡ i ,c * ) '+ <-(c * ) > = ' + "(c * ) * -( ‡ i ,c 0 ) ' + "(c * ) * <-(c 0 ) > . (4.24)
In this way, the e ect of nucleotide titrations c * is binned out into a rescaling factor "(c * ) and -( ‡ i ) again, only depends on sequence information. c 0 denotes the standard equimolar condition.

It is important to note that this factorisation is expected to work for the majority of mutations (i.e. the ones that have low enrichment in low nucleotide concentrations) but not for all mutations: Outliers like the ones in figure 4.49) are depleted in standard equimolar nucleotide concentrations but show high enrichment when being challenged by a reduced dTTP concentration. Nucleotide concentration has an e ect on these variants that is vastly di erent to the one on the population majority and can thus not be described by the trivial solution for equation 4.23. The fraction f r+1 ( ‡ i ,c) fr( ‡ i ,c) is just another expression for the enrichment from selection round r to selection round r+1. Round r template residues that manage to escape obliteration between rounds and carry over to r+1 will also express enzyme. Nonetheless, they are not a ected by the factor "(c * ) that scales enrichment according to nucleotide concentration. Equation 4.25 makes it clear why:

E = ' + "(c * ) * -( ‡ i ,c 0 ) ' + "(c * ) * <-(c 0 ) > . (4.25)
For the complete omission of a base "(c * = 0) = 0 equation 4.25 becomes

E = ' +0 * -( ‡ i ,c 0 ) ' +0 * <-(c 0 ) > =1. (4.26)
Complete omission of a nucleotide renders selection impossible, thus there will be nor negative nor positive enrichment (E =1 ). Would the scaling factor "(c * ) have an e ect on the input library the enrichment would drop to E =0 . This, however, can only be the case if the whole input library could be digested.

Figure 4.39 compares the ratio of position-dependent enrichment in standard conditions against challenging conditions. We can take equation 4.24 to write the dimensionless expression

E * E 0 = ' + "(c * ) * -( ‡ i ,c 0 ) <-(c 0 ) > * <-(c 0 ) > ' + "(c 0 ) * -( ‡ i ,c 0 ) , ( 4.27)
which simplifies to A dNTP concentration of c * =0will result in no selection occurring. Thus, "(c * = 0) = 0 and the slope of the corresponding graph will be

E * E 0 = ' + "(c * ) * -( ‡ i ,c 0 ) ' + "(c 0 ) * -( ‡ i ,c 0 ) . ( 4
E * E 0 = ' ' + "(c 0 ) * -( ‡ i ,c 0 ) . (4.29) It is ' + "(c 0 ) * -( ‡ i ,c 0 ) >> ', especially if the digestion is very e cient. Thus E * E 0 → 0 for "(c * = 0) = 0.
If the dNTP concentration is at equimolar level, then "(c * )="(c 0 ). Thus, the slope of the corresponding graph will be a diagonal with a slope of

E * E 0 =1. (4.30)
The slopes for E * E 0 with E * = E * 10-fold and E * = E * 100-fold are therefore found between the two extremes.

In order to map only the share of the dTTP titration in the slope, the following calculation method is suggested: With equations 4.24 and 4.25, the position-dependent enrichment in challenging conditions E 0 becomes

E * = ' + "(c * ) * -( ‡ i ,c 0 ) ' + "(c * ) * <-(c 0 ) > . (4.31)
In the same way, the position-dependent enrichment in standard condition becomes

E 0 = ' + "(c 0 ) * -( ‡ i ,c 0 ) ' + "(c 0 ) * <-(c 0 ) > = ' + -( ‡ i ,c 0 ) '+ <-(c 0 ) > (4.32)
which can be rewritten to

E 0 * ('+ <-(c 0 ) >) -' = -( ‡ i ,c 0 ). (4.33)
The right side of equation 4.33 can be input in equation 4.31 to give

E * = ' * (1 -"(c * )) ' + "(c * ) * <-(c 0 ) > + "(c * ) * ('+ <-(c 0 ) >) ' + "(c * ) * <-(c 0 ) > * E 0 . (4.34)
From the linear equation 4.34 the term "(c * ) * ('+<-(c 0 )>) '+"(c * ) * <-(c 0 )> denotes the slope of the positiondependent enrichment ratio of standard versus challenging conditions. It is thus

S = "(c * ) * '+ <-(c 0 ) > ' + "(c * ) * <-(c 0 ) > . (4.35)
The share that the dTTP concentration has in the slope at the molecular level is denoted by

"(S)= S * ' '+ <-(c 0 ) > * (1 -S) (4.36)
with "(S = 1) = 1 and "(S = 0) = 0.

12. Validation of identified variants E14L and R223T

This could be attributed to the fact that most of the replication is still happening, while dTTP is still available in su cient supply. Also, De Paz et al. [Paz, 2018]s h o w e di nt h e MagNIFi assay (presented in chapter 1.3.6), that A template bases had the smallest e ect on Φ29 DNAP WT fidelity profiles.

Validation of identified variants E14L and R223T

NGS sequencing results of the dTTP Titration Assay revealed several promising variants. Especially one of them -R223T -showed little selection under typical conditions, while being positively enriched in the challenging conditions. The corresponding mutation -i.e. the one that appears most often together with R223T -is E14L in the exonuclease domain. This is a very interesting result, as position 14 is part of the ExoI motif, which is highly conserved not only in Φ29 DNAP but throughout many members of the polymerase B family. The appearance of mutation E14L is thus an indication that a deleterious mutation in the exonuclease domain could indeed be restored by a mutation in the linker region. To confirm the anticipated behaviour, identified variants were synthesised: E14L and R223T as single point mutants as well as E14L/R223T as double point mutants. Seven di erent assays were considered for validation and trials were undertaken with three of these assays: [Paz, 2018].

The results of assays one to three will be presented in this chapter, section 4.12.2,w h i l e assays four to seven will be part of chapter 6 (together with suggestions to improve assays one and two).

Validation with the M13 primer extension assay

I considered using a primer extension assay to compare processivity of Φ29 DNAP variants (expressed in an IVTT system) with that of Φ29 DNAP WT (also expressed in an IVTT system). In this primer extension assay, the DNAP enzyme synthesises RCA product using a single stranded vector as template. Initial tests were carried out comparing only WT enzyme: A commercially obtained Φ29 DNAP WT and Φ29 DNAP WT expressed in an IVTT system.

Both are spiked into a replication reaction mixture to commence RCA. However, in the case of the expressed enzyme, IVTT background causes a smear that makes it di cult to discriminate the synthesised RCA product from the background. A purification of the expressed enzyme would be necessary. For this reason, the assay was not further followed for validation of the variants. More details on this assay can be found in SuM 17.

Validation with the bulk assay

A first bulk test is done by in vitro expression of Φ29 DNAP variant R223T from linear synthesised gene fragments and quantification of RCA product synthesis on a DB-shaped reporter template. A bulk validation is carried out in three independent replicates, comparing activity of the clone R223T to Q1WT and Q1DD. The enzymes are expressed from linear genes obtained by PCR (protocol in SuM 12). In the one-pot expression-replication reaction (protocol in SuM 4), expressed polymerases amplify ⁄ DNA reporter DBs and the RCA-product concentration is afterward quantified by qPCR (as described in SuM 12.2).

Figure 4.54 shows increase in DNA concentration after incubation with respect to the frozen (non-incubated) sample. The concentration is plotted for RCA product synthesised by clone R223T, Q1WT and Q1DD throughout titration conditions and for both the relative fold increase The top panel of figures 4.55 shows the di erential increase observed in figure 4.54 for clone R223T (and Q1WT) with respect to Q1DD. This is shown in terms of a) DDCq, the qPCR's log2-fold read out and b) non-logarithmic DNA fold increase (SuM 12.2). Figure 4.55 c) shows the di erential increase of clone R223T over Q1WT in terms of fold increase. In each condition ⁄ DB concentration is set in the beginning to 6.2pM . e ective molar dntp concentration in droplets remains the same as in bulk (unlike the e ective gene concentration in occupied drops). However, the absolute amount of dNTPs available to a DNAP in an occupied droplet is much smaller than in the bulk reaction volume (0.7 atto mol in one droplet of 1fL versus (vs.) 3.3 nano mol in a 5µL bulk volume).

Bulk vs. QuIK Assay analysis by qPCR

As presented in section 4.2, droplet size is a distribution with an average droplet radius of r ≈ 0.6µm and a standard deviation of sd ≈ 0.25µm As the droplets of the emulsion are thus not of the exact same size it is di cult to adjust the bulk reactions exactly to the parameters of the emulsion assay. A straight forward approach, however, is to simply validate the variants in the QuIK Assay directly. The requirements of the QuIK Assay for a qPCR analysis are nonetheless more demanding: Whereas in a bulk assay each template is treated in a separate tube, in the QuIK Assay the genes are mixed when the emulsion is broken. It is therefore important that the qPCR primers bind specifically to their respective target DNA fragments. The forward qPCR primers target the respective mutations of the clones as shown in figure 4.56 a). Only two NT make the di erence between WT and R223T variant gene. In a dilution assay I characterise the template dilution range in which the e ciency of the qPCR is in a linear range. In the same assay I quantify how the qPCR primer set for WT and variant R223T pick up the respective wrong template (figure 4.56 b)).

The template dilution / primer range assay (figure 4.56 b) and table 4.2) shows that the primer sets are not ideal, as they pick up wrong template, respectively. In addition, it is not possible to compare WT, variant R223T, variant E14L and variant R223T/E14L in the same emulsion: The double point mutant gene will be picked up equally by primer sets of single point mutant genes R223T and E14L. And there is no primer binding site available that could specifically pick up double point mutant gene R223T/E14L (compare to SuM 5). For this reason, I decided to design the gene backbone version 2 (V2)w i t has p e c i fi cp r i m e r recognition site upstream the T7 promoter (as described in SuM 5). Previously described genes will from now on be referred to as version one (V1). Unfortunately, linear fits of V2 templates primer sets are not parallel in the tested template starting quantity titration range and despite minimised sequence similarity in the primer binding sites, wrong template/primer set pick up seems to occur with a Cq value of Cq ≈ 24 for template starting quantity of 62pM (figure 4 The wrong primer template combination B) can be clearly separated from the correct matches A). Cq determination mode is set to "Regression".

A bulk comparison between V1 and V2 genes with an equimolar dNTP concentration set to 700uM revealed that much more lambda DNA could be amplified by DNAP that is expressed from V1 genes (figure 4.58). Overall, the assay did not show the same replication than the one shown in figure 4.54 but the relative findings stay the same. For both, WT and variant R223T, more DNA could be synthesised with V1 genes, compared to V2 genes.

WT V1 R223T V1 WT V2 R223T V2 E14L V2 E14LR223T V2 Fold Increase λ DB (a) WT V1 R223T V1 WT V2 R223T V2 E14L V2 E14LR223T V2 λ DB Concentration (pM) (b)
One should not be concerned by the fact that the WT seems to achieve a fold increase of 2 in the 0 dTTP condition and the variant even a fold increase of 5. This can possibly be attributed to the lower signal to noise ratio of the qPCR in high Cq regions near the NTC, rather than the actual behavior. In the standard condition, the concentration of synthesised DNA drops for both the WT and the variant compared to the 10-fold titration which is contrary to the findings from NGS analysis. The ∆∆Cq-based fold increase (SuM 12.2) shows that the variant is able to synthesise 2.2-fold more RCA product than WT in the absence of dTTP (figure 4.60). This advantage drops as dTTP concentration is increased. In 10-fold titration and standard conditions the variant has a similar advantage of fold increase FI ≈ 1.2 over WT. Overall, the emulsion assay on V1 clones shows low amplification e ciency resulting in high Cq values that are susceptible to noise. However, the data supports a core finding of the bulk assay on V1 clones: The variant seems to have a higher rate of DNA synthesis than WT, also in low and even in 0 dTTP concentrations.

dTTP input concentration (µM) DNA Fold Increase R223T over WT 0 µM 7 µM 70 µM 700 µM 1.0 1.2 1.4 1.8

2.2

Figure 4.60: Fold increase of DNA synthesised by clone R223T over Φ29 DNAP WT, as measured in qPCR, in di erent dTTP titration conditions. In the absence of dTTP, clone R223T is able to synthesise 2.2-fold more RCA product than WT. In higher dTTP concentrations, this advantage drops to FI ≈ 1.2.

Introduction

In section 5.2 of this chapter I am going to discuss ME and relate my findings to the presented in chapter 1. The Time Assay serves to study the kinetics of QuIK-based iviCSR reactions and is discussed in section 5.3 before QuIK-based assays are compared to previously conducted chip-based assays (section 5.4). An activity-based coupled selection of H025 DNAP variants has been carried out partly onchip and partly QuIK-based and will be discussed in section 5.5. Together with a random mutagenesis study the assay raised questions about the interplay of synthesis and error correction in proofreading DNAPs. Adapting base titrations from the MagNIFi assay of De Paz et al. [Paz, 2018] has lead to the development of the Titration Assay, which is discussed in section 5.6.T h i s simple idea gave rise to interesting mutations and the behaviour of the assay could be further investigated by a model. A main result of the Titration Assay was the finding of a clone with the AA substitution R223T. section 5.7 points towards open questions regarding the AA substitution and relates them to the hypothesis developed based on the results of the random mutagenesis study in chapter 4.5. Variants were synthesised to confirm the findings of the Titration Assay in orthogonal assays. A major challenge was imposed by the variants backbone, as will be discussed in section 5.8. Finally, section 5.9 deals with the derived Michaelis-Menten model and its implication on K M values from the literature.

Membrane emulsification

In figure 4.2, chapter 4.2, I demonstrated the decrease of droplet size when a used membrane was applied instead of a new one. This finding aligns with previous reports from the literature: As presented in chapter 1.5.0.1 membrane fouling is caused by deposition of particles on the membrane surface and inside the membrane pores. The hydrophobic SPG membrane is coated with a silicone resin but is not silanized. For this reason, I assume bio-molecules like protein, RNA and DNA to unfold and adsorb at the membrane surface and decrease pore size. In fact, I passivate the membrane with BSA and ⁄-DNA before application to avoid significant changes in reaction composition by membrane adsorption of bio-molecules. Adsorption could decrease pore size enough to produce smaller droplets, while keeping pores open wide enough to avoid extensive increase of the pressure needed for the premix emulsion to pass the membrane. In this way, membrane fouling -otherwise an undesirable phenomenon -might be the cause for a positive e ect: As has been reported previously, smaller membrane pores cause smaller droplet sizes [Williams, 1998], [Mine, 1996]. I have not yet compared the surface of a used membrane with respect to a new one. However, this would be important to look at, in order to quantify membrane fouling in QuIK Assays. I have not noticed any di erence between a membrane that has been used once or 10 times during my experiments in the laboratory (only e ects between new and used membranes were observed and quantified). Typically, I have not seen e ects of membrane fouling on reproducibility, as long as a passivated membrane was used. In the Titration Assay, for example, I used the same membrane to emulsify the four di erent titration conditions of R1, as each of them had the same library template (going from lowest to the highest dTTP concentration). In R2 I used for each of the dTTP titration conditions a di erent passivated membrane (as each of the samples take the R1 output library of their respective concentration as input). The results of the eight independent emulsions were consistent as shown, for example, in chapter 4.11. As mentioned in chapter 4.2, droplet size in membrane emulsification depends on the viscosity ratio of continuous and dispersed phase. A viscosity di erence of IVTT and water could thus be a possible explanation of the smaller droplet sizes observed with the optimised IVTT mix in the dispersed phase. Droplet size of an 16 h incubated emulsion was compared to a droplet size of a fresh one and no major di erence could be observed (see figure 4.3 in chapter 4.2). Yet, this result can not fully rule out the possibility of inter-droplet transfer of small molecules. It is possible that exchanges happen in such a way that the average droplet size and also droplet dispersity remains virtually unchanged. However, evidence has been found in the literature that the exchange is minimal with the fluorinated oil and fluoro-surfactant used in my assays. This oil-surfactant mixture was even used in PCRs where coalescence occurred from 80 °C on [Chowdhury, 2019].

The Time Assay

The Time Assay was developed to understand the kinetics of the iviCSR reaction via the tracking of biomolecule concentrations. Besides the DNA concentration tracking presented in chapter 4.4, I also tracked RNA concentration. As I am still working on this part of the project, the RNA data is not included in this thesis. However, the RNA data suggests that in the model system, as much RNA is transcribed from Q1WT genes as from Q1DD genes over time. This suggests that no protein is expressed from the RCA product. Protein concentration tracking would also be possible. In chapter 4.4, three regimes in the time-dependent DNA concentration of WT were observed. The second derivative of the first regime is positive, afterwards it decreases to zero and finally becomes negative. Even though DNA input concentration is set to c=1 pM, the starting concentration at t=0h is lower (C=0.85 pM). I explain this observation with adsorption of template DNA to the SPGE mebrane during emulsification. The Time Assay was developed in the beginning of this PhD project, when membrane passivation was not yet included in the protocol. The initially small increase in WT gene is likely due to low protein levels during the first time regime. IVTT is active for four hours, thus protein is expressed, while DNA replication commences during this time. During the course of the second time regime expression from IVTT stops and protein concentration is assumed to peak. It is expected that a high concentration of active protein results in a maximal DNA replication which could explain the steeper slope in the second time regime. Necessary reagents are likely abundant in the reaction. Synthesised RCA product is not multibranched and two origins of replication are present per template (at the two single stranded 4. QuIK vs. on-chip selection DB-loops), as only a forward primer is added to the iviCSR reaction. In the third regime, pyrophosphate is expected to accumulate and expressed protein could start to degrade. This could explain deceleration of DNA synthesis and the commencing plateau of DNA concentration. In figure 4.22, chapter 4.8.2, I showed that in principle, the dNTP supply in standard conditions is su cient for 320 gene copies. In the Time Assay we see at max a 10-fold di erential increase in DNA concentration, approximately. For this reason, I do not expect a depletion of dNTPs to be the reason for the negative second derivative in the third time regime. These assumptions could be the basis of a mathematical model describing the iviCSR kinetics. Fitting of the model to the data reveal if the assumptions could be plausible explanations. Such a mathematical description could, for example, help to understand whether protein is also expressed from RCA product, or only from the original template. In the case of the Titration Assay this question is particularly important: Here, RCA product is likely to contain erroneous DNA copies and expression from RCA product would be likely to give rise to mutants di erent from the ones coded for by the DNA template.

QuIK vs. on-chip selection

Droplet size investigation

QuIK is an instantaneous hand driven emulsification method. The pore size distribution of an exemplary SPGE membrane with an average 1 µM pore size is shown in chapter 1.5.0.1, figure 1.21 b). Droplet size and size distribution in emulsions produced with fast pushes through the SPGE was presented in chapter 4.2, figure 4.3. I assume that due to the size distribution of the pores, the adapter geometry and the hand-driven operation, flow rates through the individual pores vary. These flow rates cannot be individually tuned, as is the case with flow-focusing devices. The flow-focusing device, previously applied in the lab, can produce droplets as small as d=4 µM in diameter. The volumes of ∼ 30 fl are encapsulated at a rate of 10 5 Hz with a polydispersity of 5% (sd/mean). QuIK emulsification is quicker by two orders of magnitude (seconds vs. hours). The droplet radius at 10 pushes is of r=0.6 µM but the quasi instantaneous emulsification comes at the cost of an increased polydispersity of 30%. The typical DNA input concentration in the iviCSR-based assays in this work are 12.5 pM to maximal 57.5 pM. With these input concentrations and the measured droplet size and size distributions at 10 pushes through the SPGE the ⁄-value of encapsulation is still at ⁄ =0.1.T h i s means that only one in 10 droplets will be occupied with a gene on average. I accept this result as a good value to reduce co-encapsulation. The data of figure 4.6 suggests that there is no major di erence in di erential fold increase in the model system when emulsification is carried out with either slow (∼ 1 push / 2 sec) or fast pushes (∼ 1 push / 0.5 sec).

Coupled Activity Assay on-chip and with QuIK

As presented in chapter 1.5 flow focusing devices are widely used for emulsification as they allow for fine control of the droplet parameters. The emulsions of the first two rounds of selection in the Activity Assay on H0-25 variants presented in chapter 4.6 have been made with a flow focusing device. QuIK was applied in the following two rounds of selection to accelerate the emulsification process. This assay o ers the possibility to assess selection with the two techniques. Throughout figures 4.17 a) b) and c) CL-enrichment consistently increases in the on-chip selection between rounds R1 to R3. The pattern of the QuIK-based selection is less homogeneous: In figure 4.17 a) CL-enrichment stagnates or drops for many variants across rounds R3 and R4. In figure 4.17 b) CL-enrichment of the observed variants stagnates or drops from R2 to R3 for five of the seven variants before it increases again for five variants. The question arises if the homogeneity of CL-enrichment throughout rounds R3 and R4 is caused by the QuIK-based emulsification or if it is inherent to the activity-driven competition between the variants. With increased rounds of selection the competition in the population subset increases, as weaker variants drop o . The pattern in figure 4.17 c), however, is very di erent to the previous two: The seven most enriched variants are able to consistently increase CL-enrichment throughout all rounds. Even though all three sub-libraries of figures 4.17 a) b) and c) have been emulsified in the very same reaction volume, the di erent emulsification techniques seem to have not changed the CLenrichment pattern of the subsample in figure c). It should be noted that in each figure, only the most enriched variants per sublibrary are examined. They do by far not represent the entire population captured by NGS. After each round a qPCR has been carried out as a quality control before PCR amplification (see SuM 2 table 7.2). The di erential increase of the Q1WT:Q1DD model system did drop during selection round R3 by 1.7 to a value of DCq =2 .3. However, it exceeded on-chip chip values in R4, where it reaches DCq =5. The absolute CL-enrichment values, as determined by NGS, in figures 4.17 a) and b) are approximately one order of magnitude higher than the ones observed in c). Bias in the two consecutive PCR steps during library recovery could be influencing the absolute enrichment value as well as the observed enrichment patterns.

Polymerase Activity as predicted by the Coupled Activity Assay

The goal of the iviCSR directed evolution assay is to identify clones with high polymerisation activity. A DNAP clone with high polymerisation activity is able to enrich its own (DB-shaped) gene in the iviCSR reaction. Therefore, I expect the encoding gene of an active clone to have a higher read-count and higher enrichment in the NGS read-out. qPCR is a tool to measure 6. The Titration Assay relative DNA concentration quantitatively. It can be used to quantify the amount of DNA synthesised by a DNAP. Therefore, I further expect DNAP activity as measured by qPCR to correlate with gene enrichment as measured by NGS. Despite a possible PCR bias during library recovery the Activity Assay is able to predict polymerase activity quantitatively and I was able to provide a key validation: 30 genes were selected according to their CL-enrichment and synthesised. Next, their polymerase activity was determined by qPCR -measured in bulk, one gene at a time -and correlated with the result of the NGS screening (figure 7.5). The comparison shows a clear correlation which proves that the results predict quantitatively polymerisation activity. Only for the haplotypes of sublibrary 413-416 a negative correlation is derived. However, the correlation returns to a positive value on AA level and the negative haplotype correlation can probably be attributed to mentioned PCR amplification bias.

The Titration Assay

The enrichment slopes, presented in chapter 4.9.8, showed a divergence between in the slope of the 10-fold titration condition between R1 and R2: A drop of 0.69% was observed whereas for the 100-fold condition only a drop of 52% was asserted (also compare figure 4.40 a)). I explain this divergence by accumulation of enrichment throughout rounds.

The Titration Assay comprises of eight separately produced and processed emulsions. The data is very consistent and shows coherent SpS-enrichment values for the individual AA substitutions across two selection rounds as presented in chapter 4.11 and further discussed in the following.

Influence of sequencing and replication errors in the Titration Assay

The Titration Assay is based on the replication of genes by polymerase variants. Error rates can increase both due to error prone variants and challenging conditions. However, also Phi29 DNAP WT makes errors even under standard conditions. NGS sequencing is also based on replication of genetic material by high-fidelity polymerases which contribute to a part of the sequencing error. In my work I identify variants, based on their enrichment as obtained by NGS. I have also been looking at the error rates of di erent variants in the Titration Assay. Likewise, these error rates are computed from the raw NGS data and their quality is dependent on robust measurements with little error. Finally, the input of the assay itself contributes an error. The library is synthesised from oPools. These synthesised gene pools contain truncated gene fragments and biased sequences. The combination of input-replication-and sequencing error adds up to the base counts that derive from depleted or positively enriched variants and thus, blurs the observed signal. In chapter 4.9.8 I presented the RSE of the Time Assay's 0 dTTP concentration as a measure to get a better estimate of the ReSe error (i.e. the error that occurs from library reformatting, NGS library preparation and sequencing error, see also figure 4.40).

The addition of UMIs to the genes before NGS library preparation was introduced but UMI clustering still posed a challenge that could not be further addressed in the timeframe of this Figure 5.1 is a comparison of SpS-enrichment in the 10-fold dTTP titration against standard conditions. Some exemplary AA substitutions are highlighted. Similar to table 5.1, the continuous development of the SpS-enrichment in the independent experiments further illustrates coherence of the data (see also SuM 3.1 figure 7.8)

Distinction from the MagNIFi assay

The MagNIFi assay [Paz, 2018], presented in chapter 1.3.6, is an indirect measurement of DNAPs at biased deoxynucleotide concentrations that are mapped back to standard concentrations. The replication reaction is not combined with protein expression, and there is no phenotypegenotype coupling as WT protein is replicating a pool of DNA templates. The templates are 100-bp long and contain only one "rare base". The Titration Assay adapts the idea of dNTP titration and polymerase starvation. In MagNIFi the "rare base" is present on the 100 bp template only once. This structure of the MagNIFi template allows for successful replication below the K M . In the coupled expression-replication reaction of the Titration Assay an entire gene of 2.5kb has to be copied. In fact, the DB shape requires a copy to compose of forward and reverse direction. Thus, 5000 bases are copied in total, in which each of the four bases is present in virtually equal quantities of ∼25%. For Φ29 DNAP WT K M values have reported to be K M =0 .5 µM [ Saturno, 1995] and K M = 31 µM [Morin, 2015]. The Titration Assay can not provide enough polymerase activity to deliver stable read outs below these concentrations (see figure 4.22, chapter 4.8), especially as coupling with expression is inhibiting replication to some extent. The Titration Assay does not aim to provide a fidelity measurement tool, like the MagNIFi assay is. Instead, it adapts the ability to starve Φ29 DNAP variants in a directed evolution assay to increase the selection pressure beyond polymerase activity. The highly parallelised screening of large polymerase variant to identify promising variants. The assay amplifies the chance to pinpoint variants that show behaviours divergent from the variant crowd. In an ultra-high throughput manner the assay could help to identify AA substitutions that impact the activity-fidelity trade-o of proofreading DNAPs.

R223T -a more error prone variant?

The result of figures 4.60 and 4.50 in chapter 4 suggest that R223T thrives better in challenging conditions than its competitors. I find it interesting to speculate about possible mechanisms that could explain the variants activity gain in challenging conditions. Delving deeper into the NGS dataset, I could identify AA substitution E14L to be highly enriched on genes that also contain AA substitution R223T. I would like to better understand the individual and combined e ects of both substitutions. Does mutation E14L stall the polymerase once the primer strand is transferred into the exonuclease domain? And if not, is R223T nonetheless preventing primer strand switching to the exonuclease domain? What are the di erences between a variant that contains only mutation R223T and a variant that disposes of both E14L and R223T? And does the apparent activity increase of genes with AA substitution R223T come at the cost of fidelity? I would like to go through these question one by one: 7. R223T -a more error prone variant?

Does AA substitution E14L impair polymerase activity?

To shed light on mentioned questions, I pooled all NGS reads from clones with AA substitution R223T. The AA substitution that was most enriched within that pool is E14L (10-fold dTTP titration condition, R2). Position 14 is located in a beta strand of the ExoI motif in the exonuclease domain of the enzyme. As described in chapter 1.3 the metal ion A coordinates AA residues E14 as well as D169. E14L is part of the catalytic carboxylates in the exonuclease domain. The AA substitution E14A has been shown to reduce exonuclease activity by 10 5 [Kamtekar, 2004]. Glutamic acid is a negatively charged AA and alanine just as leucine is a non-polar amino acid. The question arises, if substitution to leucine has a similar e ect than substitution to alanine, but remains open. The results of the random mutagenesis study in chapter 4.5 showed that sequence sites with low PW-enrichment correlate to conserved regions found in literature. The ExoI motif is a conserved motif described in the literature [Bernad, 1989] and mutations within this region belonged to the 10% least enriched variants. It is crucial to investigate the e ects of E14L on polymerase activity and fidelity to get a better understanding of why E14L comes up enriched in the pool of variants carrying also AA substitution R223T. Does the E14L clone stall, once the template strand is switched into the exonuclease domain? Or is the e ect of this specific AA substitution on polymerase activity less disruptive as for the average mutation in the ExoI motif?

Differences between variants R223T and E14L/R223T?

Activity assays in bulk were carried out with clones in backbone V2 (See chapter 4.12). V2 backbones were designed such that di erent variants could be easily detected in qPCR. However, variants expressed from genes with V2 backbone showed little activity in the autocatalytic reaction as mentioned in section 5.8. For this reason, further tests with iviCSR and other orthogonal assays will have to be carried out in the future with V1 backbone clones (see also chapter 6.3).

Does AA substitution R223T prevent primer strand transfer to the exonuclease domain?

E14L coming up highly enriched (together with R223T) is puzzling at first sight. However, a possible (but not yet confirmed) negative impact of E14L on activity could be prevented by AA substitution R223T in the linker region. The hypothesis that mutations in the linker region could lift negative impacts on activity of mutations in the exonuclease domain was already raised after conduction of the random mutagenesis study. In fact this hypothesis was one of the core motivations to design the library for the Titration Assay with mutations in both, exonuclease domain and linker region in order to study activity-fidelity trade-o s. It is for this reason, that the actual emergence of E14L together with R223T is so exciting.

It could be speculated that AA substitution R223T is preventing the primer strand to switch to the exonuclease domain and thus might deactivate the proofreading function of the enzyme. Which in turn could be expected to lead to a variant with a more error prone replication profile.

In this case, however, another question remains: Which fitness advantage favours exactly AA substitution E14L to come up highly enriched in the pool of genes with AA substitution R223T? Also for this reason, it will be important to study the clone with AA substitution E14L individually.

However, this theory should not be used exclusively in the attempt to explain the behaviour of R223T in the Titration Assay.

It is conceivable that the mutation is responsible for a behaviour that can only be explained by epistatic e ects I could not detect in my analysis.

It is also conceivable that R223T contributes to a variant that has a higher a nity for dTTP, i.e. a lower K D,T , than its competitors in the Titration Assay. In this case, one could ask what would happen if the primer strand is transferred into a mutated exonuclease domain with AA substitution E14L. Would this cause the polymerase to stall? Was E14L able to enrich because the switch to exonuclease activity was not as often necessary as it was for the competitors in the assay?

In this context it is interesting to note that AA substitution R223I (from the positively charged Arginine to the non-polar Isoleucine) has been shown to incease a nity for dATP for the mutant with respect to the WT [Truniger, 1999] as mentioned in chapter 1.3.5. It could thus be conjectured that a change from Arginine to the polar Tyrosine might have a positive e ect on the mutants a nity for dTTP. It was further shown that R223I favoured exonucleolysis over polymerisation [Truniger, 1996].

The contrary e ect induced by R223T would be another speculation.

As presented in chapter 1.3, proofreading can enhance polymerase fidelity by a factor 10 2 to 10 3 . Φ29 DNAP WT has an error frequency of 10 -5 to 10 -6 [Paz, 2018]. Combining the two results from the literature, an exonuclease deficient Φ29 DNAP variant can be assumed to incorporate an error every 10 2 to 10 4 bases. It can thus be assumed that catalytic activity of the exonuclease domain is activated at least with a frequency of 10 -2 to 10 -4 bases. And there is evidence that switching to the exonuclease domain -or at least to a previous intermediate state -happens even more often, e.g. if the template elastic properties are modified in the absence of base pair mismatches (see chapter 1.3). An interesting question that arises from this consideration is whether I can get a better picture of the switching frequency between exonucleaseand polymerisation domain by means of the double point library screening. Do variants with mutations in the conserved regions of the exonuclease domain show a lower fitness? And if so, can I estimate the frequency of shifting to the exonuclease domain, based on the synthesised template length?

Does a possible activity increase of R223T come at the cost of fidelity?

An activity-fidelity trade-o could o er an explanation to the question why AA substitution R223T could have increased activity over its competitors in the Titration Assay. Many of the questions raised above can be approached by deeper analysis of the existing NGS data set.

8. V1 vs. V2 backbone of synthesised clones

Comparison of the SpS-enrichment on the gene-pool with substitution R223T and the gene-pool without substitution R223T was carried out in chapter 4.11.1. For this comparison only the region before the T7 promoter was examined. This aims to limit the investigation to a region where only replication errors occur. However, no significant di erence between the two subsetted gene-pools could be derived. This result, however, does not yet prove that AA substitution R223T is not a more error prone variant. It is possible that the result in chapter 4.11.1 did not show di erences in replication error as most of the replication in the Titration Assay might happen, while dTTP is still abundant. It is also important to emphasise that the analysis in chapter 4.11.1 is carried out on 1) a subgroup of genes of the R2 library that share the common AA substitution R223T and 2) a subgroup of genes of the R2 library that shares the conserved AA R223. Other AA substitutions along the individual genes of the subgroups could have additional e ects. A way to investigate replication errors is the synthesis of new variants and their individual examination in orthogonal assays (see chapter 4.12). Especially the investigation with magnetic tweezers could shed light on the fidelity impacts of AA substitutions E14L and R223T as expression and replication are decoupled and examination is carried out on the single molecule level. Chapter 6.3.5 addresses this topic.

V1 vs. V2 backbone of synthesised clones

Genes with the individual mutations E14L, R223T and E14L/R223T have been synthesised in both, the V1 and the V2 backbone (compare with chapter4.12). Genes with the V2 backbone could not be captured well in qPCR as the resulting DNA concentration after the autocatalysis reaction was too low to be reliably measured. The V2 backbone seems to have an impact on either expression or replication that is not yet understood.

In the linear V1 genes the non-coding sequence upstream the T7 promoter is 10 bp long for V1 and 331 bp long for V2.

The non-coding sequence between T7-promoter sequence and ribosome binding site is 54 bp long for V1 and 28 bp long for V2. Both, V1 and V2 genes, carry a Φ29 DNAP WT or Variant gene. However, codon optimisation was carried out slightly di erently, as required by the manufacturer. Codons are optimised not for expression or replication but for gene synthesis. Silent codon substitutions can lead translations at di erent speeds which in turn can influence correct protein folding [Spencer, 2012]. I suspect codon optimisation and/or length di erences of the sequence between T7 promoter and ribosome binding site to influence the variants expression and ultimately its replication activity.

As previously discussed, the coupled emulsion assay with V1 clones is not ideal, for example, because of the missing specific qPCR primer target sites. In chapter 6.3 I will present possible improvements for the assay and other orthogonal assays that should be carried out in the future.

Michaelis-Menten constant and model

The literature o ers di ering values for the Michaelis Menten constant K M . Figure 4.47 in chapter 4.10.0.4 shows Michaelis Menten model fits, based on K M values obtained from Morin et al. [Morin, 2015] and Saturno et al. [Saturno, 1995]. Only with the value of K M =31 µM proposed by Morin et al. the Michaelis Menten model can describe the data (at least for the 700 µM, 7 µM and 0 µM dTTP conditions). Assuming K M =0.5 µM as proposed by Morin et al., the polymerase replicates in saturating conditions throughout all conditions, except 0 µM dTTP. For both Michaelis-Menten fits, the di erent conditions between the reactions in the literature and the autocatalytic reaction in the Titration Assay have to be considered. K D in the literature is obtained in a replication reaction. iviCSR, however, is a coupled transcription, translation and replication reaction. I expect in vitro expression to inhibit replication for the following reasons:

• In case K M =0.5 µM is the more appropriate value inhibition plays a higher role in the iviCSR reaction than currently assumed.

Selection dynamics model

The discrepancy between data and model in figure 4.48 of chapter 4.10.0.4 visualises contributions to the slope that are not linked to the molecular mechanism of dTTP titration. The selection dynamics model is based on an analysis on the clone level while data is based on a position wise analysis which is a possible contribution to the o set between data and model. To increase the models accuracy, it is important to further develop the model towards a position wise representation.

Summary

The simple idea to increase selection pressure by base titration in an in vitro expressionreplication reaction gave rise to AA-substitutions that correspond to previously found critical positions along the DNAP gene. The specific AA-substitutions have not been studied before.

Chapter 6

Outlook

The developed assays in this work are a high-throughput tool in the context of direct evolution of polymerases. In this section I will address the possibilities arising from my research for continuing investigations. Further application of developed tools may reveal more clones with interesting behaviour in both DNA synthesis and proofreading. Also, extended exploration of the already found clones -and especially their investigation at the single molecule level -will further contribute to the knowledge about Φ29 DNAP in particular, and of DNAPs in general. The generalised findings might reveal principles to be applied beyond the polymerase system Objectives Contents

QuIK emulsification

Improvements in the QuIK approach are discussed in the following parts, covering modifications of the membrane and automatisation of the extrusion process. Part three sheds light on the versatility of the method.

Membrane fouling

The membrane applied in QuIK-based emulsifications is rendered hydrophobic by a coating with silicone resin. The membrane is passivated with BSA and ⁄-carrier-DNA. This treatment aims to decrease droplet size due to membrane fouling and avoids adsorption of reaction components as presented and discussed in chapters 2 and 2. Used membranes should be imaged by scanning electron microscopy to substantiate the passivation presumption by assessment of the extent of fouling.

In addition, membrane silanization could decrease adsorption of bio-particles from the reaction volume and potential interactions of the membrane with the fluorinated carrier oil and the fluoro-surfactant.

Automatisation of QuIK emulsification

QuIK emulsification is carried out with a hand driven extruder. The data of emulsion characterisation and both selection assays, i.e. Activity and Titration Assay, is largely consistent as discussed in chapter 5.4. Trials to automatise the emulsification process have been done with di erent syringe pumps as described in chapter 4.2.1 but results did not improve the emulsion's characteristics due to insu cient speed and inadequate fixation. However, an improved automation setup could be developed to maximise selection stringency.

QuIK can be applied to directed evolution of other enzymes

QuIK Assays have not only been applied to DNAPs. Successful directed evolution assays have been conducted at the LBC, ESPCI Paris Tech on recombinases and protein-binding proteins.

The Time Assay

The three mode pattern seems to follow a logistic growth function. This observation encourages the development of a model based on the concentrations of DNA, RNA and protein. The aim of the model would be to find a mathematical description of the concentration of the three biomolecules and understand their interdependencies in the iviCSR reaction. For example, it is not yet understood, if protein is expressed from RCA product or only from the To refine this model with further data, RNA and protein concentration could be determined experimentally. Due to the IVTT background of the iviCSR reaction, determination of expressed protein concentration is challenging. However, expression of a protein with a specific peptide tag (e.g., a FLAG-tag) the expressed protein could be purified by means of specific monoclonal antibodies and a nity chromatography. Alternatively, expressed protein concentration could be derived from a model based on experimental data of DNA and RNA concentration. The model could provide insight into DNA and RNA synthesis and protein expression levels in the iviCSR reaction.

Findings of the time course in emulsion could be compared with bulk time courses. Other DNA input concentrations could also be tested in bulk. This could be used to check how the observed concentrations scale after incubation and whether the developed model can reproduce this scaling. Also the assessment of a variant with decreased activity could give interesting insights into the mechanism of polymerase activity. Analogously, the Titration Assay suggests that variants exist that have a higher polymerisation activity under conditions with reduced dTTP concentrations with respect to standard equimolar dNTP conditions. Amongst others, variants with the AA substitution R223T could belong to that group. Due to the nature of the NGS read out and limitations in the developed NGS analysis pipeline, enrichment of the WT can not easily be obtained in the same way as average enrichment e ects of AA substitutions. In chapter 5.7 I raised a multitude of questions concerning the emerged new variant and the accompanying AA substitution E14L. In chapter 4.7 I presented the hypothesis that an AA substitution in the linker region could restore polymerisation of a DNAP that stalled due to a deleterious mutation in the exonuclease domain. Examination of single and doublepoint mutants at identified positions could shed light on this hypothesis. In chapter 4.12 I presented seven assays for validation of the NGS inferred findings, of which three were already tested. In the following I will suggest improvements to conducted assays and point out possible benefits of assays that I have not yet been able to conduct. To begin with, improvements for QuIK-based iviCSR reactions for validation are presented. So-called "Single Nucleotide Polymorphism Genotyping by Fragment Analysis" (SNP Genotyping by Fragment Analysis) can be an alternative to the qPCR read out of (self) replication reactions. Bulk in vitro assays already provided validation for the found H0-25 DNAP variants and will not be further covered in this chapter. The bulk primer extension assay did not deliver solid data yet which could be changed by the advancement to the single molecule level as shown in the following. Magnetic tweezer assays are another single molecule technique that could help to investigate replication activity of found variants uncoupled from the expression reaction. Finally, the MagNIFi assay is proposed to shed light on variant fidelity.

Orthogonal assays

QuIK-based iviCSR

The emulsion coupled assay with QuIK-based iviCSR supported findings from the Titration Assay, in that a Φ29 DNAP gene with AA substitution R223T showed higher activity than WT throughout tested dTTP concentrations (chapter 4.12.4). Specific target sites for qPCR primers were introduced in the non-coding region of a set of clones (V2) but their activity in qPCR remained below the previously tested variants of same genes.

In the V1 clones primer sites target the mutated NT positions. Primer specificity can be enhanced if the primer target site is located such that the primer 3' end covers the mutated NT positions but does not extend further into the conserved region of the template. In this way, primer extension is reduced in case of o -target primer annealing. Furthermore, silent mutations could be introduced in the primer target sites of the respective templates to enhance primer specificity. However, silent mutations were considered to be one possible reason why activity of V2 clones did not reach activity of V1 clones. For this reason, another approach for clone validation with QuIK-based iviCSR is considered: NGS analysis could be carried out to quantify activity of clones and WT, instead of qPCR. Due to the high read-depth of short read NGS sequencing, this validation approach is expected to be more sensitive than qPCR. An additional benefit is the possibility to access information of replication fidelity through the NGS read-out. Each of the tested clones and the WT could carry a UMI which would simplify comparison amongst the samples. SNP Genotyping by Fragment Analysis could be another alternative to qPCR analysis of both bulk and compartmentalised (self) replication reactions and is presented in the following section.

Single nucleotide polymorphism genotyping by fragment analysis

Single Nucleotide Polymorphism Genotyping by Fragment Analysis is a method developed to detect single nucleotide polymorphisms (SNPs). It could, however, also be adapted to the recognition of NT substitutions on the Φ29 DNAP gene. A primer is designed to bind just upstream the mutation to be detected. Fluorescently labeled ddNTPs are added and a DNA polymerase extends the primer by addition of a single dideoxynucleotide (ddNTP) to its 3' end.

The added nucleotide can be determined by fluorescence read-out. After ddNTP incorporation, no further primer elongation is possible: No phosphodiester bond can be formed with the next incoming nucleotide as the synthetic ddNTP lacks a hydroxyl group at the 3' carbon of its sugar moiety. This method has the advantage that it does not rely on replication of the entire gene, which could be inhibited by possible IVTT and/or oil and surfactant residues. A disadvantage is that no distinction can be made between single mutants R223T / E14L and double point mutant R223T -E14L. Moreover, the standard read-out, capillary electrophoresis, is not quantitative. However, a qPCR protocol could be developed that includes comparison to fluorophore standards in di erent dilution for each of the four fluorophores.

Bulk primer extension assay

The bulk primer extension assay presented in chapter 4.12.1 was not further followed because of a bright smear on the agarose gel of the expressed protein samples due to an IVTT background. The background could be minimised by expression of FLAG-tagged protein and purification with specific monoclonal antibodies and a nity chromatography as previously described in section 6.2. However, shifting the primer extension approach into the single molecule regime would o er a higher sensitivity. Based on this consideration, the single molecule investigation is preferred and will be described in more detail in the following section.

Primer extension on single molecule level

DNA replication speed of DNAPs can be measured on the single molecule level by visualisation of DNA synthesis in a primer extension assay [Lewis, 2017]. The ratio of number of incorporated nucleotides per time can be derived from tracked length di erences between ssDNA and dsDNA. The specific setup is explained in figure 6.1 with the example of the S. cerevisiae replisome, which could be adapted to Φ29 DNAP. In single molecule assays expression and replication are separated which o ers the possibility to study replication speed without influences of protein expression levels. Likewise, the time for enzyme-DNA binding does not a ect the read-out, either.

Single molecule assays with magnetic tweezers

Magnetic Tweezers facilitate the manipulation of individual DNA hairpin molecules that are loaded between a glass surface (DNA 3' end) and a magnetic bead (DNA 5' end). The latter is positioned in a magnetic trap by exposure to a magnetic field. Variation of the magnet distance allows for the application of forces on the bead [Mañosas, 2012b]. In this way, intra-DNA interactions can be studied via a camera based bead tracking system. Additional loading of DNA-binding proteins like helicases or DNAPs enable single-complex studies [Mañosas, 2010], [Mañosas, 2012a]. In the case of proofreading polymerases three modes can be di erentiated:

1. Strand displacement synthesis activity (figure 6.2 A) and C)). The DNAP de-hibridises the closed part of the DNA hairpin and synthesises a copy strand. The bead is displaced far and accordingly the tracking read-out shows a steep slope (figure 6.2 B), light green).

2. Primer extension activity (figure 6.2 A) and D)). The DNA hairpin has opened entirely and the DNAP synthesises a copy strand on the extended DNA template. The slope of the bead tracking read-out decreases, as dsDNA is shorter than ssDNA of equal sequence (figure 6.2 B), dark green).

3. Exonuclease activity (figure 6.2 A)). The proofreading DNAP hydrolyses bases can allow the DNA hairpin to close.

Magnetic tweezers assays provide a detailed picture of the two opposing catalytic functions of proofreading DNAPs. From the NGS data I was not yet able to infer a comparison between WT and variants. Magnetic tweezers could be a suited tool to close this gap and provide direct biological data on the single molecule level. Titration of the remaining bases could thus reveal di erent patterns in the NGS read-out. Furthermore, it would be interesting to compare enrichment of of AA substitution R223T throughout the datasets of di erent base titrations and to screen for other highly enriched variants.

Bottlenecking

By addition of UMIs to the sequenced library I aimed to get an idea of PCR bias. Currently, library recovery of emulsion based directed evolution assays involve two consecutive PCR steps at the LBC, ESPCI Paris Tech. Bottlenecking involved reduction of sequence diversity to 200.000. The initial library (R0) had a diversity of ∼ 3.5 * 10 5 variants. Without PCR bias all of the observed sequences should have the same UMI count which could be reduced by UMI consensus sequence merging. Currently, however, the processed data set has a UMI distribution with three peaks, as UMI consensus sequence merging could neot yet be carried out. Cutting o the first peak (i.e. # of UMIs < 4) did not change significantly the outcome of enrichment comparison. Further attempts for UMI consensus sequence merging should be undertaken and analysis on the merged dataset should be compared to the current analysis.

Advancing the iviCSR reaction to the single molecule level

The Activity Assay is an in vitro directed evolution approach to screen for one single parameter of DNAP variants (one-dimensional assay). The extension to a two-dimensional approach was developed with the Titration Assay, which allows for the screening of both, activity and fidelity of DNAP variants. While extension towards a three-dimensional assay may be possible, the resulting assay protocol could become highly complex. Uncoupling expression and replication could, however, provide a straight-forward path towards a single molecule, ultra-high-throughput DNAP characterisation on the multi-dimensional parameter level. Therefore the omission of one base -an idea of the Titration Assay -is taken up and thought about further. At first, DNAP variants would be expressed in an iviCSR reaction and allowed to start replication of their own gene. The omission of one base could force the polymerase to eventually stall and remain complexed with the primer-template dimer. The expressed DNAP could be engineered such that it displays a 15 amino acid AviTag peptide at either its N-or C-terminus. Highly specific enzymatic biotinylation with E. coli biotin ligase (BirA) covalently attaches biotin to the AviTag. It is known, that biotin -streptavidin binding is one of the strongest non-covalent interactions. The ternary complex of template-primer-protein could hence be fixed on streptavidin-comprising ZMW -similarly to the Φ29 DNAP WT enzyme used in ZMWs of the PacBio sequencing platform, as shown in figure 3.6, chapter 3.3.1. The geometry of parallelised ZMW nanostructure arrays allows for the isolation of complexes and simultaneous observation of incorporation events in thousands of parallelised single-molecule sequencing reactions [Eid, 2009].

IVTT background is present due to in vitro protein expression. It is expected to inhibits DNA replication and necessitates either flushing of the ZMW after streptavidin conjugation with 7. Summary the complex, or complex purification prior to biotin-streptavidin binding on the ZMW. With this approach DNAP variants are complexed with -and allowed to replicate -their own gene. ZMWs are utilised by the PacBio NGS system where DB-shaped DNA template is read via phospholinked dNTP incorporation by complexed Φ29 DNAp WT. The advancement of the technology towards DNAP variants replicating their own gene would provide insight into variant properties on the single molecule level in a massively parallelised fashion: Not only is the genotype information accessible via NGS read-out. With this approach the characteristics of the phenotype -otherwise uncovered by DNA sequencing -can also be read at the very same time as the genotype. This is due to the circumstance that the behaviour of each variantinterruptions, proofreading, dissociation, influence of sequence context, etc. -can be inferred directly from the time trace.

Summary

In this section I presented specific suggestions to further improve the understanding of iviCSR kinetics and DNAP function. On a broader level other assays could be developed to gain insight into further mechanisms of the DNA replicating enzyme. Already the Time Assay -the study of the time course of the di erential DNA increase -inspires further experiments: It would be very interesting and also of commercial interest to screen for fast expressing variants. And similar to the Titration Assay, one could imagine further set-ups that titrate bu er conditions or inhibitors. Increasing selection pressure by temperature would be another interesting directed evolution study. Data obtained could be the base for more advanced models based on enrichment and kinetic parameters to find a mathematical description of made observations. Furthermore, the coupled expression and replication reaction of iviCSR could be uncoupled and replication could be allowed for on a ZMW-based sequencing chip. This approach would allow for a ultra-high-throughout multi-dimensional characteristics screening of isothermal DNAPs on the single molecule level.

IVTT components

IVTT is obtained from NEB. The manufacturer does not indicate the ingredients of the kit. However, their system is based on the "protein synthesis using recombinant elements" (PURE) system [Shimizu, 2001]. Shimizu et al. state that the PURE system includes 32 main components:

• E.coli initiation factors: IF1, IF2, IF3

• E.coli elongation factors: EF-G, EF-Tu, EF-Ts,

• E.coli release factors: RF1, RF3,

• For termination: RRF,

• 20 aminoacyl-tRNA synthetases (ARSs)

• Methionyl-tRNA transformylase (MTF)

• T7 RNA polymerase

• Ribosomes

In addition, the system contains 46 tRNAs, NTPs, creatine phosphate, 10-formyl-5,6,7,8tetrahydrofolic acid, 20 amino acids, creatine kinase, myokinase, nucleoside-diphosphate kinase, and pyrophosphatase.

Φ29 DNAP buffer components

According to the manufacturer, the NEB Φ29 DNA polymerase reaction bu er (pH 7.5 at 25°C) contains the following components (in a 1x concentration of the bu er):

• 50 mM Tris-HCl emulsion is incubated, successively broken (SuM 9.5), the library selectively digested (SuM 11) and recovered (as described in chapter 3.2) and reformatted (SuM 8). Two QuIK-based rounds of selection were carried out. In contrast to the Activity Assay, the input library of round R1 is encapsulated in four di erent emulsions (each with a di erent dTTP concentration), resulting in four di erent input libraries for round R2.

• 10 mM MgCl 2 • 10 mM (NH 4 ) 2 SO 4 • 4m MD T T (a) (b) (c)

Supplementary figures and tables -Titration

• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •• • • • • • • • • • • • • • • • • • • • • • • • • 0.4 0.8 1.2 0.90 1.00 1.10 • •• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 0.7 0.9 1.1 1.3 • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •• • • • • • • • • • • • • • E1 • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • E01 0.8 0.9 1.0 1.1 1.2 • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 0.90 1.00 1.10 0.4 1.0 • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 0.8 1.0 1.2 • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • E0 Figure 7

Library construction

Three di erent approaches for library construction were carried out in the framework of this project:

• Random mutagenesis, using error-prone PCR, applied in the Activity Assay based random mutagenesis study of Φ29 DNAP.

• Degenerated oligos and Gibson assembly for region-specific mutagenesis applied to the H0-25 DNAP library of the Activity Assay.

• Synthetic gene fragments ordered from IDT, applied to the Φ29 DNAP library of the Titration Assay.

Library construction for Φ29 DNAP random mutagenesis study

A mutagenic PCR was carried out (GeneMorphII Random Mutagenesis Kit, 200550, Agilent Technologies, Inc., 5301 Stevens Creek Blvd, Santa Clara, CA 95051, United States) on a Φ29 DNAP WT gene to obtain the library for a random mutagenesis study, based on activity. The mutagenic rate (i.e. the average number of mutations per gene) depends on both, the amount of input DNA and the number of PCR cycles. The mutagenic rate is lower the higher the DNA concentration is and the lower the number of cycles is. The mutagenic PCR was carried out with the reaction mix and PCR program described in section SuM 12.5. An NTC was run in parallel. DpnI digestion (see section SuM 11), amplification PCR (same protocol as outPCR, see section SuM 12.4) methylation (see section SuM 7), dumbbell preparation (see section SuM 8) and the Activity Assay of chapter 4.6 (on-chip) were carried out. The random mutagenesis library contains six mutations per gene (on average, NT-level), has a diversity of approx. 10 27 , a sequencing coverage of 1725 NTs(whole gene) and a sequencing depth of 20.000 reads. One round of selection was carried out.

Library construction for H0-25 Activity Assay

The targeted H0-25 DNAP variant library consists of three multiplexed sub-libraries. Each library is obtained by site saturation mutagenesis in NNK fashion of three triplets of amino acids at positions 227-229, 413-415 and 430-432, respectively. With the NNK method all 20 AA can be obtained but the amount of possible stop codons is reduced from three to one. The first and second NT are one of either A, C, G or T and the third NT is of either G or T. Each triplet gives 8 000 mutants on AA-level, thus 24 000 mutants total. This corresponds to a diversity of 250 000 variants per library on NT-level. Per library, an oligo-pool (opool TM ) of 29 NT length with three consecutive NNK sites was ordered from IDT. Each pool was used as a forward primer in a back-to-back amplification on a PURExpress Control DHFR Plasmid (#N0424AVIAL, NEB), containing the H0-25 DNAP WT gene. After 5' phosphorilation with T4 Polynucleotide Kinase (#M0201, NEB), ligation of the The blunt ligation reaction was carried out by adding the components of table 7.12 to the test tube and incubation for two hours at 25°C. Afterwards, the reaction was ended with a heat inactivation for 10 minutes at 65 °C and cooled to 4°C. The single stranded DB adapter was obtained from IDT (high-performance liquid chromatographypurified custom DNA oligo). T4 DNA Ligase #M0202T and T4 DNA Ligase Reaction Bu er #B0202S were obtained from NEB. 9. QuIK emulsification mix is presented in table 7.14. If the iviCSR reaction was emulsified for characterisation of volume and degree of polydispersity, it was not supplied with dNTPs, loop primers and template.

Reagent

QuIK emulsification protocol

Before emulsification of template containing iviCSR reaction, fluorinated Oil HFE7500 Engineered Fluid, Novec TM , 3M TM , 3M Center Bldg 2162N07 Saint Paul, MN, 55144-1000 United States) is mixed with 0.2% Fluoro surfactant (009A-FS from RAN Biotechnologies, 100 Cummings Center Suite 434J, Beverly, MA 01915, United States) and supplied with 10 µL of BSA to wet and passivate the hydrophobic membrane by flushing. Afterwards, de-ionised water containing 1ng/µL is used to further flush and passivate the membrane. Next, the membrane is flushed with oil-surfactant-BSA mix again. Between each flushing step, the membrane is dried by tapping on a non-pilling tissue. After this membrane preparation, die emulsification is carried out as follows: Fluorinated Oil is mixed with 2% Fluoro surfactant and cooled on ice. 0.5 mL of oil and surfactant mixture (FOS) is drawn into a single use syringe of 1 mL capacity. The SPGE is connected with the syringe and the FOS is pushed through the SPGE.

FOS is added together with the iviCSR reaction of table 7.14 into a 1.5mL tube and pre-mixed by tapping on the tube. The total premix volume of 0.3 mL is drawn into a second 1mL single use syringe. Air is withdrawn and the syringe is flipped such that the premix aqueous phase is collecting below the plunger, while the syringe tip is facing towards the ground. The second syringe is connected with the SPGE such that no air is trapped inside neither syringe nor the SPGE itself. The premix is emulsified by consecutive pushes through the membrane by hand (forward push ˆ= push #1, backward push ˆ= push #2, etc.). Before each push the membrane is flipped such that the aqueous droplets can move upwards and gather in close proximity to the membrane to avoid droplets remaining in the dead volume of the SPGE during a push. The premix is coarse and the first push is carried out slowly (slow pushes: ∼ 1 push / 2 sec) to decrease shear forces acting on the aqueous phase. The following nine pushes are carried out fast (fast pushes: ∼ 1 push / 0.5 sec) to minimise droplet size and size distribution. 300 µL mineral oil is layered on top of the emulsion to avoid evaporation. Emulsion is incubated in an Eppendorf ® ThermoMixer ® (Eppendorf SE, Barkhausenweg 1, 22339 Hamburg, Germany) at 30 °C.

For the emulsion characterisation in terms of model selection, one sample is not incubated but frozen immediately and serves as a control for DNA concentration increase during the reaction. Emulsion is heat inactivated and broken as described in section SuM 9.5 and further processed according to the requirements of the respective assay.

Emulsion characterisation with static light scattering

Static Light Scattering was carried out on the Malvern Panalytical Mastersizer 3000. The instrument measures particle size as the diameter of an imaginary sphere with volume equal to the particle volume. However, in the case of dispersed aqueous droplets in oil the shape is a nearly perfect sphere and the instrument measures the volume directly. The measurement chamber (i.e. cuvette) is filled with HFE 7500 and inserted into the device. No additional surfactant is added to the oil in the cuvette to avoid bubble formation due to the stirrer which is set to 1000 rpm during measurement. Emulsion is added to the oil in the inserted cuvette with a transfer pipette until the obscuration value lies in the range between 10% -20% . The obscuration value gives the fraction of laser light that is lost to introduction of sample into the laser beam. Particle size is are calculated using Mie Theory and categorised into size classes. Diagrams are plotted in R.

Breaking of emulsion

The emulsion was heat inactivated for 10 minutes at 65 °C and put on ice for 1 min. Emulsion was broken by addition 40uL Perfluoro-1-Octanol (1H,1H,2H,2H-Perfluoro-1-octanol, #370533, Sigma-Aldrich) gentle mixing, centrifuging at 11.000 rpm for 3 min collection of aqueous phase, centrifuging of aqueous phase at 13.000 rpm for 1 min and collection of supernatant. Oil and pelleted protein were discarded.

Synthesis of H0-25 DNAP clones found in the Activity Assay

Forward Primers of 29 NT length were ordered as custom oligos from IDT. Each primer comprised of a 9 NT tail that contained the 9 NT substitutions of the respective mutation. Corresponding reverse primers were also ordered as custom oligos at IDT. Primers were used in a back-to-back amplification and the same protocol was followed as during H0-25 DNAP library synthesis (section SuM 6.2).

12.

Out polymerase chain reaction

After Gibson Assembly (figure 3.2 C)) the so-called "outPCR" is performed on non-purified Gibson Assembly product, which results in a reformatted 2.5 kb dsDNA product that contains the entire gene of the respective DNAP variants (figure 3.2 D)). Table 7.20 shows the reaction composition of the outPCR. Reagents are obtained from the same sources as in part 12.3. The M13mp18 plasmid is of length 7249 bp and appears at this height on the agarose gel of picture 7.14. In the case of primer extension with commercial Φ29 DNAP WT without digestion, two bands are observed at 7000 bp and 10000 bp. Despite the height di erence the upper band is assumed to correspond to the plasmid template and the lower band could correspond to small RCA products. The main part of the synthesised RCA product is assumed to be stuck in the well. The same 7000 bp band shows up in the corresponding digested sample. Here, bands are also observed at 4000 bp and 3000 bp as well as 1000 bp. These are assumed to correspond to the 3043 bp, 2721 bp and 1080 bp long digestion product. No band at 400 bp length is observed.

In practice slight changes in band height between lanes with similar template can occur. This is especially the case if the templates are plasmids or RCA product, causing them to migrate di erently than the linear fragments of the ladder.

In the third set of samples no commercial but expressed Φ29 DNAP WT is spiked into the primer-template preannealing reaction mix. Despite the fact that the sample is made up of only 10% expression mix, the IVTT background shows up very strongly on the gel. Identification of digestion bands is not as readily possible as for the samples spiked with commercial Φ29 DNAP WT. Faint bands that are visible in the digested sample but not in the non-digested sample include lengths: 4000 bp, 1000 bp and 400 bp. I doubt that a quantitative comparison of processivity between expressed Φ29 DNAP WT and variants to be feasible due to the prominent smear from the IVTT background. Especially in conditions with base titrations, DNA synthesis is exacerbated which could make comparison even more sensitive to background smears. A possible solution to this problem would be the purification of expressed protein before it is spiked into the reaction. Purification could be achieved by expression of a FLAG tagged polymerase that could then be purified with ANTIFLAG beads. However, this would include addition of a sequence encoding for a FLAG tag, as well as the addition of the purification and elution steps to the protocol. For this reason, validation with the M13 primer extension assay was not followed further.

H0-25 DNAP library of the Activity Assay

Library Preparation for the Pacific Biosciences (Pacific Biosciences of California, Inc., 1305 O'Brien Drive Menlo Park, CA 94025 USA, hereafter "PacBio") system was carried out using the DNA/Polymerase Binding Kit P6 v2. multiplexing of sublibraries was done according to the scheme in figure 7.17. Library Preparation for the Illumina system was carried out similarly to the procedure described in section SuM 18.1 but with the NEBNext Ultra DNA Library Prep Kit for Illumina (#E7370) and the NEBNext ® Multiplex Oligos for Illumina ® (Dual Index Primers Set 1) (#E7600) from NEB.

  ........... 1.3 Right hand analogy of bacterial and eukaryotic DNAPs. Left: Schematic representation of T7 DNAP. The fingers (orange), palm (green) and thumb (yellow) domains are highlighted. The 3'-5' exonuclease domain is coloured in gray. Figure altered, from [Bradshaw, 2015]. Right: Schematic representation of a right hand. Anatomic features are coloured according to the DNAP domains carrying the corresponding names. ............................... 1.4 Three orifices give access to the active site of Φ29 DNAP. A) Cross-sectional surface representation of the enzyme in complex with homology-modeled DNA.

  . ............................ 1.5 Multiple sequence alignment of highly conserved N-terminal regions (ExoI, ExoII, ExoII) of DNA-dependent DNAPs on AA level. The DNAPs were grouped into four: PolI-like DNAPs (group A), viral --like DNAPs (group B), cellular -like DNAPs (group C), protein-primed --like DNAPs (group C). The numbers between slashes denote the AA position relative to the N-terminal end of each respective DNAP. White letters indicate AA similarity among the di erent groups, while grey boxes indicate other similarities and stars indicate specially conserved residues and/or PolI residues involved in exonucleolytic catalysis. Figure from [Blanco, 1991]. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . and interactions of ssDNA with the exonuclease site of Φ29 DNAP. A)

  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

  Figure from [Technology Co, ]. . . . . . . . . . . . . . . . . . . . . . 31 1.22 Scanning electron micrograph of SPG membranes with a pore size of a) d = 0.262 µm" (hydrophilic), b) d = 14.8 µm (hydrophilic) and c) d = 0.262 µm( r e n d e r e d hydrophobic with a coating of silicone resin). For each SPG membrane type, two di erent magnifications of the micrograph are shown. Figure altered, from [VladisavljeviÊ, 2005] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 1.23 Principle ME strategies. a) Cross-flow system (SPG membrane, direct ME), b)

  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . round of selection with iviCSR.

  Figure altered, from Uniprot feature viewer of P03680: https://beta.uniprot.org/uniprotkb/P03680/feature-viewer . . . . . . . 3.5 One round of selection consists of a selection assay that screens for the parameter to be selected. The output library is screened by NGS and mutants for validation are identified. The output library can be reformatted to serve as input for another round of QuIK-based selection. . . . . . . . . . . . . . . . . . . . . . . . . .

  Standard deviation (green) is plotted separately to point out degree of monodispersity. . . . . . . . . . . . . . . . . . . 4.3 Mean (a) and standard deviation (b) of droplet radius as measured by SLS as a function of number of pushes through an SPGE with pore size d =5 µm.F r e s h emulsion at t =0 h (blue) is compared to emulsion after incubation of t = 16h (red). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4 Mean (a) and standard deviation (b) of droplet volume as measured under the microscope (green) and by SLS (blue and red) as a function of number of pushes through an SPGE with pore size d =5µm. In case of the SLS measurement, fresh emulsion at t =0 h (blue) is compared to emulsion after incubation of t = 16h (red). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5 Schematic QuIK emulsification profiles of three di erent operators, hand-driven emulsification. While droplet size and degree of polydispersity di er for low number of pushes (x 1 ), the parameters converge for increasing number of pushes (x 1 ). 4.6 Di erential increase of Q1WT vs. Q1DD gene after a 16 h incubation as a function of number of pushes through the SPGE. The qPCR measurements show that the optimum of e ciency of in vitro compartmentalised self replication (iviCSR) is reached at 10 pushes in the QuIK Assay. . . . . . . . . . . . . . . . . . . . . . . . 4.7 Top left panel: emulsification speed of QuIK is two orders of magnitude faster than on-chip emulsification. Bottom left panel: Comparison of sample volumes emulsified with QuIK and on-chip. Right panel: Size distribution obtained by microscope measurements and static light scattering for QuIK and on-chip emulsification.

  (a and c) and Phred scores (b and d) for sequences with low (a and b) and high (c and d) UMI counts (cuto 3). Sequences across R0, R1 and R2 and all titration conditions were pooled before UMI subgroups were generated. Data analyzed with and graphs obtained from FastQC. . 88 4.26 Average position-wise frequencies on NT level for a) the entire sequenced region of the library prior to selection (R0), b) the entire sequenced region after two rounds of selection in standard conditions, c) positions 0 to 300 and 700 to 925 of the library prior to selection (R0) and d) positions 0 to 300 and 700 to 925 after two rounds of selection in standard conditions. Positions are related to bases along the sequenced DNA fragment and do not correspond to positions along the Φ29 gene. The high frequencies observed in between positions 300 to 450 and 475 to 925 (top panel) are due to a decrease in sequencing quality. Those regions are cut out in the bottom panel to highlight frequency changes between NNK-scanned and conserved positions. Vertical grey lines represent the positions at which the data was clipped. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 4.27 Average position-wise frequencies on NT level for a) the entire sequenced region of the library prior to selection (R0) and all titration conditions after one round of selection (R1), b) the entire sequenced region of the library prior to selection (R0) and all titration conditions after one round of selection (R1), c) positions 0 to 300 and 700 to 925 of the library prior to selection (R0) and all titration conditions after two rounds of selection (R2) and d) positions 0 to 300 and 700 to 925 prior to selection (R0) and all titration conditions after two rounds of selection (R2). Positions are related to bases along the sequenced DNA fragment and do not correspond to positions along the Φ29 gene. The high frequencies observed in between positions 300 to 450 and 475 to 925 (top panel) are due to a decrease in sequencing quality. Those regions are cut out in the bottom panel to highlight frequency changes between NNK-scanned and conserved positions. Vertical grey lines represent the positions at which the data was clipped. . . . . . 90 4.28 Enrichment of the four bases after selection R2 for the three challenging conditions a) 0µM, b) 7µM and c) 70µM, as well as for the standard condition d) 700µM dTTP. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 4.29 PW-enrichment matrices (NT level, the three mutated regions of positions 586-603, ML1 in the linker region) across dTTP titrations in round 2 (R2) of the dTTP Titration Assay. Histograms show the density of each titration sample. Molarities above the histograms refer to the dTTP concentrations in the respective sample. The top right tiles show pearson correlations. Loess smooths and pearson correlation ellypses are superimposed on the data points. . . . . . . . . . . . . . . 92 4.30 PW-enrichment matrices (NT level, the three mutated regions of positions 646-690, ML2 in the linker region) across dTTP titrations in round 2 (R2) of the dTTP Titration Assay. Histograms show the density of each titration sample. Molarities above the histograms refer to the dTTP concentrations in the respective sample. The top right tiles show pearson correlations. Loess smooths and pearson correlation ellipses are superimposed on the data points. . . . . . . . . . . . . . . 93 4.31 PW-enrichment matrix across dTTP concentrations, ML1 in the linker region, NT position 586 to 600 (AA 196 to 200). In each tile the diagonal (black line) and a linear fit to the data-points (red line) are displayed. . . . . . . . . . . . . . 94 xxi List of figures 4.32 PW-enrichment matrix across dTTP concentrations, ML2 in the linker region, NT positions 646 to 690 (AA 216 to 230). In each tile the diagonal (black line) and a linear fit to the data-points (red line) are displayed. . . . . . . . . . . . . . 4.33 PW-enrichment matrices (NT level, the three mutated regions of positions 4-72, 586-603, 646-690) across dTTP titrations in round 1 (R1) of the dTTP Titration Assay. Histograms show the density of each titration sample. Molarities above the histograms refer to the dTTP concentrations in the respective sample. The top right tiles show pearson correlations. Loess smooths and pearson correlation ellypses are superimposed on the data points. a) No UMI sequences have been clipped o during preprocessing of the NGS data. b) UMI sequences < 4 have been clipped o during preprocessing of the NGS data. . . . . . . . . . . . . . . . 4.34 PW-enrichment matrices (NT level, the three mutated regions of positions 4-72, 586-603, 646-690) across dTTP titrations in round 2 (R2) of the dTTP Titration Assay. Histograms show the density of each titration sample. Molarities above the histograms refer to the dTTP concentrations in the respective sample. The top right tiles show pearson correlations. Loess smooths and pearson correlation ellypses are superimposed on the data points. a) No UMI sequences have been clipped o during preprocessing of the NGS data. b) UMI sequences < 4 have been clipped o during preprocessing of the NGS data. . . . . . . . . . . . . . . . 4.35 PW-enrichment (PW -E) matrices (NT level, positions 4 -75, 586 -600 and 646 -690) across dATP titrations in round 1 (R1) of the dATP Titration Assay. Histograms show the density of each titration sample. Molarities above the histograms refer to the dATP concentrations in the respective sample. The top right tiles show pearson correlations. Loess smooths and pearson correlation ellypses are superimposed on the data points. . . . . . . . . . . . . . . . . . . . . . . . . . 4.36 PW-enrichment (PW -E) matrices (NT level, positions 4 -75, 586 -600 and 646 -690) across dCTP titrations in round 1 (R1) of the dCTP Titration Assay. Histograms show the density of each titration sample. Molarities above the histograms refer to the dCTP concentrations in the respective sample. The top right tiles show pearson correlations. Loess smooths and pearson correlation ellipses are superimposed on the data points. . . . . . . . . . . . . . . . . . . . . . . . . . 4.37 PW-enrichment (PW -E) matrices (NT level, positions 4 -75, 586 -600 and 646 -690) across dGTP titrations in round 1 (R1) of the dGTP Titration Assay. Histograms show the density of each titration sample. Molarities above the histograms refer to the dGTP concentrations in the respective sample. The top right tiles show pearson correlations. Loess smooths and pearson correlation ellipses are superimposed on the data points. . . . . . . . . . . . . . . . . . . . . . . . . . 4.38 Conservation pattern of PW-enrichment within the mutated region in the exonuclease domain across dTTP titration conditions, selection round R2. The connecting lines do not represent continuous data but are for visualisation purposes only. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.39 Position-wise enrichment correlation on NT level for the three NNK-scanned regions on the Φ29 DNAP gene. PW-enrichment of round R2 (as determined in NGS) is plotted for each tested dTTP concentration on the y-axis against the standard condition of 700 µM dTTP on the x-axis. Linear fits are plotted in the same colour as the respective dTTP titration condition. . . . . . . . . . . . . . . xxii List of figures 4.40 Enrichment correlation for the three NNK-scanned regions on the Φ29 DNAP gene after selection (after round R2). a) Slopes of the linear fits of figure 4.39 are plotted on the y-axis against the corresponding dTTP concentration on the xaxis. b) The RSE for the the three imbalanced dNTP concentrations. No RSE can be obtained for the standard conditions, as it is the dependent and independent variable of the linear fit. The RSE of the 0 dTTP condition serves as error bar for data points in panel a). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 4.41 Position-wise enrichment correlation on NT level for the three NNK-scanned regions on the Φ29 DNAP gene after selection (round R1, dTTP titration), as determined by NGS. a) PW-enrichment is plotted for each tested dTTP concentration on the y-axis against the standard condition on the x-axis. Linear fits are plotted in the same colour as the respective dTTP titration condition. b) Slopes of the linear fits of panel a) are plotted on the y-axis against the corresponding dTTP concentration on the x-axis. c) The RSE for the three imbalanced dNTP concentrations. No RSE can be obtained for the standard conditions, as it is the dependent and independent variable of the linear fit. The RSE of the 0 dTTP condition serves as error bar for data points in panel b). . . . . . . . . . . . . . . 104 4.42 Position-wise enrichment correlation on NT level for the three NNK-scanned regions on the Φ29 DNAP gene after selection (round R1, dATP titration), as determined by NGS. a) PW-enrichment is plotted for each tested dATP concentration on the y-axis against the standard condition on the x-axis. Linear fits are plotted in the same colour as the respective dATP titration condition. b) Slopes of the linear fits of panel a) are plotted on the y-axis against the corresponding dATP concentration on the x-axis. c) The RSE for the the three imbalanced dNTP concentrations. No RSE can be obtained for the standard conditions, as it is the dependent and independent variable of the linear fit. The RSE of the 0 dTTP condition serves as error bar for data points in panel b). . . . . . . . . . . 105 4.43 Position-wise enrichment correlation on NT level for the three NNK-scanned regions on the Φ29 DNAP gene after selection (round R1, dCTP titration), as determined by NGS. a) PW-enrichment is plotted for each tested dCTP concentration on the y-axis against the standard condition on the x-axis. Linear fits are plotted in the same colour as the respective dCTP titration condition. b) Slopes of the linear fits of panel a) are plotted on the y-axis against the corresponding dCTP concentration on the x-axis. c) The RSE for the the three imbalanced dNTP concentrations. No RSE can be obtained for the standard conditions, as it is the dependent and independent variable of the linear fit. The RSE of the 0 dTTP condition serves as error bar for data points in panel b). . . . . . . . . . . 106 4.44 Position-wise enrichment correlation on NT level for the three NNK-scanned regions on the Φ29 DNAP gene after selection (round R1, dGTP titration), as determined by NGS. a) PW-enrichment is plotted for each tested dGTP concentration on the y-axis against the standard condition on the x-axis. Linear fits are plotted in the same colour as the respective dGTP titration condition. b) Slopes of the linear fits of panel a) are plotted on the y-axis against the corresponding dGTP concentration on the x-axis. c) The RSE for the the three imbalanced dNTP concentrations. No RSE can be obtained for the standard conditions, as it is the dependent and independent variable of the linear fit. The RSE of the 0 dTTP condition serves as error bar for data points in panel b). . . . . . . . . . . 107 xxiii List of figures 4.45 The sequence space of a round r (black) includes all genes of a library. NGS sequencing can only capture a sub-sample (blue) of all sequences. The function g(n r ( ‡ i )) maps the gene ‡ i , captured in NGS, to the computationally ascertainable value n r ( ‡ i ) that denotes the number of occurrences of a sequence in the sample. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.46 The NGS sampled sequence space of a round r (blue). The function e(n r ( ‡ i ))

Figure 1 . 3 :

 13 Figure 1.3: Right hand analogy of bacterial and eukaryotic DNAPs. Left: Schematic representation of T7 DNAP. The fingers (orange), palm (green) and thumb (yellow) domains are highlighted. The 3'-5' exonuclease domain is coloured in gray. Figure altered, from [Bradshaw, 2015]. Right: Schematic representation of a right hand. Anatomic features are coloured according to the DNAP domains carrying the corresponding names.

Figure 1 . 6 :

 16 Figure 1.6: Contact and interactions of ssDNA with the exonuclease site of Φ29 DNAP. A) Side chains of residues T15, N62, F65, Y148, P129 (yellow), and L567 (green sticks) contact the single stranded template DNA. The polymerase domain and two 5' bases are omitted. A segment of the thumb backbone is shown in green (positions 560-575). DNA residues are labeled T3-T5 and metal ion A is represented by a blue dot. B) Di erence electron denisty map of cocrystallised ssDNA and native protein.Figure from[Kamtekar, 2004].

  Figure 1.22: Scanning electron micrograph of SPG membranes with a pore size of a) d = 0.262 µm" (hydrophilic), b) d = 14.8 µm (hydrophilic) and c) d = 0.262 µm (rendered hydrophobic with a coating of silicone resin). For each SPG membrane type, two di erent magnifications of the micrograph are shown.Figurealtered, from [VladisavljeviÊ, 2005]

Figure 3

 3 Figure 3.3: Standard curve of qPCR primers targeting Q1WT and Q1DD templates. The two templates can easily be discriminated in an iviCSR reaction.

Figure 3

 3 Figure 3.7: Illumina NGS workflow. Top panel: The to-be-sequenced DNA templates are hybridised to the flow-cell and a polymerase creates a complement strand. The original template is then washed away. Left middle panel: A complementary strand is synthesised through bridge amplification. The double stranded bridge is denatured, leaving two single stranded copies of each template immobilised on the flow cell. Parallelisation of the bridge amplification generates clusters. Right middle panel: Reverse copy strands are cleaved and washed away. Sequencingby-synthesis is carried out on the remaining forward strands as shown in the bottom panel.This picture does not show the generation of the second read: After completion of the first read, the read product strand is dehibridised and washed o . A complementary strand is synthesised through bridge amplification. The bridge is dehibridised and the already read forward strand is cleaved and washed o , leaving the reverse strand which can then be sequenced to give read 2. This schematic representation is not to scale. Figure from[Weinstock, 1752]

  With QuIK I am able to emulsify reaction volumes up to approximately 2mL and thus perform a large number of experiments in parallel. The typical reaction volumes of the assays presented in this work are 30-210 µL, corresponding to 3 * 10 10 to 2 * 10 11 droplets of 1 fL volume.

Figure 4

 4 Figure 4.1: The QuIK setup for emulsification.An SPGE is installed between two syringes and an emulsion is conveniently created by pushing two phases (aqueous and oil phase) multiple times back and forth through the extruder.

Figure 4

 4 Figure 4.2: Emulsification of de-ionised water and 35% IVTT in water mixture. Test of di erent membrane types with 50 pushes through the SPGE. SLS measurements suggest that droplet size can be decreased by smaller pore sizes, emulsification of viscous sample and membrane passivation.Standard deviation (green) is plotted separately to point out degree of monodispersity.

Figure 4 .

 4 Figure4.3 a) shows the droplet radius as a function of number of pushes through the extruder. After five pushes the mean droplet radius is 880 nm and drops to 455 nm after 20 pushes. The decrease in radius becomes smaller with increasing number of pushes, which limits the course of the decrease curve, which plateaus after 15 pushes. The data -each value shows mean and standard error on a triplicate -indicates that both mean droplet radius and polydispersity of the aqueous droplets are reduced by increasing number of passes through the extruder. The standard deviation of droplet size (figure 4.3 b)) follows a comparable pattern: The polydispersity decreases with an increase of pushes. Standard deviation is 340 nm for five pushes. After 20 pushes the standard deviation decreased to 170 nm. Again, the negative slope of the curve increases with increasing number of pushes: An additional five pushes leads to a 1.5-fold decrease of polydispersity when starting with five pushes. However, after 15 pushes have been carried out an additional five pushes will decrease polydispersity only marginally by 1.08-fold. At 10 pushes the mean droplet radius is r =0.6µm with a standard deviation of sd =0.2µm. I envisioned the upcoming selection assays to have a maximum incubation time of 16 hours at an incubation temperature of 30 °C. There is a potential for droplets to either shrink or coalesce during this time. Comparison of fresh and incubated emulsions, however, reveals only a small increase in droplet radius (i.e., below the experimental resolution for 5 and 10 pushes, "r =0.06

Figure 4

 4 Figure4.5: Schematic QuIK emulsification profiles of three di erent operators, hand-driven emulsification. While droplet size and degree of polydispersity di er for low number of pushes (x 1 ), the parameters converge for increasing number of pushes (x 1 ).

Figure 4

 4 Figure 4.6: Di erential increase of Q1WT vs. Q1DD gene after a 16 h incubation as a function of number of pushes through the SPGE. The qPCR measurements show that the optimum of e ciency of in vitro compartmentalised self replication (iviCSR) is reached at 10 pushes in the QuIK Assay.

Figure 4 .

 4 12 shows the position-wise impact (AA-level) of amino-acid changes on the enrichment of Φ29 DNAP variants (PW-enrichment, see chapter 3.3.4.2), characterised by the polymerisation activity increase in one round of selection. With the position-wise plotting it becomes immediately evident that E has a clear biological meaning: The regions marked in red colour (figure4.12) correspond to the mutations with bottom 10% PW-enrichment (E<0.3), i.e. to the positions where mutations have the strongest negative impact on activity.

Figure 4 .

 4 Figure 4.12: Position-wise enrichment e ects of random mutagenesis on Φ29 DNAP genes. Sequence positions having a PW-enrichment E<0.3 are coloured in red.

  Figure 4.15: Frequency correlation on NT level across rounds between Pacific Biosciences and Illumina sequencing data at NT positions a) 689-687 (AA level: 227-229), b) 1,237-1,245 (AA level: 413-415) and c) 1,288-1,296 (AA level: 430-432).

  Figure 4.17: CL-Enrichment across rounds for the six top enriched rounds H025 DNAP clones with mutations at AA positions 227-229 (a), 413-415 (b) and 430-432 (c).

Figure 4 .Figure 4

 44 Figure4.27: Average position-wise frequencies on NT level for a) the entire sequenced region of the library prior to selection (R0) and all titration conditions after one round of selection (R1), b) the entire sequenced region of the library prior to selection (R0) and all titration conditions after one round of selection (R1), c) positions 0 to 300 and 700 to 925 of the library prior to selection (R0) and all titration conditions after two rounds of selection (R2) and d) positions 0 to 300 and 700 to 925 prior to selection (R0) and all titration conditions after two rounds of selection (R2). Positions are related to bases along the sequenced DNA fragment and do not correspond to positions along the Φ29 gene. The high frequencies observed in between positions 300 to 450 and 475 to 925 (top panel) are due to a decrease in sequencing quality. Those regions are cut out in the bottom panel to highlight frequency changes between NNK-scanned and conserved positions. Vertical grey lines represent the positions at which the data was clipped.

Figures 4 .Figure 4

 44 Figure 4.38: Conservation pattern of PW-enrichment within the mutated region in the exonuclease domain across dTTP titration conditions, selection round R2. The connecting lines do not represent continuous data but are for visualisation purposes only.

Figure 4 .

 4 Figure 4.39: Position-wise enrichment correlation on NT level for the three NNK-scanned regions on the Φ29 DNAP gene. PW-enrichment of round R2 (as determined in NGS) is plotted for each tested dTTP concentration on the y-axis against the standard condition of 700 µM dTTP on the x-axis. Linear fits are plotted in the same colour as the respective dTTP titration condition.

  Figure 4.40: Enrichment correlation for the three NNK-scanned regions on the Φ29 DNAP gene after selection (after round R2). a) Slopes of the linear fits of figure 4.39 are plotted on the y-axis against the corresponding dTTP concentration on the x-axis. b) The RSE for the the three imbalanced dNTP concentrations. No RSE can be obtained for the standard conditions, as it is the dependent and independent variable of the linear fit. The RSE of the 0 dTTP condition serves as error bar for data points in panel a).

  Figure 4.41: Position-wise enrichment correlation on NT level for the three NNK-scanned regions on the Φ29 DNAP gene after selection (round R1, dTTP titration), as determined by NGS. a) PW-enrichment is plotted for each tested dTTP concentration on the y-axis against the standard condition on the x-axis. Linear fits are plotted in the same colour as the respective dTTP titration condition. b) Slopes of the linear fits of panel a) are plotted on the y-axis against the corresponding dTTP concentration on the x-axis. c) The RSE for the three imbalanced dNTP concentrations. No RSE can be obtained for the standard conditions, as it is the dependent and independent variable of the linear fit. The RSE of the 0 dTTP condition serves as error bar for data points in panel b).

  8 for dTTP. The results shown in figures 4.41 a) and 4.42 a) suggest that the titration of purine Adenine shows a similar pattern than titration of pyrimidine Thymine in round one: The drop of the curves of figures 4.41 b) and 4.42 b) is not abrupt in the range of 1 to 70 muM. The round one titrations of pyrimidine Cytosine and purine Guanine on the other hand show a di erent behaviour: For both titrations the slopes of the 7 muM titration condition are rather flat (approximtely 0.7 and 0.8) before they both drop abruptly to approximately 0.2 for the 1 muM condition (see figures 4.43 b) and 4.44 b)).

  Figure 4.42: Position-wise enrichment correlation on NT level for the three NNK-scanned regions on the Φ29 DNAP gene after selection (round R1, dATP titration), as determined by NGS. a) PW-enrichment is plotted for each tested dATP concentration on the y-axis against the standard condition on the x-axis. Linear fits are plotted in the same colour as the respective dATP titration condition. b) Slopes of the linear fits of panel a) are plotted on the y-axis against the corresponding dATP concentration on the x-axis. c) The RSE for the the three imbalanced dNTP concentrations. No RSE can be obtained for the standard conditions, as it is the dependent and independent variable of the linear fit. The RSE of the 0 dTTP condition serves as error bar for data points in panel b).

  Figure 4.43: Position-wise enrichment correlation on NT level for the three NNK-scanned regions on the Φ29 DNAP gene after selection (round R1, dCTP titration), as determined by NGS. a) PW-enrichment is plotted for each tested dCTP concentration on the y-axis against the standard condition on the x-axis. Linear fits are plotted in the same colour as the respective dCTP titration condition. b) Slopes of the linear fits of panel a) are plotted on the y-axis against the corresponding dCTP concentration on the x-axis. c) The RSE for the the three imbalanced dNTP concentrations. No RSE can be obtained for the standard conditions, as it is the dependent and independent variable of the linear fit. The RSE of the 0 dTTP condition serves as error bar for data points in panel b).

  the reaction dynamics of the Titration Assay

  Nucleotide Polymorphism Genotyping by Fragment Analysis 5. Magnetic Tweezers (Single Molecule Level) 6. Leading-Strand Synthesis (Single Molecule Level) 7. The MagNIFi Assay of De Paz et al.

  (figure 4.54 a)) and the absolute final DNA concentration (figure 4.54 b)).

  Figure 4.54: A) Fold increase and B) absolute concentration of ⁄ DNA reporter DBs amplified in bulk by in vitro expressed variants in a one-pot expression-replication assay. Fold Increase of incubated with respect to frozen sample is shown. Four separate dTTP concentrations are tested: Standard condition, 10x dilution, 100x dilution, no dTTP.

Figure 4 .

 4 Figure 4.58: Bulk expression replication reaction with standard equimolar dNTP concentration set to 700uM . V1 and V2 genes are compared and di erential increase of ⁄ DNA [ID:FND] is shown in terms of a) fold increase and b) absolute DNA concentration (SuM 12.2).For both, WT and variant R223T, more DNA could be synthesised with V1 genes, compared to V2 genes. 128

  Di erent Bu ering Conditions • NTPs crowd the environment of the reaction • Binding of T7 RNA polymerase to the template DNA Morin et al. published their results in 2015 based on single molecule measurements in optical tweezers. Saturno et al. published in 1995 already and based their results on a bulk primer extension assay that was interpreted based on urea-20% polyacrylamide gel electrophoresis and autoradiography. For this reason, I assume K M =31 µM to be closer to the actual K D value of Phi29 DNAP WT. My data and the fitting of the Michaelis-Menten Model supports this assumption.

Figure 6

 6 Figure 6.1: a) Experimental setup of single molecule primer extension. DNA molecules are immobilised on a glass slide that forms a flow chamber in ensemble with an attached PDMS cover. A laminar flow in the microfluidic chip stretches the DNA molecules. Wide-field optical microscopy is utilised to image and track beads attached to the DNA molecules. b) Image of the glass cover slip. The inset shows a representative field of view with 4,000 beads. An image of beads on flow-stretched DNA (magenta) is superimposed with an image of the same DNAattached beads flow-stretched in the opposite direction (green). Improperly tethered are shown in black. black scale bar represents 150 µm. c) DNA template with attached bead (yellow) on one side and a replication fork at the other side.The 5' end of the fork is attached to the glass slide via a biotin-streptavidin binding. A primer is annealed to the 3' end of the fork to initiate DNA synthesis. d) Primer extension is schematically represented as an example using the S. cerevisiae replisome. As dsDNA of the template is converted into ssDNA during DNA synthesis, the bead moves and its trajectory can be captured with the wide-field optical microscope. This schematic representation is not to scale. Figure altered from[Lewis, 2017].

Figure 7

 7 Figure 7.4: Frequency correlation on NT level across rounds between Illumina sequencing data with and without filtering by quality and MAPQ scores. NT positions a) 689-687 (AA level: 227-229), b) 1,237-1,245 (AA level: 413-415) and c) 1,288-1,296 (AA level: 430-432).

  Figure 7.8: SpS-Enrichment of AA substitutions across rounds 1 and 2 of the dTTP Titration Assay. Standard Conditions are plotted against challenged conditions with 10-fold and 100-fold titrated dTTP, respectively. a) AA positions 2 to 24 in the exonuclease domain. b) and c) AA positions 196 to 200 in the linker region. d) and e) AA positions 216 to 230 in the linker region.

  Figure 7.18: Read counts after pre-processing of the Illumina NGS data of the Titration Assay for a) R0 and R1 as well as b) R0 and R2

  ). RNA concentration is derived by multiplication of initial DNA concentration (reverse transcribed from RNA) with fold increase (fold increase is calculated by ∆Cq = Cq incubated -Cq non-incubated ). . . . . . . . . . . . . 70 4.12 Position-wise enrichment e ects of random mutagenesis on Φ29 DNAP genes.Sequence positions having a PW-enrichment E<0.3 are coloured in red. . . . . 71 4.13 Left: The PW-enrichment displayed in figure4.12 is mapped onto the 3D protein Schematic representation of DNA synthesis and error correction. a) Φ29 DNAP is bound to a template. The enzyme's polymerisation domain (green), exonuclease domain (blue) and linker region (red) are highlighted. b) The DNAP synthesises the primer strand by incorporation of dNTPs (cyan), which are present in the reaction mixture. A nucleotide, which does not correspond to the base coming up next in the template strand, is coloured in red. c) The non-corresponding nucleotide has been incorporated resulting in a mismatching base pair. Consequently, the physical confirmation of the nascent strand is shifted from the polymerisationto the enzyme's exonuclease domain, where the misincorporation is excised by hydrolysis of the mismatched base. Afterwards, the physical conformation of the nascent strand is switched back into the polymerisation domain, where synthesis is resumed (not shown). Figure inspired by Kamtekar et al.[Kamtekar, 2004]. . 4.20 One round of selection with the Titration Assay. The workflow is adapted from the Activity Assay of section 4.6. A library with mutations in the exonuclease domain and the linker region serves as input. Q1WT and Q1DD serve as positive and negative controls. The environment in the compartments is changed such that dTTP is present in varying concentrations. Created with Biorender.com. . . 4.21 Position-wise enrichment e ects of random mutagenesis on Φ29 DNAP genes.

	List of figures
	4.19
	76
	4.17 CL-Enrichment across rounds for the six top enriched rounds H025 DNAP clones
	with mutations at AA positions 227-229 (a), 413-415 (b) and 430-432 (c). . . . . 77
	4.18 Activity of H025 DNAP variants as determined by fold increase over WT activity
	in qPCR. a) In library 227-229 nine variants show activity, four of which are
	able to outcompete WT. Variant VQT shows 3.2-fold higher enrichment than
	WT. b) In library 413-430 eight variants show activity, six variants are able to
	outcompete WT. Variant VHR shows 4.14-fold higher enrichment than WT. c)
	With nine mutants showing activity increase over WT, region 430-432 contributes
	to most of the 19 found improved variants. Variant VKS shows highest activity
	gain over WT (4.4-fold). Variant GIK shows activity -1.8-fold lower than WT. . 78
	xix

structure of Φ29 DNAP (Protein Data Bank: 2pyj). The main catalytic domains (exonuclease and polymerisation domain as well as the linker subdomain) are highlighted. Right: Inset of the 3D structure on the left. The polymerase domain and the conserved Pol A, B and C motifs are highlighted. . . . . . . . . . . . . . 72 4.14 Position-wise enrichment e ects of random mutagenesis on Φ29 DNAP genes. Sequence positions having a PW-enrichment E<0.3 are coloured in red. The regions selected for the H025 variant library are highlighted in yellow. The regions corresponding to the 3' → 5' exonuclease domain, the polymerase domain as well as linker and TPR2 subdomains are indicated below the conservation pattern. . . 73 4.15 Frequency correlation on NT level across rounds between Pacific Biosciences and Illumina sequencing data at NT positions a) 689-687 (AA level: 227-229), b) 1,237-1,245 (AA level: 413-415) and c) 1,288-1,296 (AA level: 430-432). . . . . . 74 4.16 Schematic flow chart representation of the bulk iviCSR assay. Variants are expressed in individual tubes from linear genes and replicate a ⁄-DNA reporter DB. After digestion of the template by the DpnI enzyme the replication activity is quantified by qPCR. Created with Biorender.com . . . . . . . . . . . . . . . . . . Sequence positions having a PW-enrichment E < 0.3 are coloured in red. The regions selected for the Φ29 DNAP library of the titration assay are highlighted in yellow. The double-point library disposes of one codon substitution in the exonuclease domain and one codon substitution in either of two regions within the linker region. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.22 A 1 fL sized droplet contains 400.000 deoxynucleotides if the reaction volume is at

  .23 The template of iviCSR-based QuIK Assays are DB shaped. Replication is primed with a ssDNA fragment ("Dumbbell Primer", green) that targets the single stranded loops. The RCA product synthesised by active DNAP variants consists of consecutive copies of the DB template in forward. Representation of UMIs in the NGS dataset. 0.71% of the UMIs occur only once.UMIs that occur one or two times make 0.75% of all UMIs and so on. . . . . . . 87 4.25 Per Base Sequence Quality Scores

	List of figures
	4.24
	One ssDNA copy contains
	two parts: 1) a forward sense strand and 2) a reverse complementary of the tem-
	plate's coding strand. PCR primers target and amplify both parts of the copy
	(blue box). In the dTTP Titration Assay misincorporations (red) can occur at
	sites corresponding to "A" sites and "T" sites of the template's coding strand,
	depending on the region within the RCA copy. The sequences in this schematic
	xx

mentioned the presence of residual dNTPs in individual dATP, dCTP, dGTP and dTTP vials due to the manufacturing process. This could explain minor activity in 0 µM and 7 µM conditions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4representation are not the actual sequences of the templates applied, but serve demonstration purposes only. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

xxix Abbreviation list K M Michaelis Menten Constant AA Amino Acid bp Basepair CL-Enrichment Clone-Level-Enrichment CL-Frequency Clone-Level-Frequency Cq Quantification Cycle CSR Compartmentalised Self Replication CTRL Control D Digested DB Dumbbell DNA Deoxyribonucleic Acid DNAP DNA Polymerase dNTP Deoxynucleoside Triphosphate E Enrichment EES Error-Enriched Site f Frequency FAC S Fluorescence-Activated Cell Sorting FI Fold Increase

  ). Michaelis-Menten theory based models with K M = 31±3 µM (blue) and K M = 0.5±0.2 µM (green) are fitted and superimposed. A best fit was derived by Monte-Carlo method giving K M = 45 µM (cyan). . . . . . . . . . 4.48 Slope of the linear fits of enrichment correlations in the three mutated regions(R1, nt level, in black). The share that the dTTP concentration has in the slope at the molecular level is modelled (red). . . . . . . . . . . . . . . . . . . . . . . . . . 4.49 PW-enrichment at NT level in the mutated linker region ML2, round two. PW-.52 Protein model of Φ29 DNAP. Blue arrows and bolt font indicate positions R223 on an alpha helix in the linker region and E14 as well as V19 on a beta sheet in the exonuclease domain. Figure altered, from Uniprot feature viewer of P03680: https://beta.uniprot.org . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.53 Position-wise amino acid substitution enrichment on all genes with substitution R223T and all genes without substitution R223T. R2, dTTP concentration c T = 6.7µM , sequence before T7 promoter. . . . . . . . . . . . . . . . . . . . . . . . . 4.54 A) Fold increase and B) absolute concentration of ⁄ DNA reporter DBs amplified in bulk by in vitro expressed variants in a one-pot expression-replication assay. Di erential increase for clone R223T (and Q1WT) with respect to Q1DD. This is shown in terms of a) DDCq, the qPCR's log2-fold read out (DDCq X:DD = [DCq[DD FND -X FND ] -DCq[DD ID -X ID ]], where "X" is either clone R223T or Q1WT, see also SuM 12.2) and b) non-logarithmic DNA fold increase (FI = 2 DDCq ). c) Di erential increase of clone R223T over Q1WT in terms of fold increase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123 4.56 a) The 5' end of Φ29 DNAP WT gene (top), the two single point variants (middle) and the double point variant (bottom) are shown. The qPCR primer binding sites are annotated. qPCR primers target AA substitutions of the clones. For this reason, primer sets for R223T and E14L can not discriminate between the single point mutant clones and the double point mutant clone. b) Template dilution / primer range assay on the first set of synthesised variants and WT. Cq values and linear fits as determined by qPCR over a range of seven 10-fold template dilutions starting with 62pM . A) The right template-primer set combination shows a linear and parallel Cq distribution throughout the whole titration range for both, WT and variant R223T. 4.59 Comparison of the R223T V1 clone to WT in the QuIK Assay. Fold increase and absolute synthesised DNA for non-digested (left panels) and digested (right panel) samples. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 4.60 Fold increase of DNA synthesised by clone R223T over Φ29 DNAP WT, as mea-sured in qPCR, in di erent dTTP titration conditions. In the absence of dTTP, clone R223T is able to synthesise 2.2-fold more RCA product than WT. In higher SpS-Enrichment on AA level standard conditions are plotted against the challenging condition of 10-fold diluted dTTP for R1 (blue) and R2 (black). In the R1 dataset exemplary amino acid substitutions are highlighted in magenta. The same substitutions are highlighted in red in the R2 dataset. . . . . . . . . . . . . 6.1 a) Experimental setup of single molecule primer extension. DNA molecules are immobilised on a glass slide that forms a flow chamber in ensemble with an attached PDMS cover. A laminar flow in the microfluidic chip stretches the DNA molecules. Wide-field optical microscopy is utilised to image and track beads attached to the DNA molecules. b) Image of the glass cover slip. The inset shows a representative field of view with 4,000 beads. An image of beads on flow-stretched DNA (magenta) is superimposed with an image of the same DNA-attached beads flow-stretched in the opposite direction (green). Improperly tethered are shown in black. black scale bar represents 150 µm. c) DNA template with attached bead(yellow) on one side and a replication fork at the other side. The 5' end of the fork is attached to the glass slide via a biotin-streptavidin binding. A primer is annealed to the 3' end of the fork to initiate DNA synthesis. d) Primer extension is schematically represented as an example using the S. cerevisiae replisome. As dsDNA of the template is converted into ssDNA during DNA synthesis, the bead moves and its trajectory can be captured with the wide-field optical microscope. Kinetic rates, equilibrium constants and force dependencies proposed by Morin et al.[Morin, 2015] for the nucleotide incorporation cycle based on Brownian ratched model. k on denotes the rates at which the enzyme binds the nucleotide, k cat the catalytic rate constant, k T and k -T the forward and backward translocation rates, d T and d -T their respective associated characteristic distances, K " the equilibrium constant of the translocation step and " the distance between the pre-and posttranslocated states. . . . . . . . . . . . . . . . . . . . . . . . . . . . Sequences mentioned in this work and their base content. . . . . . . . . . . . . . 168 7.9 The in vitro methylation reaction. . . . . . . . . . . . . . . . . . . . . . . . . . . 171 7.10 The DNA damage repair reaction. . . . . . . . . . . . . . . . . . . . . . . . . . . 171 7.11 The end repair reaction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171 7.12 The blunt ligation reaction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172 7.13 The exonuclease reaction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172 7.14 Composition of the iviCSR reaction for QuIK Assays. . . . . . . . . . . . . . . . 173 7.15 Composition of digestion and non-digestion control reactions . . . . . . . . . . . 175 7.16 The qPCR reaction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175 7.17 qPCR program. Steps 2)-5) are cycled 40 times. . . . . . . . . . . . . . . . . . . 176 7.18 The recPCR Reaction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179 7.19 The recPCR program. Steps 2)-4) are cycled 30 times. . . . . . . . . . . . . . . . 179 7.20 The recPCR Reaction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

		List of figures List of figures List of tables Abbreviation list
	7.9 Activity Increase (∆Cq Incubated:Frozen), as determined in qPCR, of the Φ29 DNAP library in bulk in the standard condition after one round of selection. . . 166 7.10 Enrichment, as determined in qPCR, of the Φ29 DNAP library in emulsion after one round of selection. Enrichment is measured as ∆∆Cq of Library:DD. . . . . 166 7.11 Enrichment, as determined in qPCR, of the Φ29 DNAP library in emulsion after round 2. Enrichment is measured as ∆∆Cq of Library:DD. . . . . . . . . . . . . 167 7.12 Circularised product (Φ29 DNAP gene cloned into a PURExpress Control DHFR Plasmid (#N0424AVIAL, NEB)) of Gibson Assembly between recPCR and out-PCR. outPCR primer target sites and Φ29 DNAP gene with mutated regions in exonuclease domain and linker region are shown. To amplify a linear Φ29 DNAP gene fragment, PCR primers are used that target the shown "PCR primer" sites. 168 7.13 Typically, the outputs of assays in this work are a) a frozen, non-incubated ref-List of tables 7.4 Left: CL-Enrichment of AA substitutions at positions 413-415 on the H0-25 DNAP gene across rounds R0 to R4. PDI is the WT. Right: Corresponding Haplotypes with the highest respective CL-enrichment. . . . . . . . . . . . . . . . 160 7.5 Left: CL-enrichment of AA substitutions at positions 430-432 on the H0-25 DNAP gene across rounds R0 to R4. LTL is the WT. Right: Corresponding Haplotypes with the highest respective CL-enrichment. . . . . . . . . . . . . . . . . . . . . . 161 7.6 PW-enrichment correlation matrix across dTTP concentrations, exonuclease do-main, NT positions 4 to 75 (AA 2 to 25, MEx). . . . . . . . . . . . . . . . . . . . 163 7.7 WT sequence context 20 NT positions upstream and 10 NT positions downstream the AA mutation positions E14 and R223 in the Phi29 DNAP gene. Base content on the right refers only to the 20 NT sequence upstream the red codon. Codon exchanges at the two examined positions are E14L: GAA→CTG and R223T: iviCSR In Vitro Isothermal Compartmentalised Self Replication IVTT In Vitro Transcription Translation kbp Kilo Basepair L-dTG Tri-Glycerol-Based Dendritic Polar Head with PFPE Polymer Chains of Low Molecular Weight Fluorine Chains ME Membrane Emulsification MEx Mutated Exonuclease AA Positions 2-25 ML1 Mutated Linker AA Positions 196 to 200 4.55 a) This schematic representation is not to scale. Figure altered from [Lewis, 2017]. . 6.2 Schematics of a magnetic tweezers assay on a DNAP. A) A DNA hairpin template is tethered between a glass slide on one end and a magnetic bead on the other end. The bead is exposed to a magnetic field. A DNAP is bound to the tem-plate and polymerisation as well as exonuclease rates can be obtained by position tracking of the bead. Three di erent modes can be distinguished. B) Read-out of bead position tracking results in characteristic traces for the DNAP. C) Strand displacement mode. D) primer extension mode. This schematic representation is not to scale. Figure altered from [Mañosas, 2012b]. . . . . . . . . . . . . . . . . . 7.3 Mutation Rate in R0 along the DNA fragment sequenced on the PacBio system. The gene starts after NT position 301. The site saturated regions at gene positions (NT level) 681-690, 1239-1248 and 1290-1299 are sticking out. . . . . . . . . . . . 7.4 Frequency correlation on NT level across rounds between Illumina sequencing data with and without filtering by quality and MAPQ scores. NT positions a) 689-687 (AA level: 227-229), b) 1,237-1,245 (AA level: 413-415) and c) 1,288-1,296 (AA level: 430-432). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5 CL-Enrichment in R4 as determined by NGS sequencing analysis is plotted against WT DCq value). Left side: NGS CL-enrichment as determined on haplotype level. Right side: NGS enrichment as determined on amino acid level. . . . . . . 7.6 PW-enrichment Matrix across dTTP Concentrations, Exonuclease Domain, NT 7.7 PW-enrichment matrix across dTTP concentrations, exonuclease domain, NT Assay. Standard Conditions are plotted against challenged conditions with 10-exonuclease domain. b) and c) AA positions 196 to 200 in the linker region. d) and e) AA positions 216 to 230 in the linker region. . . . . . . . . . . . . . . . . . subsequent NGS analysis (C). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191 7.3 Left: CL-enrichment of AA substitutions at positions 227-229 on the H0-25 DNAP with the highest respective Cl-enrichment. . . . . . . . . . . . . . . . . . . . . . . IVC In Vitro Compartmentalisation gene across rounds R0 to R4. LGF is the WT. Right: Corresponding Haplotypes IDT Integrated DNA Technologies, Inc. fold and 100-fold titrated dTTP, respectively. a) AA positions 2 to 24 in the the templates. Bottlenecking to 200.000 molecules reduces PCR bias in the PCR for NGS library preparation (C) and limits the library to a reasonable size for over Q1DD. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ID Incubated, Digested Sample rounds of selection. Bulk enrichment of the library before selection was ∆Cq=1.88 7.8 SpS-Enrichment of AA substitutions across rounds 1 and 2 of the dTTP Titration and outPCR). B) A UMI sequence of 11 nt (∼4 Mio possible UMIs) is ligated to in qPCR. Enrichment is given in terms of ∆Cq as determined by qPCR over four FOS Oil and Surfactant Mixture positions 4 to 75 (AA 2 to 25). . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.20 Workflow from library recovery after selection over bottlenecking to NGS library preparation. A) Recovery of the selected library involves two PCR steps (recPCR 7.1 Composition of the iviCSR reaction. . . . . . . . . . . . . . . . . . . . . . . . . . 7.2 Enrichment of Q1 and the H0-25 DNAP variant library over Q1DD, as determined FND Frozen, Non-Digested Sample positions 4 to 75 (AA 2 to 25, MEx). . . . . . . . . . . . . . . . . . . . . . . . . . for a) R0 and R1 as well as b) R0 and R2 . . . . . . . . . . . . . . . . . . . . . . 189 depth, PacBio has a larger coverage on the gene. . . . . . . . . . . . . . . . . . . 190 the concentration of dTTP. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . selection on the H0-25 DNAP library. While Illumina has much higher sequencing tions belonging to the codon forming outliers in figure 4.49. Molarities refer to 7.19 Illumina and PacBio sequencing read depth illustrated by the first two rounds of 5.1 SpS-Enrichment across rounds and titrations of the two dominant AA substitu-the DCq values as obtained in a bulk replication assay by qPCR (normalised to erence sample and b) an incubated sample. Selective digestion of the methylated DB-shaped template with the DpnI restriction enzyme, as described in section SuM 11, gives two additional samples: c) The frozen, digested sample and d) the incubated, digested sample. The author would like to thank PacMan for the embodiment of the DpnI restriction enzyme. . . . . . . . . . . . . . . . . . . . . . 177 7.14 Agarose Gel of a M13 Primer Extension Assay with commercial and expressed Φ29 DNAP WT. Lane 1: Single Stranded M13 Vector with pre-annealed forward and reverse primers (ssM13FWRV), non digested. Lane2: ssM13FWRV, digested. Lane 3: ssM13FWRV and RCA product synthesised by commercial Φ29 DNAP WT, non-digested. Lane 4: ssM13FWRV and RCA product synthesised by com-mercial Φ29 DNAP WT, digested. Lane 5: ssM13FWRV and RCA product synthesised by expressed Φ29 DNAP WT, non-digested. Lane 6: ssM13FWRV and RCA product synthesised by expressed Φ29 DNAP WT, digested. Ladder: Thermo Scientific GeneRuler 1 kb Plus DNA Ladder. The picture of the ladder is taken from Thermo Scientific and modified. Numbers denote gene length in bp. 184 7.15 The fragment of the Φ29 DNAP library that is sequenced. From each side only maximally 450bp can be read. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185 7.16 The pooled library of the dTTP Titration Assay: R0, the four libraries of R1 and the four libraries of R2 without (lane B1) and with (lane C1) ligated adapters 7.18 Read counts after pre-processing of the Illumina NGS data of the Titration Assay "Regression". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . barcodes on the 5' end (left) and round specific barcodes on the 3' end (right). . 188 clearly separated from the correct matches A). Cq determination mode is set to 7.17 Multiplexing for the PacBio system has been carried out with library specific primer set and vice versa. The wrong primer template combination B) can be to sequencing by size selection with SPRI-beads (lane D1). . . . . . . . . . . . . 186 template mismatch tests have been conducted: WT template with variant R223T 48.502 kb in length as carrier DNA. The sample was purified from ⁄-DNA prior binding sites are designed to have minimal sequence similarity, only two primer-for NGS sequencing. The library was supplied with H2O containing ⁄-DNA of 1.1 The nine theoretically conceivable residue-by-residue transfers of sequential infor-mation as described by the central dogma of Crick [Crick, 1970]. ......... 1.2 3.1 ⁄ values for di erent droplet sizes and DNA input concentrations. . . . . . . . . 3.2 Comparison of short (PacBio system) and long (Illumina system) read NGS. Read length and sequencing depth are ranges for comparison. The exact number de-pends on template length and the specific platform of each system. . . . . . . . . CGT→ACG. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 ML2 Mutated Linker AA Positions 216 to 230 ND Non-Digested NEB New England Biolabs Inc. NGS Next-Generation Sequencing NT Nucleotide NTC Non-Template Control ORF Open Reading Frame outPCR Out Polymerase Chain Reaction 7.8 . . 179 7.21 The outPCR program. Steps 2)-4) are cycled 25 times. . . . . . . . . . . . . . . . 180 PacBio Pacific Biosciences of California, Inc. 4.1 Enrichment values across dTTP titrations and rounds are displayed for a selection of AA substitutions at position 223. . . . . . . . . . . . . . . . . . . . . . . . . . 7.22 The mutPCR Reaction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180 PFPE Perfluorinated Polyethers 7.23 The mutPCR program. Steps 2)-4) are cycled 25 times to give up to 5 mutations. 180 4.2 Characterisation of the template dilution / primer assay of figure 4.56.C qd e t e r -4.3 Characterisation of the template dilution / primer assay of figure 4.56. As primer 7.25 The read1, read2, P7, P5 and P7 index primers. . . . . . . . . . . . . . . . . . . . 187 mination mode is set to "Regression". . . . . . . . . . . . . . . . . . . . . . . . . . 7.24 The Gibson Assembly reaction composition. . . . . . . . . . . . . . . . . . . . . . 182 PW-Enrichment Position-Wise-Enrichment
	xxiv xxvi xxviii xxx	xxvii

enrichment in challenging condition of a 10-fold dTTP titration (y-axis) is plotted against typical dTTP conditions (x-axis). Blue ellipses 1 and 2 highlight outliers that appear to thrive better in the challenging condition. NTs in ellipse 1 form a codon at AA position 230; NTs in ellipse 2 form a codon at AA position 223. A regression line is added (grey). . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.50 A) SpS-Enrichment of single point mutations at amino acid (AA) level, positions

2 -25, 196 -200, 216 -230, round

two. For each mutation the SpS-enrichment in an environment with dTTP in abundance (700 uM dTTP, x-axis) is plotted against the SpS-enrichment in a dTTP-deprived environment (7 uM and 70 uM dTTP, y-axis). B) A heat map representation of SpS-enrichment at position 223 (the outlier in panel A). SpS-enrichment is shown for di erent AA substitutions for each environment tested throughout rounds one and two. Substitution R → T is enriched in dTTP deprived conditions but not in conditions with an abundant dTTP concentration, suggesting that a variant with this mutation prospers in low dTTP conditions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.51 AA-substitutions in the exonuclease domain that are co-enriched with mutation R223T in a) 10-fold titration (70 uM dTTP, blue) and b) 100-fold (7 uM dTTP, green) titration of dTTP. Mutation E14L comes up highest in 10-fold dTTP titration and mutation V19L comes up highest in 100-fold dTTP titration. . . . . 4Fold Increase of incubated with respect to frozen sample is shown. Four separate dTTP concentrations are tested: Standard condition, 10x dilution, 100x dilution, no dTTP. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B) Neither WT nor variant R223T are picked up by primer sets for negative (NCTRL) or positive control (PCTRL) template. All dilutions for the four conditions are in the range of the non template CTRL (NTC, not shown). C) Both, the WT and variant R223T gene are being picked up by the non corresponding primer set, respectively. The di erence between A) and C) is in the range of ∆Cq ≈ 7. Cq determination mode is set to "Regression". . . . . . 125 4.57 a) The 5' end of Φ29 DNAP genes in the V2 backbone is shown: Φ29 DNAP WT gene, the two single point variants and the double point variant. The qPCR primer binding sites are annotated. qPCR primers target unique sequences before the T7 promoter sequence aiming for discrimination of WT, single-and double point clone sequences in qPCR. b) Template dilution / primer range assay on the second set of syntesised variants and WT. Cq values and linear fits as determined by qPCR over a range of six 10-fold template dilutions starting with 62pM . A) The linear fits of right template-primer set combination intersect at 0.62pM . C) WT and variant R223T gene are being picked up by the non corresponding primer set, respectively. But the di erence between correct and wrong templateprimer set matches are in the range of ∆Cq ≈ 12 for the 62pM template starting quantity. Linear fit of R223T V2 template/WT V2 primer set crosses the correct template/primer curves in the range of 6.2 * 10 03 pM template starting quantity. Cq determination mode is set to "Regression". . . . . . . . . . . . . . . . . . . . . 126 4.58 Bulk expression replication reaction with standard equimolar dNTP concentration set to 700uM . V1 and V2 genes are compared and di erential increase of ⁄ DNA [ID:FND] is shown in terms of a) fold increase and b) absolute DNA concentration (SuM 12.2). For both, WT and variant R223T, more DNA could be synthesised with V1 genes, compared to V2 genes. . . . . . . . . . . . . . . . . . . . . . . . . 128 dTTP concentrations, this advantage drops to FI ≈ 1.2. . . . . . . . . . . . . . . 130 xxv List of figures 5.1

PW-Frequency Position-Wise-Frequency Q1DD Φ29 DNAP Gene with a Mutation Coding for an Inactive Phenotype Q1WT Φ29 DNAP WT Gene qPCR Quantitative Polymerase Chain Reaction

	Chapter 1
	Introduction
	Objectives
		This chapter will first introduce the Φ29 DNA polymerase -a fascinating enzyme
		that forms the basis for the model system presented in the course of this work.
		Subsequently, techniques are presented that allow for the artificial engineering of en-
		zymes in the wet laboratory. As emulsions are a tool to massively scale up mentioned
		enzyme engineering techniques, they will be addressed thereafter.
	Contents
	1	The central dogma of molecular biology ...............................
	2	Polymerases: The central component of the DNA replication machinery .............
	3T h e Φ29 DNA polymerase ......................................
		3.1	DNA replication by Φ29 DNAP can be protein or DNA primed .............
		3.2	Φ29 DNAP topology contributes to its high processivity and intrinsic helicase activity
		3.3	Φ29 DNAP translocation ...................................
		3.4	Φ29 DNAPs exonuclease activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
		3.5	Balancing Φ29 DNAPs polymerisation and exonuclease domain . . . . . . . . . . . . .
		3.6	The MagNIFi assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
		3.7	Summary Φ29 DNA polymerase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
	4	Directed evolution ...........................................
	4.1 QuIK Quasi-Instantaneous Emulsification for directed Evolution and Kinetics Studies Droplet-based microfluidics as a tool for directed evolution . . . . . . . . . . . . . . . . 5 Emulsification .............................................
	RCA Rolling Circle Amplification 6 Summary ................................................
	recPCR Recovery Polymerase Chain Reaction
	ReSe Error Errors Occurring from Reformatting, NGS Library Preparation and Sequencing
	RNA Ribonucleic Acid
	RSE Residual Standard Error
	sd Standard Deviation
	SLS Static Light Scattering
			xxxi

Table 3

 3

			Length Sequencing Depth Preferred Applications
	PacBio	10 4	10 4	Random Mutagenesis on an entire gene
	Illumina 10 2	10 6	Localised Mutagenesis

.2: Comparison of short (PacBio system) and long (Illumina system) read NGS. Read length and sequencing depth are ranges for comparison. The exact number depends on template length and the specific platform of each system.

Table 4 .

 4 .57, table 4.3). 3: Characterisation of the template dilution / primer assay of figure 4.56. As primer binding sites are designed to have minimal sequence similarity, only two primer-template mismatch tests have been conducted: WT template with variant R223T primer set and vice versa.

			12. Validation of identified variants E14L and R223T
	Template Primer	Efficiency % Slope Y-Intercept R 2
	R223T V1 PCTRL V1	702.28	-1.106 33.140	0.946
	R223T V1 NCTRL V1 -99.73	0.390	33.374	0.762
	R223T V1 R223T V1	90.94	-3.560 7.633	1.000
	R223T V1 WT V1	148.46	-2.530 16.860	0.919
	WT V1	PCTRL V1	7925.40	-0.525 34.735	0.632
	WT V1	NCTRL V1 -100.00	0.127	32.568	0.422
	WT V1	R223T V1	95.21	-3.442 14.974	0.996
	WT V1	WT V1	94.91	-3.450 7.212	0.997
	Table 4.2: Characterisation of the template dilution / primer assay of figure 4.56.C qd e t e r m i -
	nation mode is set to "Regression".
	Template Primer	Efficiency % Slope Y-Intercept R 2
	R223T/ E14L V2	R223T/ E14L V2	97.60	-3.381 8.840	0.981
	E14L V2	E14L V2	84.38	-3.763 7.780	0.993
	WT V2	R223T V2 146.21	-2.556 11.055	0.913
	WT V2	R223T V2 166.13	-2.352 23.924	0.839
	R223T V2 R223T V2 78.82	-3.962 7.523	0.992
	R223T V2 WT V2	20877.58	-0.431 25.288	0.198

Table 5 .

 5 project. The low cycle number of library prep PCR suggest that PCR bias is rather low in this step. It would rather be advisable to develop strategies for PCR bias quantification in rec-and outPCR, either by introduction of UMIs or by mapping from library prep PCR bias. Error introduced by oligo synthesis could be assessed by sequencing of the ordered gene fragment prior to assay input. In chapter 4.11 six NT positions were identified that thrive better in challenging conditions (figure 4.49). The fact that these six positions form two non-contiguous codons is a first indication that the outliers are not an accumulation of noise: I would expect noise not to produce such systematic outliers, but to elevate individually scattered NTs rather than triplets that translate into amino acids. Both predominant AA substitutions at the corresponding positions show a continuous increase of SpS-enrichment throughout rounds in the challenging conditions (table5.1). Each round and each condition is an entirely independent experiment from encapsulation to NGS library preparation. This result supports the assumption that the outliers are genuine behavior in the assay. 1: SpS-Enrichment across rounds and titrations of the two dominant AA substitutions belonging to the codon forming outliers in figure4.49. Molarities refer to the concentration of dTTP.

	R1	R1	R1	R1	R2	R2	R2	R2
	0µM	7µM	70µM	700µM	0µM	7µM	70µM	700µM
	R223T 0.99	0.99	4.03	0.74	1.05	4.94	10.00	1.03
	F230Y 0.93	1.03	3.71	0.89	1.07	2.37	3.10	1.28

1

  QuIK emulsification ..........................................147 1.1 Membrane fouling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147 1.2 Automatisation of QuIK emulsification . . . . . . . . . . . . . . . . . . . . . . . . . . . 147 1.3 QuIK can be applied to directed evolution of other enzymes . . . . . . . . . . . . . . . 147 2 The Time Assay ............................................147 3 Orthogonal assays ...........................................148 3.1 QuIK-based iviCSR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149 3.2 Single nucleotide polymorphism genotyping by fragment analysis . . . . . . . . . . . . 149 3.3 Bulk primer extension assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150 3.4 Primer extension on single molecule level . . . . . . . . . . . . . . . . . . . . . . . . . . 150 3.5 Single molecule assays with magnetic tweezers . . . . . . . . . . . . . . . . . . . . . . . 152 3.6 MagNIFi assay to assess Φ29 DNAP variant fidelity . . . . . . . . . . . . . . . . . . . 153 4 dATP-,dCTP-and dGTP-titration .................................153 5 Bottlenecking .............................................154 6 Advancing the iviCSR reaction to the single molecule level ....................154 7 Summary ................................................155

  Both directed evolution assays presented in this work, the Activity Assay and the Titration Assay revealed new variants with possibly beneficial activities with respect to the WT. In case of the Activity Assay 30 variants have been identified after four rounds of selection and validated in a bulk Activity Assay of which 19 showed an increased activity over WT. Especially the AA substitutions LGF → VQT at positions 227-229, PDI → VHR at positions 413-415 and LTL → VKS at positions 430-432 would be promising candidates to study on the single molecule level.

  .6: PW-enrichment Matrix across dTTP Concentrations, Exonuclease Domain, NT positions 4 to 75 (AA 2 to 25, MEx).

		700 µM	70 µM7 µM0 µM
	700 µM 1	0.8792030 0.86377219 -0.05862058
	70 µM	0.87920298 1	0.89305277 -0.24058082
	7 µM	0.86377219 0.8930528 1	-0.07491698
	0 µM	-0.05862058 -0.2405808 -0.07491698 1
	Table 7.6: PW-enrichment correlation matrix across dTTP concentrations, exonuclease domain,
	NT positions 4 to 75 (AA 2 to 25, MEx).

Table 7 .

 7 9: The in vitro methylation reaction.The input for DB preparation was in the range of 1-2 µg methylated template. DNA damage repair reaction was carried out by adding the components of table 7.10 in a test tube and incubation for 20 minutes at 37°C. Afterwards, the reaction was kept at 4°C for 1 minute. PreCR ® Repair Mix #M0309L, ThermoPol ® Reaction Bu er #B9004SVIAL and --Nicotinamide adenine dinucleotide (NAD+) #B9007SVIAL were obtained from NEB.

	8. Dumbbell preparation

Table 7 .

 7 10: The DNA damage repair reaction.The end repair reaction was carried out by adding the components of table 7.11 to test tube and incubation for 30 minutes at 20°C. Afterwards, the reaction was kept at 4°C until a 0.6X magnetic bead purification was carried out (see section SuM 14.1). NEBNext ® End Repair Enzyme Mix and bu er #E6050 were obtained from NEB.

	10X End Repair Reaction Bu er 6 µL
	End Repair Enzyme Mix	5 µL
	Total volume	61 µL

Table 7 .

 7 11: The end repair reaction.

Table 7 .

 7 14: Composition of the iviCSR reaction for QuIK Assays.

	%

Table 7 .

 7 19: The recPCR program. Steps 2)-4) are cycled 30 times.

	Polymerase chain reactions

Table 7 .

 7 21 shows the PCR program. NTC was run in parallel. Figure3.2 E) shows an exemplary 1% agarose E-gel picture of the dsDNA backbone, NTC controls (NTC CTRL), recPCR product and outPCR product.

	Reagent	%
	Q5 Polymerase Bu er 5X	20
	Primer Fw 10 uM	2
	Primer Rv 10 uM	2
	10 mM each dNTP mix	2
	Q5 Polymerase	1
	Template (Gibson Product) 2
	H2O	71

Table 7 .

 7 20: The recPCR Reaction. 17. M13 primer extension assay reason, synthesised DNA is expected to be produced as a multibranched RCA product which allows NdeI digestion. Digestion results in four bands of sizes 3043 bp, 2721 bp, 1080 bp and 405 bp.

Table 7 .

 7 25: The read1, read2, P7, P5 and P7 index primers.

	18. NGS library preparation

Next generation sequencing

In vitro isothermal compartmentalised self replication

Acknowledgements

Chapter 3

Materials and Methods

The focuses of this chapter lies on the most important tools, which -partly already available in the laboratory, partly (further) developed -form the basis for the results presented in chapter 4. Detailed wet laboratory protocols can be found in the Supplementary Material and are referenced where necessary.

Modelling the reaction dynamics of the Titration Assay

In this section I will present two approaches to describe the function that maps sequences of a gene library to the measurements from NGS sequencing. The first presented model is based on Michaelis-Menten theory that describes the rate of enzymatic reactions.

The second approach is based on considerations on transmission of sequences throughout rounds of selection.

13. Summary

Summary

The developed QuIK Assay opens new possibilities for polymerase evolution, as it allows for quasi-instantaneous emulsification producing droplets with size in the range of ∼ 1 fL. This work involved scaling the droplet diameter, droplet dispersity and gene concentration to ensure encapsulation of less than one gene per droplet. With the Time Assay I was able to draw a more detailed picture of the kinetics of iviCSR.

A random mutagenesis study and the Activity Assay on H0-25 DNAP variants lead to the finding of clones with improved activity. Furthermore, I could show that there is a significant correlation between enrichment as determined by NGS data from the Activity Assay and as determined by qPCR.

Mutations in conserved regions of the Φ29 DNAP's exonuclease domain raised questions about the enzyme's balance of synthesis and error correction. The findings motivated the development of the Titration Assay which revealed evidence that some clones with subtitutions in the linker region thrive better in challenging dTTP conditions than under standard conditions. Orthogonal assays were developed to further investigate found clones and a model was derived to describe the NGS results of the Titration Assay.

In the next section I will discuss the mentioned findings and relate them to the literature presented in chapter 1.

Chapter 5

Discussion

In the previous chapter I could present novel QuIK-and iviCSR-based assays and insights into their kinetics. With those assays I found DNAP clones with higher processivity than Φ29 DNAP WT and evidence that amino acid position 223 contributes to the coordination of the enzyme's activity-fidelity trade-o . In this section I will discuss the results -connecting them with the existing literature presented in chapter 1 -and address questions arising from my findings.

Summary

The behaviour of the variant library, as seen in NGS read-out, presented the opportunity to model the system's behaviour. Improvements of this model will aim to gain a better understanding of both, the behaviour of the average mutant and the outliers with beneficial variations. Further investigation of found outliers is still ongoing and ideas for orthogonal assays will be presented in the next chapter.

Supplementary Material 1 In vitro isothermal compartmentalised self replication

Dumbbell shaped dsDNA is added to a reaction composed of the reagents in

The Titration Assay

The Titration Assay has so far been carried out with the titration of dTTP. Similarly to the Activity Assay, the iviCSR protocol, presented in chapter 3.2 is used and modified in the following way: The aqueous phase thus contains the components of table 7.14 with varying dTTP concentrations (700 µM, 70 µM, 7 µM, and 0 µM total concentration). dNTPs were taken from individual vials of the Deoxynucleotide (dNTP) Solution Set NEB #N0446 and the three non titrated bases were kept constant at 700 µM.

Replication is initiated with a primer targeting the single stranded loops of the DB template.

Active DNAP variants can make successive copies of the template in an RCA but are eventually forced to stall or make mis-matching basepairs.

Supplementary Material

PCR product was carried out with T4 DNA Ligase (#M0202T, NEB). The methylated template plasmid was digested with DpnI. The circularised product was transformed in E. coli (section SuM 16, a sample was sent to sanger sequencing for quality control and the remaining sample was used as template for PCR (section SuM 12.4) to obtain the gene in the right configuration for DB preparation (see section SuM 8). Four rounds of selection were carried out and the selection and NGS library preparation on the H0-25 library has been carried out by Dr. Adeline Pichard-Kostuch. The sequencing coverage in the case of the H0-25 variant library is 80 bp and sequencing depth is 12.5 Mio. reads (0.8 Mio. per sample).

Library construction for Φ29 DNAP Titration Assay

A Φ29 DNAP WT gene (custom-oligo, IDT) was cloned into the PURExpress Control DHFR plasmid (NEB# N0424AVIAL) by Gibson Assembly (see section SuM 15 with 100ng backbone and a 2-fold molar excess of insert vs. backbone (from here on called "Φ29 DNAP plasmid").

The library was synthesised from four fragments: Two conserved fragments were obtained by PCR on the Φ29 DNAP plasmid. Two fragments were ordered as oligo-pools at IDT. The latter are pooled synthesised single stranded DNA fragments, each containing a mutation of one to three consecutive nucleotides (3-NT mutation) at a position along a defined region of its sequence. Each 3-NT mutation was introduced in NNK fashion.

In vitro methylation

The components listed in table 7.9 were mixed in a test tube. S-adenosylmethionine (SAM) #B9003S was obtained from NEB and diluted 40-fold in de-ionised H2O to obtain a concentration of 800 µM. dam Methyltransferase and bu er #M0222 were obtained from NEB.

The reaction was incubated for 2h at 37°C, followed by a 10 minutes heat inactivation at 65°C. 1 µL of the reaction was then DpnI digested (see section SuM 11) and loaded on an E-Gel TM EX Agarose Gel, 1%, G401001, Invitrogen TM , Thermo Fisher, and run for 10 minutes on an E-Gel TM Power Snap Electrophoresis Device, G8100, Invitrogen TM , Thermo Fisher, together with the 1 Kb Plus DNA Ladder (10787018, Invitrogen TM , Thermo Fisher). A gel image was taken to ensure that the entire template is methylated and can be digested entirely.

The in vitro methylation reaction is subsequently gel purified (see section SuM 14.2) and magnetic beads purified (see section SuM 14.1).

Supplementary Material

Blunt The synthesised DB templates are purified by two consecutive 0.6X magnetic beads purifications (see section SuM 14.1).

QuIK emulsification

Shirasu porous glass membrane

The "SPG Pumping Connector" (PC05U Hydrophobic, Shirasu Porous Glass Technology Co.,Ltd 16079-41 Higashikaminaka Sadowaracho, Miyazaki-city, Miyazaki-pref. 880-0303. JAPAN, hereafter "SPGE") was used for all QuIK emulsifications. The membrane of the extruder is made of Shirasu (volcanic ash deposit), CaCO3, and boric acid. The silicone resin coated, hydrophobic membrane has a surface area of 0.4cm 2 . If not otherwise stated, a membrane with 5 µm average pore size is used. Further membrane characteristics are mentioned in chapter 1.5.0.1.

Optimised iviCSR reaction

The iviCSR reaction presented in SuM 1 is optimised for QuIK emulsification: ⁄-DNA (Lambda DNA, #SD0011, Thermo Fisher Scientific) and BSA (Bovine Serum Albumin replacement was used: Recombinant Albumin, Molecular Biology Grade, #B9200S, NEB) is added to passivate the membrane. ⁄-DNA serves as a carrier DNA to prevent adhesion of template DNA to the mebrane. BSA prevents adhesion of protein to the membrane. The optimised iviCSR reaction

Quantitative polymerase chain reaction analysis

The qPCR read out is given in terms of the quantification cycle (Cq value). It is the fractional number of cycles needed until the detected fluorescence reaches a quantification threshold.

In a reaction of e ciency E = 100% each thermal cycle of qPCR will double the amount of DNA which leads to a value of ∆Cq =1. Cq values were determined as an average of four consecutive 10-fold dilutions loaded per sample.

∆Cq between two 10-fold dilutions is ideally 3.3 PCR cycles apart: ∆Cq = log 2 (10) = 3.3. In all qPCR experiments only dilutions in a range of ∆Cq =3.3 ± 0.6 and a Cq value below the one of the respective NTC were considered for average enrichment calculation.

Typically, the outputs of assays in this work are an incubated sample which is compared (figure 7.13 b)) to a frozen, non-incubated reference sample (figure 7.13 a)). The methylated template can be selectively digested with the DpnI restriction enzyme (section SuM 11), leading to two additional samples ((figure 7.13 c) and d)).

Supplementary Material

high Cq values and digestion:

In this calculation, X can be WT, for example, and Y a variant. In the case of the model selection (chapter 3.2.1), equation 12.2 becomes:

Fold increase (FI) between two data points is calculated by FI =2 ∆Cq . In the case of ∆∆Cq, fold increase is obtained by

If the ∆Cq is calculated between a standard with known DNA concentration and another sample, than the absolute DNA concentration in the sample can be obtained by multiplication of the standard's DNA concentration with the fold increase. Cq determination mode in qPCR analysis is set to "regression".

Recovery polymerase chain reaction

After breaking, pooling and digestion the output library of the coupled assay is loaded on an E-Gel TM EX Agarose Gel, 1%, G401001, Invitrogen TM , Thermo Fisher, and run for 10 minutes on an E-Gel TM Power Snap Electrophoresis Device, G8100, Invitrogen TM , Thermo Fisher, together with the 1 Kb Plus DNA Ladder, 10787018, Invitrogen TM ,ThermoFisher. R CAproduct is not expected to run in the gel like linear dsDNA. Gel pictures of RCA product purification (figure 3.2 A) always showed smears which is in line with this assumption. Large RCA product is expected to be retained in the well walls. For these reasons, the product is purified from a gel cut-out that comprises the well and reaches down until ∼ 2.5 kb on the ladder. Purification is carried out as described in section SuM 14.2 (The initial PCR clean up was omitted and the RCA product was loaded directly onto the gel). Recovery polymerase chain reaction is carried out with primers flanking a ∼ 0.9kb region on the DNAP gene, that contains the mutated regions of the library (figure 3.2 B)). The reagents of the PCR are shown in table 7.18 and the PCR program is shown in table 7.19.Q 5 ® High-Fidelity DNA Polymerase, #M0491, Q5 ® Reaction Bu er, #B9027, and Deoxynucleotide (dNTP) Solution Mix #N0447 was obtained from NEB. Primers were custom DNA oligos from IDT. NTC was run in parallel. recPCR product was PCR-and Gel-purified as described in SuM 14.2.

Supplementary Material

Step Temperature Time 1) 98°C 30 sec 2) 98°C 10 sec 3) 60°C 20 sec 4) 72°C 1.15 min 5) 72°C 2m i n Table 7.21: The outPCR program. Steps 2)-4) are cycled 25 times.

Mutagenic polymerase chain reaction

Agarose gel preparation

1X TAE bu er (TAE bu er (50X) for molecular biology, #A4686,1000, ITW PanReac Ap-pliChem, 155 Harlem Ave, Glenview, IL 60025, USA) was prepared by dilution with de-ionised water. 1 tablet of agarose (TopVision Agarose Tablets, #R2801, Thermo Fisher Scientific) was dissolved per 50 mL of 1X TAE bu er and heated in a microwave for 5 minutes until boiling.

The mix was poured in a mould and supplied with 1 µL intercalating dye (GelGreen ® Nucleic Acid Gel Stain, 10.000X in water, #41005, Biotium, Biotium, Inc. 46117 Landing Parkway Fremont, CA 94538, USA) per 10 mL of agarose mix. A rack was placed in the mould and the assembly was stored until solidification. The gel was put in an agarose gel electrophoresis system containing 1X TAE bu er. Sample was supplied with loading dye (TriTrack DNA Loading Dye 14. Purifications (6X), #R1161, Thermo Fisher Scientific) and loaded on the gel together with the GeneRuler 1kb plus ladder. The sample was run for 40 minutes at 100V.

Purifications

Magnetic beads purification

If not otherwise stated, sample was mixed in a 1:1 volume ratio with SPRI beads (SPRIselect 5mL, #B23317, Beckman Coulter, 5350 Lakeview Parkway S Drive Indianapolis, IN 46268 United States) and incubated for 10 minutes at 1200 rpm in an Eppendorf ® Thermomixer C at room temperature. Samples were placed on a magnetic rack and left for 3 minutes until beads separated. Supernatant was discarded and beads were washed two times with 1mL of 70% ethanol (#1117270500 Sigma-Aldrich) in de-ionised water. Samples were incubated for one to two minutes to let excess ethanol and water evaporate. 10 µL to 30 µL de-ionised water was added to the beads and the mix was incubated for 10 minutes at 1200 rpm in an Eppendorf ® Thermomixer C at room temperature. Samples were placed again on magnetic rack for 5 minutes until beads pelleted. The supernatant was retrieved and DNA concentration was measured on a Nanodrop spectrophotometer system.

Agarose gel preparation

Gel purifications were carried out by loading of sample on a gel prepared according to section SuM 13. After cut out of the corresponding band, the sample was purified according to section SuM 14.2.1.

PCR-product and agarose gel purification

Both, PCR and agarose gel clean-ups were carried out with the Macherey-Nagel NucleoSpin Gel and PCR Clean-up, Mini kit for gel extraction and PCR clean up kit, #740609.50.

Gibson assembly

In a 20 µL reaction the reagents of table 7.24 are added. The dsDNA backbone was obtained by PCR on the plasmid presented in section SuM 5, figure 7.12. The insert was obtained by PCR on the respective template. Gibson Assembly ® Master Mix, #E2611, was from NEB. The reaction is incubated at 50°C for 20 minutes. For library synthesis 467pM of 3.3kb backbone and 1.31 nM of each insert was used.

Supplementary Material

Reagent µL dsDNA Backbone, 100ng total 1 ds DNA Insert (Purified recPCR Product), 50ng total 1 Gibson Assembly Master Mix, 2X 10 H2O 8

Table 7.24: The Gibson Assembly reaction composition.

Transformation

Per sample 25 µL 5-alpha competent cells (NEB ® 5-alpha Competent E. coli (High E ciency), #C2987H) were thawed on ice for 10 minutes. 2 µL of Gibson Assembly product (∼ 2ng of plasmid) were added per sample and the mix was carefully homogenised by pipetting. The cells were incubated for 40 minutes on ice and heat shocked at 42 °C for exactly 30 seconds. Cells were subsequently left to rest on ice for 5 minutes and supplied with 200 µL lysogeny broth (LB Broth, #10855001, Thermo Fisher Scientific) at room temperature. The mix is placed in an Eppendorf ® Thermomixer C and incubated for 30 minutes at 500 rpm and 37 °C. 100 µL of the mix is spread on pre-warmed selection plates containing ampicillin supplied agar (1000x diluted).

For each sample and the control (containing non circularised fragments) three 10-fold dilutions of the cell-LB mix were plated. The petri-dishes were sealed and incubated upside-down over night at 37°C. Colonies were picked. LB Broth was mixed with 1000x diluted ampicillin solution. Either 100 µL of cell-LB mix or a cell colony was mixed with 7 mL of ampicillin supplied LB and incubated over night at 100 rpm and 37°C in a not tightly sealed falcon tube. OD value was obtained (>1) and the mix was pelleted by centrifuging at 11000 rpm.

M13 primer extension assay

Application of the M13 primer extension assay aims to compare the processivity of Φ29 DNAP variants with Φ29 DNAP WT. Forward and reverse primers are pre-annealed to a single stranded M13 plasmid and the DNAPs are allowed to complex with the ternary complex of FW primer -RV primer -M13 template. During complexing the DNA synthesis is suppressed by the absence of one of the four nucleotides. Synthesis is initialised by addition of the missing nucleotide and incubation (protocol in section SuM 17.1). Subsequently the reaction is loaded on an agarose gel to quantify the amount of synthesised DNA. Φ29 DNAP WT is reported to replicate the circular genome of the M13 phage more than tenfold leading to single strands of more than 70 kb in length [Blanco, 1989]. RCA products of this length will not be able to migrate in a 2% agarose gel which necessitates digestion of RCA product into smaller fragments. The applied primers are forward and reverse complementary primers to the single stranded plasmid. For this Supplementary Material The sequencing primers supplied with the sequencing preparation kits can be found in table 7.25. The read structure is as follows: P7-indexP7-SBS12-geneinsert-SBS3-P5. 10 µL of 20 nM DNA was provided for sequencing (as determined by qPCR and Qubit) which corresponds to ∼ 10 1 1 molecules. The sample was spiked with 30% PhiX prior to loading on the system.

Supplementary Material

Bottlenecking is the selection of a random subset of molecules from a larger molecule pool, thus the reduction of its genetic diversity. After selection the output library is amplified in two subsequent PCR steps (figure 7.20). Both PCR steps generate a multitude of copies of each gene. However, amplification is subject to bias and not every template of the output library is amplified to the same extent. In the next step 11 nt long UMI sequences are ligated. The UMI diversity of ∼ 4Mio possible sequences exceeds the library diversity by a factor 4. For this reason, a subset of the ligated library-UMI pool is picked to represent the original population as closely as possible. This population ideally consists only of unique template-UMI ligations. In practice it can be expected that some templates share a common UMI. It should be noted that bias of rec-and outPCR is expected to be represented in the bottlenecked population as well. A third PCR is needed in order to add the P5 and P7 adapters to the genes. Thanks to the ligated UMI sequences amplification bias of the NGS library preparation PCR can be eliminated: Virtually all unique template-UMI ligates are amplified during the PCR step. Afterwards, the PCR products are sequenced. As the NGS read-out will not only be limited to the actual gene sequence, but includes also the 11 nt long UMI sequence, it is possible to trace the amplification bias of the last PCR and combine multiplied sequences into one consensus sequence.

In concrete terms the library was reduced to a targeted diversity of 200.000 unique genes. Therefore, a 11 NT long UMI (Unique Dual Index UMI Adaptors DNA Set 1, NEB #E7395) with a diversity of 4,194,304 was ligated to the pool of to-be-sequenced DNA fragments. A 25-cycle library preparation PCR was carried out to amplify the library to a size of ∼ 6 * 10 1 2 molecules. 10 sublibraries were pooled and the NGS facility was provided with a pooled library sample of 5uL at 20 nM (2nM ˆ= 6 * 10 9 molecules per sublibrary).