Pierre Crozet

Francisco Navarro

Felix Willmund

Payam Mehrshahi

Kamil Bakowski

Kyle Lauersen

Maria-Esther Pérez-Pérez

Pascaline Auroy

Aleix Gorchs

Susana Sauret-Gueto

Crozet Pierre

Willmund Felix

Mehrshahi Payam

Bakowski Kamil

Lauersen Kyle

Pérez -Pérez

Auroy Pascaline

Rovira Gorchs

Aleix

Sauret-Gueto Susana

Niemeyer Justus

Spaniol Benjamin

Theis Jasmine

Trösch Raphael

Westrich Lisa-Desiree

Vavitsas Konstantinos

Baier Thomas

Hübner Wolfgang

Carpentier Felix

Cassarini Mathieu

Danon Antoine

Henri Julien

Marchand Christophe

Mia Marcello

Sarkissian Kevin

Baulcombe David

Peltier Gilles

José-Luis Crespo

Kruse Olaf

Jensen Poul Erik

Schroda Michael
email: schroda@bio.uni-kl.de

Alison G Smith

Lemaire Stéphane
email: stephane.lemaire@ibpc.fr

S D Lemaire

Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii

Keywords: Algal biotechnology, Chlamydomonas reinhardtii, modular cloning, synthetic biology

établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

There is an urgent need to decarbonize the world economy due to depletion of fossil fuel reserves coupled with accumulation of greenhouse gases produced by their combustion. One alternative to the use of fossil fuels is to use photosynthetic microorganisms, such as microalgae, as green cell factories to produce fuels and chemicals from atmospheric CO 2 in a sustainable process driven by sunlight [START_REF] Zargar | Leveraging microbial biosynthetic pathways for the generation of 'drop-in' biofuels[END_REF][START_REF] Georgianna | Exploiting diversity and synthetic biology for the production of algal biofuels[END_REF] . The fixed carbon can be redirected towards compounds that can be used in the fuel, food, cosmetic and pharmaceutical industries, such as proteins, alcohols, alkanes, lipids, sugars, pigments or terpenes [START_REF] Wijffels | Potential of industrial biotechnology with cyanobacteria and eukaryotic microalgae[END_REF][START_REF] Gimpel | Metabolic Engineering of Eukaryotic Microalgae: Potential and Challenges Come with Great Diversity[END_REF][5] . By contrast with land plant-based photoproduction, microalgae do not compete with agriculture and can be grown at high yields even at large scale [START_REF] Gimpel | Metabolic Engineering of Eukaryotic Microalgae: Potential and Challenges Come with Great Diversity[END_REF][START_REF] Gangl | Biotechnological exploitation of microalgae[END_REF] , including on waste streams, thus minimizing inputs [START_REF] Wijffels | Potential of industrial biotechnology with cyanobacteria and eukaryotic microalgae[END_REF] . The green microalga Chlamydomonas reinhardtii (referred to hereafter as "Chlamydomonas") has been extensively engineered for basic research and industrial biotechnology [START_REF] Gimpel | Metabolic Engineering of Eukaryotic Microalgae: Potential and Challenges Come with Great Diversity[END_REF][START_REF] Gangl | Biotechnological exploitation of microalgae[END_REF][START_REF] Lauersen | Efficient phototrophic production of a high-value sesquiterpenoid from the eukaryotic microalga Chlamydomonas reinhardtii[END_REF][START_REF] Wichmann | Tailored carbon partitioning for phototrophic production of (E)-alpha-bisabolene from the green microalga Chlamydomonas reinhardtii[END_REF] . Its nuclear and organellar genomes are sequenced and annotated, molecular biology techniques and culture conditions are highly developed, and its physiology and metabolism are well understood [9][START_REF] Gallaher | Chlamydomonas Genome Resource for Laboratory Strains Reveals a Mosaic of Sequence Variation, Identifies True Strain Histories, and Enables Strain-Specific Studies[END_REF][START_REF] Scaife | Establishing Chlamydomonas reinhardtii as an industrial biotechnology host[END_REF][START_REF] Li | An Indexed, Mapped Mutant Library Enables Reverse Genetics Studies of Biological Processes in Chlamydomonas reinhardtii[END_REF][START_REF] Jinkerson | Molecular techniques to interrogate and edit the Chlamydomonas nuclear genome[END_REF] . Moreover, the metabolic plasticity and cellular compartments of Chlamydomonas offer great potential for advanced metabolic engineering strategies [START_REF] Agapakis | Natural strategies for the spatial optimization of metabolism in synthetic biology[END_REF][START_REF] Chen | Designing biological compartmentalization[END_REF] .

Chlamydomonas has already been engineered for production of the biodiesel precursor bisabolene [START_REF] Wichmann | Tailored carbon partitioning for phototrophic production of (E)-alpha-bisabolene from the green microalga Chlamydomonas reinhardtii[END_REF] , the terpene patchoulol [START_REF] Lauersen | Efficient phototrophic production of a high-value sesquiterpenoid from the eukaryotic microalga Chlamydomonas reinhardtii[END_REF] , and recombinant proteins as well as enzymes such as an HIV antigen [START_REF] Barahimipour | Efficient expression of nuclear transgenes in the green alga Chlamydomonas: synthesis of an HIV antigen and development of a new selectable marker[END_REF] and xylanase [START_REF] Rasala | Robust expression and secretion of Xylanase1 in Chlamydomonas reinhardtii by fusion to a selection gene and processing with the FMDV 2A peptide[END_REF] . Despite these proofs of concept however, engineering of Chlamydomonas is still slow due to a lack of standardized resources and tools [START_REF] Scaife | Establishing Chlamydomonas reinhardtii as an industrial biotechnology host[END_REF] . Development of the field of algal synthetic biology offers the means to enable design and construction of microalgal cells with defined and predictable properties [START_REF] Scaife | Towards developing algal synthetic biology[END_REF] . Besides biotechnological applications, the transition from empirical to synthetic approaches also provides the opportunity to answer fundamental biological questions using new concepts and approaches based on understanding by construction rather than deconstruction.

Synthetic biology approaches, predicated on the Design-Build-Test-Learn cycle [START_REF] Endy | Building a new biology[END_REF] , make organisms easier to engineer through the use of standardized parts and their assembly to simplify the building of designed DNA molecules [START_REF] Endy | Building a new biology[END_REF] . Among available standards [START_REF] Casini | Bricks and blueprints: methods and standards for DNA assembly[END_REF] , the Golden Gate Modular Cloning (MoClo) technology, based on Type IIS restriction enzymes, offers extensive standardization and allows the assembly of complex multigenic DNA from basic gene parts (e.g. promoters, CDS, terminators) in just two steps [START_REF] Engler | A one pot, one step, precision cloning method with high throughput capability[END_REF][START_REF] Weber | A modular cloning system for standardized assembly of multigene constructs[END_REF] . The method accelerates and multiplies the possibilities to permute multiple genetic elements, and makes facile the building of multigene constructs for full metabolic pathways [START_REF] Smanski | Functional optimization of gene clusters by combinatorial design and assembly[END_REF] . MoClo is efficient and versatile, but relies on intensive upfront generation of a standardized library of basic building blocks, the gene parts, that have been domesticated to remove Type IIS sites, and codon optimized for the host as appropriate. MoClo toolkits have already been developed for a few model organisms [START_REF] Celinska | Golden Gate Assembly system dedicated to complex pathway manipulation in Yarrowia lipolytica[END_REF][START_REF] Engler | A golden gate modular cloning toolbox for plants[END_REF][START_REF] Iverson | CIDAR MoClo: Improved MoClo Assembly Standard and New E. coli Part Library Enable Rapid Combinatorial Design for Synthetic and Traditional Biology[END_REF][START_REF] Lee | A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly[END_REF][START_REF] Martella | EMMA: An Extensible Mammalian Modular Assembly Toolkit for the Rapid Design and Production of Diverse Expression Vectors[END_REF][29] although not yet for microalgae.

Here, we report the generation of a MoClo toolkit composed of more than 100 gene parts codon-optimized for the Chlamydomonas nuclear genome. These genetic parts were designed to provide maximum modularity to end-users, and to facilitate the development of engineered strains for fundamental and green biotechnological applications, through iterative design and testing. We provide functional validation and characterization of many gene parts in several Chlamydomonas strains. This kit is available to the community, to allow Chlamydomonas to become the next chassis for sustainable synthetic biology approaches.

RESULTS

Standard and content of the Chlamydomonas MoClo kit

Standardization is the key to efficient building. The Chlamydomonas MoClo kit adopts the syntax proposed by the plant synthetic biology community including the OpenPlant Consortium 30 (Fig. 1). This syntax is defined for level 0 plasmids containing standard gene parts (promoters, coding sequences, untranslated regions, etc.) and assigns strict fusion sites for 10 cloning positions. In a single step, standardized parts can be assembled into modules (Transcriptional Unit, TU, level 1) and modules into devices (multigenic construct, level M or 2) according to the original MoClo syntax 22 (Supplementary Fig. 1). Our Chlamydomonas MoClo toolkit is composed of a set of 119 parts representing 67 unique genetic elements available at different positions within the standard, thereby providing maximum modularity to designers (Fig. 1, Fig. 2). The kit recapitulates most of the standard genetic elements previously developed for Chlamydomonas which we "domesticated" by removing BpiI and BsaI restriction sites (the two enzymes used by the MoClo strategy [START_REF] Weber | A modular cloning system for standardized assembly of multigene constructs[END_REF] , Supplemental Figure 1) from their sequences by DNA synthesis or PCR-based mutagenesis. The available gene parts encompass 7 promoters coupled or not to their original 5'UTR, the corresponding 5'UTR and the CrTHI4 riboswitch, 8 immunological or purification tags in positions leading to N-or Cterminal translational fusions, 9 signal and targeting peptides, 12 reporters, 5 antibiotic resistance genes, the foot and mouth virus (FMDV) 2A peptide which allows expression of two or more proteins from a single transcriptional unit [START_REF] Rasala | Robust expression and secretion of Xylanase1 in Chlamydomonas reinhardtii by fusion to a selection gene and processing with the FMDV 2A peptide[END_REF][START_REF] Lopez-Paz | Identification of Chlamydomonas reinhardtii endogenous genic flanking sequences for improved transgene expression[END_REF][START_REF] Plucinak | Improved and versatile viral 2A platforms for dependable and inducible high-level expression of dicistronic nuclear genes in Chlamydomonas reinhardtii[END_REF] , 2 micro RNA (miRNA) backbones and associated controls, and six 3'UTR-terminators (Fig. 1b, Fig. 2 and Supplementary Table 1). All sequences and plasmids are available through the public Addgene repository (http://www.addgene.org/).

Constitutive promoters and reporter genes

Five antibiotic resistance genes are used as selectable markers for Chlamydomonas but also can function as reporter genes [START_REF] Lumbreras | Efficient foreign gene expression in Chlamydomonas reinhardtii mediated by an endogenous intron[END_REF][START_REF] Strenkert | Heat shock factor 1 counteracts epigenetic silencing of nuclear transgenes in Chlamydomonas reinhardtii[END_REF] . We assembled three modules that allow control of the expression of the aadA gene, conferring spectinomycin resistance, by three constitutive promoters: P PSAD and P βTUB2 with or without the first intron of βTUB2 (pCM1-1 to 3, Supplementary Table 3). The transformation efficiency of the three modules in UVM4 [START_REF] Neupert | Generation of Chlamydomonas strains that efficiently express nuclear transgenes[END_REF] cells was estimated by counting spectinomycin resistant colonies and showed resistance frequencies within the same range (Fig. 3a). The presence of the first TUB2 intron significantly increased the transformation efficiency as previously observed with the presence of RBCS2 introns in the ble marker [START_REF] Lumbreras | Efficient foreign gene expression in Chlamydomonas reinhardtii mediated by an endogenous intron[END_REF][START_REF] Schroda | Sequence elements within an HSP70 promoter counteract transcriptional transgene silencing in Chlamydomonas[END_REF][START_REF] Eichler-Stahlberg | Strategies to facilitate transgene expression in Chlamydomonas reinhardtii[END_REF] . Alternative reporters are bioluminescent proteins, which allow more sensitive and quantitative analysis of gene expression. The kit contains Gaussia princeps luciferase, the brightest luciferase established in Chlamydomonas [START_REF] Shao | A codon-optimized luciferase from Gaussia princeps facilitates the in vivo monitoring of gene expression in the model alga Chlamydomonas reinhardtii[END_REF] , as well as the redesigned Nanoluciferase (NanoLuc) which provides a stable and strong luminescence signal [START_REF] Hall | Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate[END_REF] . Chlamydomonas NanoLuc was specifically developed for our MoClo kit through recoding to match the codon bias of Chlamydomonas, and cloned at 6 different positions within the standard. This new part was first tested with the most widely used promoter/terminator combination (P AR promoter / T RBCS2 terminator) for strong constitutive expression in Chlamydomonas. The corresponding module (pCM1-04) was assembled with another module conferring paromomycin resistance (Supplementary Fig. 2) into a device (pCMM-1) that was introduced into the genome of the D66 strain (CC-4425, Fig. 3b). Among paromomycin resistant colonies, 34.8% ± 8.3 (N=48, mean ± SEM) were luminescent. The signal was variable between clones due to genomic position effects [START_REF] Lodha | A new assay for promoter analysis in Chlamydomonas reveals roles for heat shock elements and the TATA box in HSP70A promoter-mediated activation of transgene expression[END_REF][START_REF] Barahimipour | Dissecting the contributions of GC content and codon usage to gene expression in the model alga Chlamydomonas reinhardtii[END_REF] but was linear from 50 to 5 x 10 5 cells (Fig. 3b and Supplementary Fig. 2). By contrast, non-expressing transformants (resistant to paromomycin only) or the D66 recipient strain displayed only a faint signal, 3 orders of magnitude lower, and saturating swiftly (Fig. 3b,inset). The modularity of the MoClo strategy allows rapid assessment of combinations of multiple parts. For example, we assembled 4 modules where NanoLuc expression is controlled by all possible combinations of the two most common constitutive promoters (P AR and P PSAD ) and terminators (T RBCS2 and T PSAD ) (Fig. 3b, pCM1-4 to 7, Supplementary Table 3). Each module was assembled with the paromomycin resistance module (pCMM-1 to 4, Supplementary Table 4) and introduced into the Chlamydomonas genome. Bioluminescence levels were averaged over hundreds of transformants to account for the genome position effect [START_REF] Lodha | A new assay for promoter analysis in Chlamydomonas reveals roles for heat shock elements and the TATA box in HSP70A promoter-mediated activation of transgene expression[END_REF][START_REF] Barahimipour | Dissecting the contributions of GC content and codon usage to gene expression in the model alga Chlamydomonas reinhardtii[END_REF] . The strengths of the two promoters were found to be comparable, whilst T PSAD appeared to confer robust expression from both promoters, 10-fold higher than T RBCS2 (Fig. 3c). In a distinct context (strain, reporter sequence, culture conditions, etc.), the same genetic element may perform differently [START_REF] Lopez-Paz | Identification of Chlamydomonas reinhardtii endogenous genic flanking sequences for improved transgene expression[END_REF][START_REF] Neupert | Generation of Chlamydomonas strains that efficiently express nuclear transgenes[END_REF] .

Such context sensitivity can be overcome by taking advantage of the modularity of the Chlamydomonas MoClo kit, which allows for the rapid characterization of all possible parts combinations. These results also confirmed the performance of the Chlamydomonas NanoLuc reporter and its employability for detailed understanding and characterization of genetic circuits especially if coupled with automated cell-sorting microfluidic devices [START_REF] Best | Label-Free Analysis and Sorting of Microalgae and Cyanobacteria in Microdroplets by Intrinsic Chlorophyll Fluorescence for the Identification of Fast Growing Strains[END_REF] .

Control of gene expression

To build genetic circuits, the fine-tuning of gene expression is a prerequisite. Multiple parts enabling controlled gene expression have therefore been implemented. The activity of the P NIT1 promoter can be controlled by switching the nitrogen source since it is strongly repressed by ammonium and highly induced on nitrate [START_REF] Strenkert | Heat shock factor 1 counteracts epigenetic silencing of nuclear transgenes in Chlamydomonas reinhardtii[END_REF][START_REF] Ohresser | Expression of the arylsulphatase reporter gene under the control of the nit1 promoter in Chlamydomonas reinhardtii[END_REF][START_REF] Schmollinger | An inducible artificial microRNA system for Chlamydomonas reinhardtii confirms a key role for heat shock factor 1 in regulating thermotolerance[END_REF] . A module where P NIT1 controls expression of the ble-GFP gene (pCM1-8) conferred strong zeocin resistance in the CC-1690 strain but only when ammonium was replaced by nitrate as nitrogen source. By contrast, the P PSAD promoter (pCM1-9) conferred strong antibiotic resistance on both nitrogen sources (Fig. 4a and Supplementary Fig. 3a-c). The vitamin B 12 -repressible promoter P METE [START_REF] Helliwell | Unraveling vitamin B12-responsive gene regulation in algae[END_REF] allowed conditional functional complementation of the photosynthetic mutant nac2-26 (CC-4421), which lacks photosystem II due to the absence of the TPR-like protein NAC2 required for stability of the psbD mRNA encoding the D2 reaction center protein [START_REF] Boudreau | The Nac2 gene of Chlamydomonas encodes a chloroplast TPR-like protein involved in psbD mRNA stability[END_REF] . nac2-26 mutant cells engineered with a module harboring the NAC2 coding sequence under the control of the P METE promoter (pCM1-10) could grow photoautotrophically in the absence of vitamin B 12 , but growth was compromised by increasing its concentration by amounts as low as 5 ng/L (Fig. 4b).

Regulation of gene expression can also be controlled by vitamin B 1 (thiamine) at the level of the transcript through riboswitches [START_REF] Croft | Thiamine biosynthesis in algae is regulated by riboswitches[END_REF][START_REF] Moulin | Analysis of Chlamydomonas thiamin metabolism in vivo reveals riboswitch plasticity[END_REF] . Binding of thiamine pyrophosphate to the THI4 riboswitch (RS) results in alternative splicing and retention of an 81 bp upstream open reading frame, ultimately interfering with translation [START_REF] Croft | Thiamine biosynthesis in algae is regulated by riboswitches[END_REF][START_REF] Moulin | Analysis of Chlamydomonas thiamin metabolism in vivo reveals riboswitch plasticity[END_REF] . The RS also responds when cells are grown in the presence of the thiamine biosynthetic intermediate 4-methyl-5-(2-hydroxyethyl) thiazole (HET), but not with 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) [START_REF] Croft | Thiamine biosynthesis in algae is regulated by riboswitches[END_REF] . A module combining P AR and THI4 (RS) to drive expression of the ble-GFP gene (pCM1-11) conferred conditional zeocin sensitivity in the UVM4 strain [START_REF] Neupert | Generation of Chlamydomonas strains that efficiently express nuclear transgenes[END_REF] . Resistance was compromised by thiamine or HET but not HMP (Fig. 4c and Supplementary Fig. 3d), thereby demonstrating the efficient repression of the transgene through the THI4 riboswitch.

Finally, to allow targeted repression of gene expression, a microRNA precursor sequence derived from the pre-miR1157 and used for the generation of artificial miRNAs (amiRNA) [START_REF] Molnar | miRNAs control gene expression in the single-cell alga Chlamydomonas reinhardtii[END_REF] was re-designed for compatibility with the Golden Gate cloning method. To demonstrate its effectiveness in driving gene repression, a specific amiRNA sequence directed against the MAA7 gene, whose repression provides resistance to 5'-fluoroindole (5'-FI) [START_REF] Palombella | Identification of the gene encoding the tryptophan synthase beta-subunit from Chlamydomonas reinhardtii[END_REF] , was inserted into the microRNA precursor. A control random sequence ("scrambled") amiRNA was inserted into the same backbone. These parts were placed under the control of P PSAD and T PSAD (pCM1-12 and 13) and assembled with a paromomycin resistance module (pCM1-27). The same amiRNA sequences were introduced into the previously established pChlamiRNA3 vector [START_REF] Molnar | miRNAs control gene expression in the single-cell alga Chlamydomonas reinhardtii[END_REF] as controls. After transformation of the CC-1690 strain, 36% of paromomycin-resistant cells displayed resistance to 5'-FI with the device targeting MAA7 (pCMM-5) but not with the scrambled amiRNA (pCMM-6) (Fig. 4d and Supplementary Fig. 3f). A modified 5' rapid amplification of cDNA ends (5'-RACE) assay revealed that the MAA7 transcript was most frequently cleaved at a site corresponding to positions 10 and 11 of the amiRNA, as expected for a specific action of the miRNA (Fig. 4d). The properties of controllable parts can also be combined as shown for P NIT1 control of amiRNA-dependent gene repression [START_REF] Strenkert | Heat shock factor 1 counteracts epigenetic silencing of nuclear transgenes in Chlamydomonas reinhardtii[END_REF] . An amiRNA strategy recently proved useful for concerted metabolic engineering of a biodiesel precursor in Chlamydomonas 8 . The versatility of the MoClo kit opens new possibilities for sophisticated metabolic engineering strategies, e.g. the specific downregulation of up to six target genes with one level M assembly.

Multiple fusion tags for detection and purification of gene products.

Protein fusion tags are indispensable tools used to improve protein expression yields, enable protein purification, and accelerate the characterization of protein structure and function [START_REF] Young | Recombinant protein expression and purification: a comprehensive review of affinity tags and microbial applications[END_REF] .

Our MoClo kit includes multiple epitope and affinity tags known to be functional in Chlamydomonas. The modularity of the MoClo assembly allows rapid assessment of the best tagging strategy through a rapid design/build/test/learn cycle. We took advantage of the well characterized rap2 mutant (FKBP12), which is insensitive to rapamycin [START_REF] Crespo | Inhibition of target of rapamycin signaling by rapamycin in the unicellular green alga Chlamydomonas reinhardtii[END_REF] , to test the functionality of five tags (Fig. 5a,b). We designed and built 5 devices allowing strong constitutive expression of N-or C-terminal tagged FKBP12 coupled to a paromomycin module (pCMM-7 to 11, Fig. 5c-h and Supplementary Table 4). The engineered strains were selected on paromomycin and the functionality of the fusion protein was tested by assessing sensitivity to rapamycin. Protein extracts were probed by immunoblotting using FKBP12-specific and tag-specific antibodies (Fig. 5d-h). All tags allowed detection (Fig. 5d-h) or purification (Fig. 5i) of FKBP12 even though some were not functional for restoring rapamycin sensitivity. The test revealed that pCMM-9 outperforms other devices since it provides a WT-like phenotype and expression level coupled to a strong and specific Myc signal with no significant processing of the protein. These results demonstrate the importance of the modularity provided by the Chlamydomonas MoClo toolkit for designing optimal fusion proteins.

Visualization and targeting of proteins in living cells

Fluorescent protein tags allow the temporal and spatial monitoring of dynamic expression patterns at cellular and subcellular scales [START_REF] Crivat | Imaging proteins inside cells with fluorescent tags[END_REF] . Natural and synthetic metabolic pathways can be optimized through spatial organization since cell compartments offer many advantages, such as isolation of metabolic reactions and generation of concentration gradients [START_REF] Agapakis | Natural strategies for the spatial optimization of metabolism in synthetic biology[END_REF] . In a eukaryotic chassis like Chlamydomonas, organelles such as microbodies, mitochondria and chloroplasts can be engineered to implement or improve metabolic pathways [START_REF] Chen | Designing biological compartmentalization[END_REF] . The Chlamydomonas MoClo kit includes 11 targeting and signal peptides that allow the targeting of fusion proteins to mitochondria, chloroplast, nucleus, secretory pathway, ER and peroxisome-like microbodies. The functionality of the targeting and signal peptides and of the five fluorescent proteins (mVenus -yellow, mCherry -red, mRuby2 -red, Clover -green, mCerrulean3 -cyan) included in the toolkit was tested. Eight modules (pCM1-19 to 26, Supplementary Table 3) combining diverse fluorescent proteins and targeting sequences were assembled into devices with an antibiotic resistance module (pCMM-12 to 19). All devices were found to behave as expected and provided the expected fluorescent signal in the targeted compartment (Fig. 6).

The fluorescent and targeting parts of the Chlamydomonas MoClo toolkit, most of which have been validated here, enable engineering in the third dimension [START_REF] Agapakis | Natural strategies for the spatial optimization of metabolism in synthetic biology[END_REF] i.e. isolation and organization in multiple cellular compartments, and offer new tools for biological design/build/test cycles.

Discussion

The Chlamydomonas MoClo toolkit presented here provides more than 100 domesticated gene parts to allow advanced synthetic biology in microalgae. Numerous parts of multiple types have been characterized and validated in different genetic backgrounds [START_REF] Gallaher | Chlamydomonas Genome Resource for Laboratory Strains Reveals a Mosaic of Sequence Variation, Identifies True Strain Histories, and Enables Strain-Specific Studies[END_REF] and culture conditions, and can be readily used for biological design without further development. With the efficiency and modularity of the MoClo strategy, molecular cloning is no longer a limiting step for engineering Chlamydomonas cells. Indeed, from design to building, a complex device of up to six different genes/modules can be obtained within a week using the standardized parts provided in our kit. The modularity will also enable combinatorial assembly by shuffling part libraries [START_REF] Engler | Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes[END_REF] and determine a posteriori which combination is the most relevant. The development of gene-editing technologies in Chlamydomonas, including Zinc-finger nucleases [START_REF] Greiner | Targeting of Photoreceptor Genes in Chlamydomonas reinhardtii via Zinc-finger Nucleases and CRISPR/Cas9[END_REF]56 and several CRISPR-Cas9 approaches [START_REF] Greiner | Targeting of Photoreceptor Genes in Chlamydomonas reinhardtii via Zinc-finger Nucleases and CRISPR/Cas9[END_REF][START_REF] Shin | CRISPR/Cas9-induced knockout and knock-in mutations in Chlamydomonas reinhardtii[END_REF][START_REF] Baek | DNA-free two-gene knockout in Chlamydomonas reinhardtii via CRISPR-Cas9 ribonucleoproteins[END_REF][START_REF] Ferenczi | Efficient targeted DNA editing and replacement in Chlamydomonas reinhardtii using Cpf1 ribonucleoproteins and singlestranded DNA[END_REF] , together with the development of high-throughput microfluidics [START_REF] Best | Label-Free Analysis and Sorting of Microalgae and Cyanobacteria in Microdroplets by Intrinsic Chlorophyll Fluorescence for the Identification of Fast Growing Strains[END_REF] are beginning to gather pace. Coupling these resources to our standardized MoClo toolkit will facilitate the use of Chlamydomonas as the photosynthetic chassis for innovative synthetic biology approaches aimed at fundamental and biotechnological applications. We expect that the creativity of designers, released from the time constraints associated with classical cloning strategies, will allow rapid expansion of the standard gene parts, modules and devices through open distribution, notably using the Addgene repository. We invite the community to openly share their parts through Addgene and/or our consortium (contact M. Schroda). The development of the Chlamydomonas MoClo toolkit constitutes a complete step-change in the fields of microalgal biology and biotechnology. The parts developed for the MoClo toolkit may also be employed in other microalgal species since the orthogonality of several Chlamydomonas transcriptional units has been demonstrated in multiple hosts, including the industrially relevant species Chlorella ellipsoidea, Nannochloropsis sp. and Dunaliella salina [START_REF] Doron | Transgene Expression in Microalgae-From Tools to Applications[END_REF] . Synthetic approaches will allow engineering of microalgae in a predictable and efficient manner and thereby offer great potential to couple environmental protection, energy transition and bioeconomic growth [START_REF] Gimpel | Metabolic Engineering of Eukaryotic Microalgae: Potential and Challenges Come with Great Diversity[END_REF] .

Methods

All chemicals were obtained from Sigma-Aldrich, unless otherwise specified.

Escherichia coli and Chlamydomonas reinhardtii strains, transformation and growth conditions.

Bacterial growth was performed at 37°C in LB broth supplemented with agar (20% m/V), spectinomycin (50 μg/mL), ampicillin or carbenicillin (50 or 100 μg/mL, respectively) and X-gal (40 µg/mL) when required. Chemically competent E. coli DH10β (New England Biolabs) were used for transformation (by heat shock following the manufacturer's instructions) and maintenance of plasmids. All plasmids of the kit were maintained and amplified in TOP10 E. coli strain prior to submission to Addgene.

C. reinhardtii strains [START_REF] Neupert | Generation of Chlamydomonas strains that efficiently express nuclear transgenes[END_REF][START_REF] Boudreau | The Nac2 gene of Chlamydomonas encodes a chloroplast TPR-like protein involved in psbD mRNA stability[END_REF][START_REF] Crespo | Inhibition of target of rapamycin signaling by rapamycin in the unicellular green alga Chlamydomonas reinhardtii[END_REF][START_REF] Sager | Inheritance in the Green Alga Chlamydomonas Reinhardi[END_REF][START_REF] Kuchka | Mutation at the Chlamydomonas nuclear NAC2 locus specifically affects stability of the chloroplast psbD transcript encoding polypeptide D2 of PS II[END_REF] , culture and transformation conditions are recapitulated in Supplementary Table 5. They were grown in Tris-Acetate-Phosphate (TAP) medium [START_REF] Gorman | Cytochrome f and plastocyanin: their sequence in the photosynthetic electron transport chain of Chlamydomonas reinhardi[END_REF] supplemented with agar (1.6 % m/V), spectinomycin (100 µg/mL), paromomycin (15 µg/mL), zeocin (ThermoFisher Scientific, 10 to 15 µg/mL), 5-fluoroindole (20 µM) or rapamycin (LC Laboratories, 1 µM) when required. For NIT1 promoter characterization (Figure 4a), a modified TAP medium lacking nitrogen source (TAP-N) was used instead, and was supplemented with 4 mM KNO 3 (nitrate) or 7.5 mM NH 4 Cl (ammonium). For NAC2 autotrophy test (Figure 4b), cells were grown in minimal media (HSM) for selection of complemented strains. The responsiveness to B12 was assessed on plate and then in liquid. Cells were grown for 15 days in HSM until 1-5 x10 7 cells/mL concentration prior to inoculation in a 96-well plate at a concentration of 10 5 cells/mL in 200 μL of HSM. For response assays (Figure 4c) thiamine (Melford Laboratories Ltd.), 4-methyl-5-(2-hydroxyethyl) thiazole (HET) and 4-amino-5hydroxymethyl-2-methylpyrimidine (HMP, Fluorochem UK) were added to TAP media at a final concentration of 10 µM.

For transformation by electroporation (see Supplementary Table 5), a TAP culture of 1-5 x 10 6 cells/mL was concentrated 100 times in TAP complemented with 60 mM sucrose or the MAX Efficiency Transformation reagent for Algae (ThermoFisher scientific) and 25-250 µL were incubated with 80-300 ng of DNA for 10-30 min on ice in a 0.4 cm gapped cuvette (BioRad) prior to electroporation (BioRad Gene Pulser Xcell). The cells were then left to recover in TAP complemented with 40-60 mM sucrose for 16 h under appropriate light and shaking conditions (typically 50 µmol photon m -² s -1 at 100 rpm) prior to plating on TAP-agar plates with adapted antibiotics. Transformation by glass-beads method followed previously published protocols [START_REF] Lauersen | Efficient phototrophic production of a high-value sesquiterpenoid from the eukaryotic microalga Chlamydomonas reinhardtii[END_REF][START_REF] Kindle | High-frequency nuclear transformation of Chlamydomonas reinhardtii[END_REF] . Briefly, after growth in TAP until 5 x 10 6 cells/mL, cells were concentrated 30 times and 5 x 10 7 cells were mixed with DNA using glass beads. After 2-fold dilution with TAP, 2.5 x 10 7 cells were spread onto TAP agar plates containing 100 µg ml -1 spectinomycin. Plates were incubated for 16 h in the dark prior to light exposition (30 µmol photon.m -2 .s -1 ). When colony counting was performed (Figure 3a), it was 8 days after the beginning of light. In both cases, the transformation protocol leads to insertion of a linear DNA in a random location within the nuclear genome.

Design. All in silico sequence designs and analysis were performed with Serial Cloner, Benchling, SnapGene, ApE or Genome Compiler. For exogenous parts, reverse translation was performed with Serial Cloner using C. reinhardtii nuclear genome codon frequency (http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=3055). amiRNAs can be generated using DNA parts pCM0-068 and pCM0-069. Both are derived from the endogenous pre-miR1157 [START_REF] Molnar | Highly specific gene silencing by artificial microRNAs in the unicellular alga Chlamydomonas reinhardtii[END_REF] , but differ in the way in which the amiRNA specific sequence is introduced. pCM0-069/pCMM-20 is analogous to pChlamyRNA3 [START_REF] Molnar | Highly specific gene silencing by artificial microRNAs in the unicellular alga Chlamydomonas reinhardtii[END_REF] , and a dsDNA fragment containing the amiRNA/loop/amiRNA* sequence is introduced into a SpeI site inside the miRNA precursor sequence. pCM0-068 presents two divergently oriented BpiI sites, allowing the cloning of the dsDNA fragment by Golden Gate. In this last case, the dsDNA fragment is formed by the annealing of two oligos with the following sequence: 1) sense oligo (5' AGTA-

(MIRNA*SEQ)-TCTCGCTGATCGGCACCATGGGGGTGGTGGTGATCAGCGCTA-(MIRNA SEQ)-T 3'), 2)
anti-sense oligo (5'CAGT-A-(rev com MIRNA SEQ)-TAGCGCTGATCACCACCACCCCCATGGTGCCGATCAGCGAGA-(rev com MIRNA*SEQ) 3'). There are online tools that help with the design of the amiRNA sequence (http://wmd3.weigelworld.org/cgi-bin/webapp.cgi).

Parts repository.

All sequences listed in Supplementary Table 2 were deposited in Addgene.

Physical distribution of the DNA is performed through Addgene. We invite the community to share their future parts through Addgene and/or with our consortium (contact M. Schroda) which will make them available to the community. (NEB) in a medium containing the NEB CutSmart buffer and 1 mM ATP (with stock of 10 mM solubilized in 0.1 M Tris-HCl, pH 7.9). Typical ratio between destination plasmid (100 fmol) and entry plasmid/parts was 1:2. To facilitate handling of the kit for end-users, we provide detailed protocols and reaction mix calculators for each type of assembly: level 0 for parts (Supplementary Table 6), level 1 for modules (Supplementary Table 7) and level M for devices (Supplementary Table 8).

Parts

Quality Control of generated DNAs. All plasmids were controlled by differential restriction. In addition, all level 0 plasmids were sequenced with specific primers. Sequencing was performed by Eurofins Genomics, Source BioSciences UK, Seqlab, Macrogen, Microsynth, GATC Biotech or Core Facility (CeBiTec, Bielefeld University).

NanoLuc activity determination.

Reagents were purchased from Promega (ref. N1110) and activity was determined as previously described [START_REF] Hall | Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate[END_REF] . For screening, C. reinhardtii colonies were transferred into a 96-well plate containing 100 µL of TAP in each well. After gentle resuspension, 50 µL was transferred into a solid white 96-well plate to which 50 µL of Nano-Glo substrate diluted in the provided buffer (2% V/V) was added and gently mixed by pipetting.

Luminescence was measured at 460 nm with a CLARIOstar plate reader (BMG Labtech). For promoter/terminator combination assessment experiment (Figure 3c), all C. reinhardtii colonies from a transformation event were pooled and resuspended in TE buffer (50 mM Tris-HCl pH 7.9, 1 mM EDTA) complemented with anti-protease (1 tablet per 50 mL, Sigma-Aldrich: S8830). The cells were lysed by vortexing 10 seconds twice in the presence of glass beads (about 1:5 ratio beads/cells V/V) prior to two centrifugations (20000 g for 10 min at 4°C) to clarify the supernatant. The protein concentration was then determined using Bradford reagent with a Bovine Serum Albumin standard curve and the concentration was standardized to 0.5 g/L. The activity was determined in a 96-well plate in a final volume of 50 µL (1:1 with nano-Glo resuspended in provided lysis buffer) per well. NanoLuc activity was determined on 6 different increasing protein quantities (0.1 to 2.5 µg) for each assay, allowing to assess linearity of the signal.

Absorbance measurement of cultures growing in microtiter plates

Growth in microtiter plates was determined by measuring the optical density of each well at 730 nm. Microtiter plates containing 180-200 μL culture were incubated under constant light (125 µmol photon.m -2 .s -1 ) at 25°C and 40 rpm orbital shaking. For density determination, cultures were resuspended by pipetting and 100 µL of cell suspension was transferred to a new microtiter plate containing 50 µL TAP 0.03% Tween-20. Optical density of each well was determined at 730 nm in a CLARIOstar plate reader (BMG Labtech). Plates were shaken for 6-10 sec at 600 rpm before measurement.

RNA extraction and miRNA-mediated cleavage mapping

RNA isolation was carried out as previously described [START_REF] Molnar | miRNAs control gene expression in the single-cell alga Chlamydomonas reinhardtii[END_REF] (a detailed protocol can be found at http://www.plantsci.cam.ac.uk/research/davidbaulcombe/methods/downloads/smallrna.pd f/view), with the following modifications: Cells were centrifuged and resuspended in 0.25 mL of water and mixed with 0.25 mL Lysis buffer (50 mM Tris-HCl pH 8.0, 200 mM NaCl, 50 mM EDTA, 2% SDS, 1 mg/mL proteinase K). Lysis buffer was incubated at 50°C for 5 min prior mixing with cells. Cell suspension was then incubated at 25°C for 20 min. Finally, 2 mL of PureZol (Biorad) was added and samples were snap-frozen. RNA quality was assessed in gel and quantified in Nanodrop (ThermoFisher scientific).

miRNA cleavage site determination was performed as previously described [START_REF] Llave | Cleavage of Scarecrow-like mRNA targets directed by a class of Arabidopsis miRNA[END_REF] . Briefly, 10 µg of total RNA was ligated with an RNA oligo (5' CGACUGGAGCACGAGGACACUGACAUGGAC UGAAGGAGUAGAAA 3') using T4 RNA ligase for 1 h at 37°C. RNA was extracted with phenol:chloform and precipitated with ethanol and sodium acetate. The precipitated RNA was retrotranscribed into cDNA by SuperScript IV reverse transcriptase (ThermoFisher scientific), using random hexamers and following manufacturer's recommendations. Two µL of the cDNA was used as template of a PCR using primers FJN456 (5'-CGACTGGAGCACGAGGACACTGA) and FJN495 (5'-TGGGGTAGGGGTGGGGGCCAG). Two µL of this PCR was used as template of a second PCR with primers FJN457 (5'-GGACACTGACATGGACTGAAGGAGTA) and FJN496 (5'-TGACCCAGTCGCGGATGGCCT). PCR was resolved in a 2% agarose gel and the specific band was isolated from the gel and cloned into pGEM-T easy (Promega) for sequencing.

Immuno-blotting. Chlamydomonas cells expressing FKB12 fusion proteins from liquid cultures

were collected by centrifugation 4000 g for 5 min at room temperature (RT), washed in 50 mM

Tris-HCl pH 7.5, and resuspended in a minimal volume of the same solution. Cells were lysed by two cycles of slow freezing to -80°C followed by thawing at RT. The soluble cell extract was separated from the insoluble fraction by centrifugation (15000 g for 20 min at 4°C). Total protein extracts (15 µg) were then subjected to 15% SDS-PAGE.

mCherry-expressing cells were harvested at 3500 rpm for 2 min (4 °C) and resuspended in 60 µL of DTT-carbonate buffer (0.1 M DTT, 0.1 M Na 2 CO 3 ). After freezing at -20 °C and thawing, 55 µL of SDS-Sucrose buffer were added (5 % SDS, 30 % sucrose). Samples were then boiled for 45 s at 95 °C, followed by 2 min incubation on ice and 13000 g centrifugation for 2 min at RT. Protein extracts corresponding to 2 µg of Chlorophyll were then separated using 12% SDS-PAGE.

For immunoblot analyses, proteins were then transferred to nitrocellulose membranes (Bio-Rad, 162-0115 or Amersham Protran). After blocking with 3 to 5% low-fat Milk in PBS for 1 h at RT, membranes were incubated with primary antibody in 5% low-fat Milk in PBS for 16 h at 4°C. After 4 washes in PBS -0.1% tween-20 (TPBS), the membranes were incubated with secondary antibody in 5% low-fat Milk in PBS for 1 h at RT, and subsequently washed 4 time in TPBS prior to chemi-luminescence revelation using ECL. Primary antibodies used were anti-FKBP12 [START_REF] Crespo | Inhibition of target of rapamycin signaling by rapamycin in the unicellular green alga Chlamydomonas reinhardtii[END_REF] (1/5000 dilution; secondary was anti-rabbit 1/10000), anti-FLAG (Sigma-Aldrich F1804, 1/5000 dilution; secondary was anti-mouse 1/5000), anti-STREP (IBA, Catalog N. 2-1509-001, 1/5000 dilution; conjugated to HRP), anti-cMYC (Sigma-Aldrich M4439, 1/2500 dilution; secondary was anti-mouse 1/5000), anti-HA (Sigma-Aldrich H9658, 1/5000 dilution; secondary was anti-mouse 1/2500) and anti-PRPL1 [START_REF] Ries | Structural and molecular comparison of bacterial and eukaryotic trigger factors[END_REF] . For mCherry serum, rabbits were immunized against purified full-length mCherry protein containing an N-terminal His 6 -tag.

Microscopy.

For mCherry experiments (Figure 6b-e), images were taken at 100x magnification with a BX53F microscope (Olympus). Fluorescence images for the detection of mCherry were When the SBOL2.0 standard was not existing for a part type, the symbol proposed before [START_REF] Martella | EMMA: An Extensible Mammalian Modular Assembly Toolkit for the Rapid Design and Production of Diverse Expression Vectors[END_REF] was used, or defined here. 2). For amiRNA (amiR) backbones, b and s mean that BpiI and SpeI site are within the backbone for amiR cloning, respectively, while M and sc mean that the target amiR sequence for MAA7 and the control scrambled sequence were introduced into the miR1157 backbone, respectively (cf. Fig. 4). mSTOP stands for multi-STOP. (c) Average NanoLuc activity of D66 (CC-4425) cells transformed with 4 devices (pCMM-1 to 4) harboring promoter/terminator combinations to drive NanoLuc expression coupled to a paromomycin resistance module (represented as P R , left, Supplementary Fig. 2). Luminescence levels are represented as mean ± SEM (average of a total of more than 400 clones from 3 biological replicates). The negative and positive controls are the recipient strain and the pCMM- (i) Purification through ion-metal affinity chromatography (IMAC) of 6His-FKBP12 expressed from the same device as in h.

P R represents the paromomycin resistance module (pCM1-27, Supplementary Fig. 2a). rap2 cells transformed with pCMM-X are indicated as M-X in each panel. Data are representative of 3 biological replicates.

  cloning. All PCR reactions were performed using the Phusion DNA polymerase, KOD Xtreme Hot Start DNA polymerase (Merck) or Q5 DNA polymerase purchased from New England Biolabs (NEB) following the manufacturer's instructions adapted to GC-rich DNA, typically duration of hybridization and polymerization was doubled and/or GC enhancer solution was used. Molecular biology kits were purchased from Macherey-Nagel, peqLab, NEB or QIAgen (gel extraction and miniprep kits). Primers were produced by Eurofins Genomics or Sigma-Aldrich while synthesized parts were obtained from Genecust, DC Biosciences, IDTDNA or Sigma-Aldrich. MoClo Assembly Conditions. All Restriction/ligation reactions were performed using BbsI or BbsI-HF (BpiI is an isoschizomer) or BsaI-HF (NEB or ThermoFisher) together with T4 ligase

Figure 1 .

 1 Figure 1. Overview of the Chlamydomonas MoClo toolkit. (a) Type and position of parts used following the Plant MoClo Syntax 30 . Symbols correspond to the SBOL2.0 visual 69 representation described in b. Each of the 11 fusion sites defining a part position is represented with a color and a number. Positions presented are representative of the whole set of each part type. Parts in italicized letters are non-transcribed, parts in regular letters are transcribed and parts in bold letters are transcribed and translated. (b) Table summarizing unique and total gene parts available. The SBOL2.0 symbols are indicated for each type.

Figure 2 .

 2 Figure 2. List of parts in function of their type and assembly position.(a) Plant MoClo syntax 30 indicating the color code for fusion sites used in this figure. (b) All parts in the Chlamydomonas MoClo kit are classified primarily by their function, indicated by SBOL2.0 visual code 69 as in Fig.1(from left to right: promoters, promoter+5'UTR, 5'UTR, introns, antibiotic resistance genes, reporter genes, artificial microRNA, immunological and purification tags, 2A peptide, and 3'UTR+terminators). Colored stripes on the left and right sides of each box represent the fusion sites in 5' and 3' of the position, respectively, and follow the color code on top. AR and A'R stand for HSP70A/RBCS2 and HSP70A467/RBCS2, respectively[START_REF] Schroda | Sequence elements within an HSP70 promoter counteract transcriptional transgene silencing in Chlamydomonas[END_REF] . A star (*) indicates that the part contains extra restriction sites as in pOpt vectors[START_REF] Lauersen | Targeted expression of nuclear transgenes in Chlamydomonas reinhardtii with a versatile, modular vector toolkit[END_REF] while the same part unmarked does not. An (i) indicates the presence of an intron within the part (cf. Supplementary Table2). For amiRNA (amiR) backbones, b and s mean that BpiI and SpeI site are within the backbone for amiR cloning, respectively, while M and sc mean that the target amiR sequence for MAA7 and the control scrambled sequence were introduced into the miR1157 backbone, respectively (cf. Fig.4). mSTOP stands for multi-STOP.

Figure 3 .

 3 Figure 3. Constitutive promoters and reporter genes. (a) Average number of spectinomycin resistant colonies after transformation of UVM4 cells (mean ± SD, N=11) for the three modules (pCM1-3, where pCM stands for plasmid Chlamydomonas Moclo). Representative transformation plates are shown. (b) Linearity of NanoLuc activity as a function of cell number. NanoLuc activity for 4 independent clones transformed with the pCMM-1 device (pCMM-1.X), one non-expressing clone (NC) and the recipient strain (CC-4425 noted as control) are presented (N=3, mean ± SEM). Linear regression and correlation coefficient (R²) are shown. The NC and control are shown in the inset on a different scale.

  1.9 strain (shown in b), respectively. a,c *p<0.05; **p< 0.01 assessed by Student's t-test, SBOL2.0 69 visual of module designs are shown above the devices.

Figure 4 .

 4 Figure 4. Control of gene expression. (a) Control of gene expression by the nitrogen source. Zeocin resistant colonies (conferred by Ble(i)-GFP)selected after transformation of CC-1690 cells with each of the two represented modules ("1-8" for pCM1-8 and "1-9" for pCM1-9) were grown in TAP-nitrogen ± zeocin (15 µg/mL) supplemented with either 7.5 mM (NH 4 )Cl (ammonium, blue) or 4 mM KNO 3 (nitrate, black) and their growth was followed (Absorbance at 730 nm). The plot shows the ratio between the growth in the presence and absence of zeocin (C is the nontransformed CC-1690 strain). The right panel shows cells grown in similar conditions but on solid media. Results presented (N=16 for control CC-1690 and N=86 for each other conditions) correspond to one out of three independent transformations (for the other two, see Supplementary Fig.3). (b) Control of gene expression by vitamin B 12 . Conditional complementation of nac2-26 cells with the pCM1-10 module expressing NAC2 under P METE control. Complemented strains were selected for photoautotrophic growth on solid minimal medium and the cells were grown in liquid minimal medium supplemented with the indicated amount of vitamin B 12 . Data are mean ± SD (N=3). (c) Control of gene expression by vitamin B 1 . Average growth (absorbance at 730 nm after 7 days of growth, N=40) of UVM4 cells transformed with the pCM1-11 module designed to express constitutively Ble(i)-GFP transcripts containing the THI4 riboswitch in the 5'UTR. After culture in TAP, the cells were transferred to TAP+zeocin (10 µg/mL) supplemented with 10 µM thiamine (Thi), 10 µM 4-methyl-5-(2-hydroxyethyl) thiazole

Figure 5 .

 5 Figure 5. Design, build and test of five fusion tags (a) Phenotype of recipient (WT) and ΔFKBP12 (rap2) strains in the presence (Rap) or absence (-) of 1 µM rapamycin. (b) Molecular mechanism underlying the rap2 phenotype. Target Of Rapamycin (TOR) is inhibited by rapamycin only in the presence of FKBP12 (mutated in rap2). Upon formation of the tripartite TOR/FKBP12/rapamycin complex, TOR is inhibited and growth is arrested 52 . (c) SBOL2.0 visual 69 of module designs for functional complementation of rap2.(d-h) Phenotype in the presence (Rap) or absence (-) of 1 µM rapamycin and detection of tagged proteins in soluble extracts by immunoblotting with antibodies against either FKBP12 (αFKBP12) or the appropriate tag (indicated within each panel). Each device is indicated in the upper part of the panel. CBB: Coomassie Brilliant Blue staining of a duplicate gel loaded with the same samples and shown as loading control.

TUB2: Tubulin 2, PSAD: Photosystem I reaction center subunit II, HET: 4-methyl-5-(2hydroxyethyl) thiazole, HMP: 4-amino-5-hydroxymethyl-2-methylpyrimidine, amiRNA: artificial micro RNA, TAP: Tris Acetate Phosphate Acknowledgments. The authors would like to thank Karin Gries and Vincent Assoun for their technical help. This work was supported in part by Agence Nationale de la Recherche Grant ANR-17-CE05-0008 and LABEX DYNAMO ANR-LABX-011 (to PC, AD, FdC, JH, CHM, MdM, KS, MC and SDL), by the DFG-funded TRR175 and FOR2092 (to FW, BS, JN, JT, LW, RT and MS), by OpenPlant

taken using a TRITC filter. For other fluorescent proteins (Figure 6f-h), microscopy was performed as previously described [START_REF] Lauersen | Efficient phototrophic production of a high-value sesquiterpenoid from the eukaryotic microalga Chlamydomonas reinhardtii[END_REF][START_REF] Lauersen | Targeted expression of nuclear transgenes in Chlamydomonas reinhardtii with a versatile, modular vector toolkit[END_REF] .

Accession numbers. All parts accession numbers and the corresponding references are listed in Supplementary Table 2.

ASSOCIATED CONTENT Supporting Information.

Supplementary Figure 1 -MoClo assembly workflow reflecting the abstraction hierarchy Supplementary Figure 2 -Variability of Nanoluc expression in pCMM-1 transformants. Supplementary Figure 3 -Control of gene expression, complementary data. Supplementary Table 1 -list of all unique parts of the Chlamy MoClo kit Supplementary Table 2 -list of all parts of the Chlamy MoClo kit: level 0 plasmids Supplementary Table 3 -list of all modules used for the Chlamy MoClo kit validation: level 1 plasmids Supplementary S R and P R represent respectively modules conferring resistance to spectinomycin (S R =pCM1-1, Fig. 3a and Supplementary Fig. 2a) and paromomycin (P R =pCM1-27, Supplementary Fig. 2a). Supplementary Figure 1. MoClo assembly workflow reflecting the abstraction hierarchy.

We followed the original MoClo syntax 22 updated 30 . After design of a gene part controlled in silico for full compatibility until level M assembly, the part is cloned with BpiI (equivalent to BbsI) into the appropriate level 0 plasmid (Spectinomycin bacterial resistance). After quality control (QC) by restriction and sequencing, the resulting clone is registered in the part database as a pCM0-xxx (where pCM stands for plasmid Chlamydomonas Moclo). To generate the desired Transcriptional Unit (TU), the compatible parts are assembled with BsaI into the appropriate level 1 plasmid (Ampicillin bacterial resistance). After QC by restriction, the clone is registered as pCM1-xxx in the module database. Finally, to assemble a device, up to 6 modules at a time are assembled with the corresponding end-linker [START_REF] Weber | A modular cloning system for standardized assembly of multigene constructs[END_REF] by BpiI into a level M or 2 plasmid (Spectinomycin or kanamycin bacterial resistance, respectively).

After QC by restriction, the clone is registered as pCMM-xxx in the device database. New assembly from this device can be performed to assemble more modules to the device [START_REF] Weber | A modular cloning system for standardized assembly of multigene constructs[END_REF] . Parts are represented in SBOL2.0 visual code [START_REF] Roehner | Sharing Structure and Function in Biological Design with SBOL 2.0[END_REF] (see also Fig. 1).


Alison G Tebo

Frederico M Pimenta

Martha Zoumpoulaki

Carlos Kikuti

Helena Sirkia

Marie-Aude Plamont

Anne Houdusse

Arnaud Gautier
email: arnaud.gautier@ens.fr

Circularly permuted fluorogenic proteins for the design of modular biosensors

Cells respond to external and internal stimuli by a series of biochemical events that are tightly controlled in space and time. These downstream processes consist of interactions between macromolecules as well as between macromolecules and analytes. Detecting these interactions involves coupling a measurable output to a nanometer-scale molecular recognition event, typically by linking a reporting module to a sensing domain that responds to an input by undergoing a conformational change. Fluorescent proteins (FP) such as the green fluorescent protein (GFP) and its variants have been essential in the development of genetically encoded optical biosensors. FP-based biosensors typically function either by Förster resonance energy transfer (FRET), in which an analyte-binding domain is flanked with two FPs acting as donor and acceptor for FRET studies, or by fluorescence intensity change, in which conformational changes induced by analyte binding change the local environment of the chromophore of circularly permuted FPs. [START_REF] Okumoto | Imaging approach for monitoring cellular metabolites and ions using genetically encoded biosensors[END_REF][2][3][START_REF] Vanengelenburg | Fluorescent biosensors of protein function[END_REF] Chemical-genetic fluorogenic reporters provide an attractive alternative to FPs as they display many of the same advantages, while capitalizing on the specific interaction of a fluorogenic chromophore (so-called fluorogen) with its cognate genetically encoded receptor in order to generate fluorescence. [START_REF] Bruchez | Dark dyes-bright complexes: fluorogenic protein labeling[END_REF][START_REF] Jullien | Fluorogen-based reporters for fluorescence imaging: a review[END_REF][START_REF] Li | Fluorogenic Labeling Strategies for Biological Imaging[END_REF] Fluorogens are chromophores that display no fluorescence until they interact with their cognate tag, resulting in systems with low background signal and little to no maturation time. The modular nature of these reporters opens exciting prospects for the design of genetically encoded biosensors in which fluorogen binding, and thus fluorescence, is conditioned to the recognition of a given analyte.

An elegant illustration of this principle was obtained using Spinach, an RNA aptamer that binds analogs of the GFP chromophore [START_REF] Paige | RNA Mimics of Green Fluorescent Protein[END_REF] . The coupling of metabolite-specific RNA aptamers to Spinach led to biosensors in which metabolite binding promote Spinach fluorescence [START_REF] Paige | Fluorescence imaging of cellular metabolites with RNA[END_REF][START_REF] You | Imaging metabolite dynamics in living cells using a Spinach-based riboswitch[END_REF] .

To validate this approach with protein-based fluorogenic systems (Figure 1A), which have the advantages of being genetically targetable to any subcellular localization and in principle implementable in all model cells and organisms, we evaluated the use of the Fluorescence-Activating and absorption-Shifting Tag (FAST) [START_REF] Plamont | Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo[END_REF] , a variant of the 14 kDa photoactive yellow protein (PYP) evolved to bind and activate (through conformational locking) the fluorescence of various hydroxybenzylidene rhodanine (HBR) derivatives [START_REF] Plamont | Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo[END_REF][START_REF] Li | Dynamic multicolor protein labeling in living cells[END_REF] . As fluorogen binding affinity depends on the protein structure, we anticipated that conformational change would affect fluorogen binding and thus fluorescence.

To maximize conformational coupling and facilitate insertion of FAST as reporting module into multidomain biosensors, we circularly permuted the protein. Circular permutation is a method used both in the laboratory and by nature whereby the amino acid sequence is altered such that the N and C termini are close in space but the protein retains the same (or similar) 3D structure (and hence function). Relocating the N-and C-termini within the structure of a protein to be spatially close has proved to be effective in increasing conformational sensitivity. This technique has been successfully used in the development of most intensiometric FP-based biosensors [START_REF] Nakai | A high signal-to-noise Ca(2+) probe composed of a single green fluorescent protein[END_REF][START_REF] Zhao | An expanded palette of genetically encoded Ca²⁺ indicators[END_REF][START_REF] Marvin | An optimized fluorescent probe for visualizing glutamate neurotransmission[END_REF] . We hypothesized that FAST would tolerate circular permutation in much the same way as its parent protein, PYP. [START_REF] Kumar | A circularly permuted photoactive yellow protein as a scaffold for photoswitch design[END_REF] Furthermore, we reasoned that such a development could provide a strategy to create FAST-based biosensors by co-opting previously well-characterized sensor designs. For instance, calcium sensors composed of a circularly permuted FP and a calcium-binding motif comprised of calmodulin and M13 have been extensively explored. [START_REF] Nakai | A high signal-to-noise Ca(2+) probe composed of a single green fluorescent protein[END_REF][START_REF] Zhao | An expanded palette of genetically encoded Ca²⁺ indicators[END_REF] In these sensors, the calciumbinding moieties calmodulin and M13 are appended to the N and C termini of the circularly permuted fluorescent protein and binding of Ca 2+ results in the formation of the calmodulin and M13 ternary complex, which in turn induces a conformational change in FP, and results in higher fluorescence. This design, which has been implemented with Aequorea GFP or its variants, has not yet been successfully adapted to other fluorescent reporting modules. Herein, we describe the development of a series of circularly permuted FAST (cpFAST) variants with fluorogen affinities spanning an order of magnitude and illustrate using calcium sensors as a proof of principle that these cpFASTs can replace classic FPs for the design of intensiometric biosensors.

We designed seven circularly permuted FAST (cpFAST) variants by linking the original termini with peptide linkers of various lengths, and created new N-and C-termini at positions 115 and 114, respectively, as previously reported for circularly permuted PYP. [START_REF] Kumar | A circularly permuted photoactive yellow protein as a scaffold for photoswitch design[END_REF] All cpFASTs showed good expression in E. coli (Figure S1) and formed green-yellow fluorescent complexes with 4-hydroxy-3-methylbenzylidene rhodanine (HMBR), indicating that the circular permutation did not perturb the three-dimensional topology and function of FAST. Absorption and emission wavelengths, fluorescence quantum yields, and absorption coefficients were nearly identical to those of non-permuted FAST (Table 1). Interestingly, we observed that the shorter the linker inserted within cpFAST, the lower the affinity for HMBR (Table 1 and Figure S2). This trend may result from shorter linkers introducing a physical constraint in the three-dimensional structure of the protein, thus inducing a lower binding affinity. All cpFAST were shown to activate HMBR fluorescence in live cells (Figure S3).

To validate the use of our cpFASTs for biosensor design, we created a sensor able to selectively detect Ca 2+ by taking advantage of the Ca 2+ -dependent interaction of calmodulin (CaM) and the Ca 2+ -CaM interacting peptide M13, which has been successfully used in the past to generate genetically encoded FP-based calcium indicators (Figure S4) [START_REF] Nakai | A high signal-to-noise Ca(2+) probe composed of a single green fluorescent protein[END_REF][START_REF] Zhao | An expanded palette of genetically encoded Ca²⁺ indicators[END_REF][START_REF] Chen | Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo[END_REF] . Ca 2+ is a highly versatile secondary messenger that regulates many different cellular functions over a wide temporal range 18 . The concentration Ca 2+ at resting state and the amplitude of the concentration change can vary significantly as a function of the intracellular localization and the cell type, therefore Ca 2+ biosensors with various dissociation constants (ideally from nM to mM) are required to fully address the physiological role of Ca 2+ signaling. Because of the dynamic and transient nature of calcium signaling, Ca 2+ was an ideal model analyte for us to validate the use of cpFASTs for the design of dynamic fluorogenic biosensors. We connected the N-terminus of cpFASTs to the M13 peptide and their C-terminus to calmodulin (CaM). The corresponding sensors were expressed in E. coli with high yields (Figure S5), allowing full in vitro characterization. For all sensors, the presence of Ca 2+ increased HMBR binding affinity (Figures 1B and S6 and Table 2), demonstrating that Ca 2+ promoted fluorogen binding. We observed that the shorter the linker used within the cpFAST, the larger the Ca 2+ -induced increase in affinity. In agreement with the circular permutation providing conformational sensitivity, most cpFAST-based sensors displayed a larger Ca 2+ -induced change in fluorogen affinity than a sensor in which the original FAST was inserted (Table 2).

An important feature of FAST is the ability to change its color from green-yellow to orangered by using 4-hydroxy-3,5-dimethoxybenzylidene rhodanine (HBR-3,5-DOM) instead of HMBR [START_REF] Li | Dynamic multicolor protein labeling in living cells[END_REF] . We therefore tested HBR-3,5-DOM with the cpFAST-based sensors. Ca 2+ -dependent increases in fluorogen affinity were also observed in that case (Figures 1C andS7 and Table 2), demonstrating that the color of the sensors could be tuned at will from green-yellow to orange-red by choosing the appropriate fluorogen. This unprecedented feature provides an experimental versatility not encountered with FP-based biosensors, for which changing the color has required significant reengineering efforts [START_REF] Zhao | An expanded palette of genetically encoded Ca²⁺ indicators[END_REF] . With FASTbased sensors, sensor optimization is effectively decoupled from color choice, allowing plug-an-play color change.

In intensiometric fluorogen-based sensors, the dynamic range of the sensors is dependent on the conditions of the experiment, notably the fluorogen concentration used (See SI Text 1 and Figure S8). To evaluate the dynamic range of our sensor, we measured the fluorescence fold increase for cpFAST2-based sensor in a range of fluorogen concentrations sampling a large range of conditions and expected fluorescence fold increases (Figure 1D-G). The observed fluorescence fold increase was slightly higher (~ 1.2) than predicted based solely on the change in fraction of sensor:fluorogen complex, suggesting that the brightness of the sensor was also influenced by Ca 2+ , which we attributed to an increased rigidity of the sensor in the presence of Ca 2+ . Similar results were obtained with sensors based on other cpFASTs (Figure S9).

Next, we determined the Ca 2+ affinity of our sensors. We fixed the concentration of fluorogen so that to maximize the change in fraction of sensor:fluorogen complex (see SI Text 1). The tested sensors displayed tight Ca 2+ binding with dissociation constants (KD,Ca) of about 60 to 100 nM, in good agreement with that of the CaM and M13 fusion protein linked with two amino acid linkers previously reported (KD,Ca of 80 nM) 19,20 2). Neither the fluorogen used (HMBR or HBR-3,5-DOM) nor the cpFAST chosen had a significant influence on the affinity for Ca 2+ . Furthermore, we examined the dependence of KD,Ca on fluorogen concentration for the sensor based on cpFAST2. We found that Ca 2+ affinity is only slightly dependent on the fluorogen concentration (Figure S10 We next evaluated the function of our cpFAST-based Ca 2+ sensors in living cells. When the sensors were expressed in HeLa cells and labeled with HMBR or HBR-3,5-DOM, the fluorescence was uniformly distributed in cells (Figure 2). This observation was in agreement with the sensors being small (33 kDa), monomeric proteins as confirmed by sizeexclusion chromatography coupled to multiangle light scattering (Figure S14). Treatment with histamine, which triggers Ca 2+ release from the endoplasmic reticulum to the cytoplasm, induced a significant increase of fluorescence (2 to 3-fold), in accordance with an increase of cytosolic free Ca 2+ (Figure 2, and movies S1-S3). This initial peak was followed by oscillations and eventually desensitization (Figure 2 and movies S1-S3). Our sensors did not display the striking oscillations reported with recently, highly-optimized sensors very likely because of their high affinity for Ca 2+ . However, the observed response was in good agreement with the known behavior of HeLa cells to histamine stimulation [START_REF] Nakai | A high signal-to-noise Ca(2+) probe composed of a single green fluorescent protein[END_REF][START_REF] Zhao | An expanded palette of genetically encoded Ca²⁺ indicators[END_REF]20,21 , demonstrating our ability to image Ca 2+ levels in living cells in real-time.

In this study, we report the design of functional circularly permuted FAST variants (cpFAST). We demonstrate using Ca 2+ sensors as a proof-of-principle that cpFASTs can be an alternative to FPs as reporting module in biosensors. In cpFAST-based sensors, the intensiometric response results from conditioning fluorogen binding (and thus fluorescence) to the recognition of an intracellular analyte. We could generate functional Ca 2+ biosensors with minimal engineering efforts. The successful design of FP-based sensors of the GCaMP family -made of circular permuted GFP flanked with CaM and M13results from the serendipitous specific interaction between CaM and GFP in presence of Ca 2+ that strongly affects GFP conformation and photophysical properties 22 . Engineering efforts to implement this design to generate genetically encoded calcium indicators with fluorescent reporter modules other than FPs have not been successful so far. 23 In addition, extension of this serendipity-based design principle to analytes other than Ca 2+ has turned out to be highly challenging. [START_REF] Marvin | An optimized fluorescent probe for visualizing glutamate neurotransmission[END_REF][24][25][26][27] FAST-based Ca 2+ sensors display Ca 2+ -dependent fluorescence without the need for a specific direct interaction between FAST and the Ca 2+ sensing domains, which should thus facilitate the extension of this design principle to other analytes using various sensing modules. The ability to change the color by changing the fluorogen used allows the design of biosensors with various spectral properties without the need for reengineering, providing an unprecedented experimental versatility for multicolor imaging.

Although conformationally coupling of FAST and the sensing unit allows one to condition fluorogen binding to analyte recognition, it also makes the dynamic range of the sensor and the affinity for the analyte dependent on the fluorogen concentration. Consequently, the fluorogen concentration is a control parameter that must be chosen with care to optimize measurements with the sensors. Despite this, the use of FAST for sensor design opens exciting perspectives. Its robustness and tolerance to insertions make FAST a particularly interesting scaffold for sensor design at once taking advantage of its relatively small size and the ability to change its color at-will. The use of cpFASTs could prove useful in the screening of new sensing modules for which current development is stifled by intrinsic limitations of FPs such as maturation time, tendency to oligomerize, and oxygen sensitivity. The simplicity and tractability of such an approach should allow the design of new plug-and-play biosensors for imaging in real-time analytes, endogenous biomolecules and cellular processes for which no systems currently exist. ** Molar absorption coefficients and quantum yields for cpFAST1, cpFAST2 and cpFAST3 are approximate numbers due to restrictions imposed on the maximum amount of complex that can be formed in solution given the reported KD (i.e., only at appreciably high protein concentrations will ~100% of complex be formed).

SI Text 1

When the total concentration of fluorogen (Ftot) is large compared to the total concentration of sensor (Stot), one can write the fraction of sensor:fluorogen complex (a) as:

, where KD is the thermodynamic dissociation constant of the sensor:fluorogen complex.

To characterize the response of a fluorogen-based sensor upon analyte binding, one can either compute the variation of complex fraction upon analyte binding:

or the increase of bound fraction upon analyte binding:

, where and are respectively the fraction of sensor:fluorogen complex in excess and in absence of analyte, and KD,+ and KD,-are respectively the thermodynamic dissociation constants of the sensor:fluorogen complex in excess and in absence of analyte.

One can easily show that is optimal when Ftot = (KD,+ KD,-) 1/2 , while is maximal at small Ftot.

By noting IF,+ and IF,-the fluorescence intensities in presence and in absence of

the analyte, the fluorescence dynamic range of the sensor is given by:

α = 1 K D F tot + 1 α + -α -= 1 K D,+ F tot + 1 - 1 K D,- F tot + 1 α + / α -= K D,- F tot + 1 K D,+ F tot + 1 α + α - α + -α - α + / α -
where and are respectively the brightness of the sensor:fluorogen complex in presence and in absence of analyte. The dynamic range depends thus on the concentration in fluorogen Ftot and is maximal at low Ftot, approaching the value However, Ftot must be high enough to allow the detection of the fluorescent complex, (See Figure S8). We propose that the ideal compromise to obtain both good dynamic range and satisfactory detection sensitivity is to choose Ftot in the range:

KD,+/2 £ Ftot £ (KD,+KD,-) 1/2
The fluorogen concentration may be eventually tuned relative to the needs of the experiment -lower, when the signal ratio must be optimized, or higher when overall fluorescence signal is important.

To evaluate the dynamic range of our sensor, we measured the fluorescence fold increase in a range of fluorogen concentrations -KD,+/2, KD,+, (KD,+KD,-) 1/2 , KD,-, and 2KD,--chosen so as to sample a large range of conditions and expected fluorescence fold increases Data represent mean ± sem (n = 3). Least square fit (line) gave the thermodynamic dissociation constants KD,-and KD,+ provided in Table 2. Excitation was fixed at 520 nm and emission at 600 nm. Data represent mean ± sem (n = 3). Least square fit (line) gave the thermodynamic dissociation constant KD,Ca and Hill coefficient nH provided in Table 2.

I F,+ I F,- = B + α + B -α - B + B - B + B -- K D,- K D,+ SI Figures Figure S1.

MATERIALS AND METHODS

General FAST is a variant of the photoactive yellow protein (PYP) containing the mutations C69G, Y94W, T95M, F96I, D97P, Y98T, Q99S, M100R, T101G. HMBR and HBR-3,5-DOM were prepared as previously described [START_REF] Okumoto | Imaging approach for monitoring cellular metabolites and ions using genetically encoded biosensors[END_REF]2 .

Molecular Biology

  , and Hill coefficients (nH) of about 3 in agreement with cooperative Ca 2+ binding to CaM (Figures 1H,I, S10-S13 and Table

  and S11), indicating that experimental conditions may be tuned at will without affecting the basics of sensor function. The absence of strong dependence of the Ca 2+ affinity on the fluorogen concentration in this case is linked to the cooperative binding of Ca 2+ . With their high Ca 2+ affinity, our cpFAST-based biosensors can allow the detection of subtle changes in Ca 2+ concentration and complement the current suite of widely used single-FP-based Ca 2+ indicators 21 .

Figure 1 .

 1 Figure 1. cpFAST-based biosensors. (A) Fluorogenic biosensors composed of a cpFAST variant coupled to analyte-recognition modules in which the binding of the fluorogenic ligand, and thus the fluorescence, is conditioned to the binding of the analyte. KD,-and KD,+ are the thermodynamic dissociation constants of the sensor:fluorogen complex in the absence and the presence of analyte, respectively. (B,C) Titration curves of M13-cpFAST2-CaM in the absence or in presence of Ca 2+ with either HMBR (B) or HBR-3,5DOM (C). Data represent mean ± sem (n = 3). Least square fit (line) gave the thermodynamic dissociation constants KD,-and KD,+ provided in Table 2. (D,E) Excitation and emission spectra of M13-cpFAST2-CaM:HMBR (D) and M13-cpFAST2-CaM:HBR-3,5-DOM (E) in the absence (dotted line) and presence (solid line) of Ca 2+ . The concentration of HMBR was 0.7 µM and that of HBR-3,5-DOM 1.95 µM. Sensor concentration was fixed at 0.1 µM. (F,G) Dependence of the I+/I-emission ratio on concentration of (F) HMBR and (G) HBR-3,5-DOM. (F) I+ and I-were obtained by integrating spectra recorded at a sensor concentration of 100 nM and HMBR concentrations of 0.7 µM, 1.4 µM, 2.7 µM, 5.2 µM, and 10.4 µM at 25 o C. (G) I+ and I-were obtained by integrating spectra recorded at a sensor concentration of 100 nM and HBR-3,5-DOM concentrations of 1.95 µM, 3.9 µM, 8.4 µM, 18 µM and 36 µM at 25 o C. (H,I) Ca 2+ titration curve of M13-cpFAST2-CaM in the presence of 2.7 µM HMBR (H) and 8.4 µM HBR-3,5DOM (I). Data represent mean ± sem (n =3). Least square fit (line) gave the thermodynamic dissociation constant KD,Ca and Hill coefficient nH provided in Table 2. Sensor concentration was fixed at 0.01 µM.

Figure 2 .

 2 Figure 2. Ca 2+ imaging in HeLa cells. M13-cpFAST2-CaM was expressed in HeLa cells and labeled with 5 µM HMBR (A,D,G) or 10 µM HBR-3,5DOM (B,E,H). M13-cpFAST3-CaM was expressed in HeLa cells and labeled with 5 µM HMBR (C,F,I). Cells were stimulated with 50 µM histamine. Movies S1, S2 and S3 were recorded by confocal microscopy. (A-C) Average fluorescence images showing homogenous distribution of the sensors. (D-F) Fluorescence intensity changes in response to histamine addition. Time of histamine addition is indicated by the black arrow. (G-I) Low fluorescence was observed in resting state (low Ca 2+ ). Histamine addition (50 µM) induced a significant increase of fluorescence (high Ca 2+ ), in accordance with an increase of cytosolic free Ca 2+ . (A-I) Scale bars 20 µm.

  Figure S1. Bacterial expression and purification of the circularly permuted FAST variants. Coomassie-stained SDS-PAGE of purified 1: FAST, 2: cpFAST1, 3: cpFAST2, 4: cpFAST3, 5: cpFAST4, 6: cpFAST5, 7: cpFAST6, 8: cpFAST7.

Figure S2 .

 S2 Figure S2. HMBR titration curves of (A) FAST, (B) cpFAST1, (C) cpFAST2, (D) cpFAST3, (E) cpFAST4, (F) cpFAST5, (G) cpFAST6, (H) cpFAST7. Titrations were done at a protein concentration of 50 nM in pH 7.4 PBS buffer at 25 °C. Data represent mean ± sem (n = 3). Least square fit (line) gave the thermodynamic dissociation constants KD provided in Table 1.

Figure S3 .

 S3 Figure S3. Characterization of cpFAST in mammalian cells. Confocal images of HeLa cells expressing the circularly permuted FAST variants fused to the red fluorescent protein mCherry. The green channel shows the fluorescence signal from the FAST variants labeled with concentrations of HMBR ensuring full labeling of FAST (50 µM for cpFAST1, 40 µM for cpFAST2, 15 µM for cpFAST3, 10 µM for cpFAST4 and 6 µM for cpFAST5, and 5 µM for FAST5), while the red channel shows the fluorescence signal from mCherry. Side-by-side images were recorded with the same settings for direct comparison. Scale bars 20 µm.

Figure S4 .

 S4 Figure S4. FAST-based Ca 2+ biosensors. FAST-based Ca 2+ biosensors were constructed by connecting the N-terminus of FAST or cpFAST to the M13 peptide, and its C-terminus to Calmodulin.

Figure S5 .

 S5 Figure S5. Bacterial expression and purification of the FAST-based Ca 2+ biosensors. Coomassie-stained SDS-PAGE of purified 1: M13-FAST-CaM, 2: M13-cpFAST1-CaM, 3: M13-cpFAST2-CaM, 4: M13-cpFAST3-CaM, 5: M13-cpFAST4-CaM, 6: M13-cpFAST5-CaM.

Figure S6 .

 S6 Figure S6. HMBR titration curves of the FAST-based Ca 2+ biosensors in the presence and absence of Ca 2+ . (A) M13-FAST-CaM, (B) M13-cpFAST1-CaM, (C) M13-cpFAST3-CaM, (D) M13-cpFAST4-CaM, (E) M13-cpFAST5-CaM. Titrations were done at a sensor concentration of 100 nM in pH 7.4 HEPES buffer containing 10 mM CaCl2 (+ Ca 2+ ) or 10 mM EGTA (-Ca 2+ ) at 25 °C. Excitation was fixed at 480 nm and emission at 540 nm. Data represent mean ± sem (n = 3). Least square fit (line) gave the thermodynamic dissociation constants KD,-and KD,+ provided in Table2.

Figure S7. HBR- 3 , 5 -

 35 Figure S7. HBR-3,5-DOM titration curves of the FAST-based Ca 2+ biosensors in the presence and absence of Ca 2+ . (A) M13-cpFAST1-CaM, (B) M13-cpFAST3-CaM, (C) M13-cpFAST4-CaM, (D) M13-cpFAST5-CaM. Titrations were done at a sensor concentration of 100 nM in pH 7.4 HEPES buffer containing 10 mM CaCl2 (+ Ca 2+ ) or 10 mM EGTA (-Ca 2+ ) at 25 °C. Excitation was fixed at 520 nm and emission at 600 nm.

Figure S8 .

 S8 Figure S8. FAST-based optical biosensors. Simulated fluorogen binding curves in the presence and absence of analyte (with KD,-= 10 KD,+). a+ and a-are the fluorogen bound fractions in presence and absence of the analyte, respectively. Choosing the concentration of fluorogen between KD,+/2 and (KD,+KD,-) 1/2 (contrasted rectangles) is a good compromise to obtain both good dynamic range and satisfactory detection sensitivity.

Figure S9 .

 S9 Figure S9. Excitation and Emission spectra of (A) M13-cpFAST3-CaM:HMBR and (B) M13-cpFAST3-CaM:HBR-3,5DOM in the absence (dashed lines) and presence of Ca 2+ (solid lines). Spectra were recorded at a sensor concentration of 100 nM and a fluorogen concentration of (KD,+KD,-) 1/2 at 25 o C. Excitation was scanned from 380 to 530 nm at 540 nm emission andemission was scanned from 490 to 650 nm at 480 nm excitation.

Figure S10 .

 S10 Figure S10. (A) Dependence of KD,Ca on HMBR concentration for M13-cpFAST2-CaM (B-F).Titrations were done at a sensor concentration of 10 nM and HMBR concentrations of (B) 0.7 µM (C) 1.4 µM (D) 2.7 µM, (E) 5.4 µM and (F) 10.4 µM. Excitation was fixed at 480 nm and emission at 540 nm. Data represent mean ± sem (n = 3). Least square fit (line) gave the thermodynamic dissociation constant KD,Ca and Hill coefficient nH.

Figure S11 .

 S11 Figure S11. (A) Dependence of KD,Ca on HBR-3,5DOM concentration for M13-cpFAST2-CaM (B-F).Titrations were done at a sensor concentration of 10 nM and HBR-3,5DOM concentrations of (B) 1.95 µM (C) 3.9 µM (D) 8.4 µM, (E) 18 µM and (F) 36 µM. Excitation was fixed at 520 nm and emission at 600 nm. Data represent mean ± sem (n = 3). Least square fit (line) gave the thermodynamic dissociation constant KD,Ca and Hill coefficient nH.

Figure S12 .

 S12 Figure S12. Ca 2+ titration curves of M13-cpFAST-CaM in presence of HMBR. (A) M13-cpFAST1-CaM. (B) M13-cpFAST3-CaM. Titrations were done at a sensor concentration of 100 nM and a HMBR concentration of (KD,+KD,-) 1/2 at 25 o C. Excitation was fixed at 480 nm and emission at 540 nm. Data represent mean ± sem (n = 3). Least square fit (line) gave the thermodynamic dissociation constant KD,Ca and Hill coefficient nH provided in Table 2.

Figure S13 .

 S13 Figure S13. Ca 2+ titration curves of M13-cpFAST-CaM in presence of HBR-3,5-DOM. (A) M13-cpFAST1-CaM. (B) M13-cpFAST3-CaM. Titrations were done at a sensor concentration of 100 nM and a HBR-3,5-DOM concentration of (KD,+KD,-) 1/2 at 25 °C.

Figure S14 . 5 .

 S145 Figure S14. SEC-MALS analysis of recombinant M13-cpFAST2-CaM in 10 mM HEPES, 50 mM NaCl, 2 mM CaCl2, pH 7.5. Continuous line: static light scattering, normalised from 3 angles; dotted line: molecular mass in kDa. Concentration at the monomer peak was ~ 5 mg/ml = 161 µM. In the conditions used, 96% of M13-cpFAST2-CaM (33.4 kDa) was monomeric and 4% was found to be dimeric. Note that no dimers were detected when the SEC column was equilibrated in buffer containing 1 mM EGTA instead of 2 mM CaCl2 (i.e. in absence of Ca 2+ ).

  The genes encoding the circularly permuted FAST variants were constructed by PCR assembly from the gene encoding FAST optimized for human codon usage[START_REF] Okumoto | Imaging approach for monitoring cellular metabolites and ions using genetically encoded biosensors[END_REF] . The plasmids for bacterial expression of cpFAST1-7 were generated by inserting the cpFAST1-7 DNA sequence in the plasmid pET18a using the Nhe I and Xho I restriction sites. The plasmids for mammalian expression of mcherry-cpFAST1-5 were constructed by replacing FAST DNA sequence by the cpFAST1-5 DNA sequence in pAG96 encoding mCherry-GGGS-FAST[START_REF] Okumoto | Imaging approach for monitoring cellular metabolites and ions using genetically encoded biosensors[END_REF] . The calcium sensors were generated from a synthetic sequence (Eurofins) coding for M13-linker-CaM and inserted in the plasmid pET28a using Nhe I and Xho I. The sequence for M13 used was various cpFAST DNA sequences were amplified by PCR and inserted using Mfe I and Sac I restriction sites present in the linker between M13 and CaM. The plasmids for mammalian expression of the circularly permuted FAST variants and the cpFAST-based calcium biosensors were generated by inserting the corresponding DNA sequences into the previously reported pAG104 plasmid 1 using BamH I and Bgl II Protein expression and purification Expression vectors were transformed in Rosetta (DE3) pLysS E. coli (New England Biolabs). Cells were grown at 37°C in Lysogeny Broth (LB) medium complemented with 50 μg/ml kanamycin and 34 μg/ml chloramphenicol to OD600nm 0.6. Expression was induced for 4 h by adding isopropyl β-D-1thiogalactopyranoside (IPTG) to a final concentration of 1 mM. Cells were harvested by centrifugation (4,000 × g for 20 min at 4°C) and frozen. The cell pellet was resuspended in lysis buffer (phosphate buffer 50 mM, NaCl 150 mM, MgCl2 2.5 mM, protease inhibitor, DNase, pH 7.4) and sonicated (5 min at 20 % of amplitude, 3 sec on, 1 sec off). The lysate was incubated for 2 h at 4 °C to allow DNA digestion by DNase. Cellular fragments were removed by centrifugation (9200 × g for 1h at 4°C). The supernatant was incubated overnight at 4°C under gentle agitation with Ni-NTA agarose beads in phosphate buffered saline (PBS) (sodium phosphate 50 mM, NaCl 150 mM, pH 7.4) complemented with 10 mM imidazole. Beads were washed with 20 volumes of PBS containing 20 mM Imidazole, and with 5 volumes of PBS complemented with 40 mM Imidazole. His-tagged proteins were eluted with 5 volumes of PBS complemented with 0.5 M Imidazole. The buffer was exchanged to PBS (50 mM phosphate, 150 mM NaCl, pH 7.4) for cpFAST or HEPES buffer (HEPES 50 mM, NaCl 259 mM, pH 7.4) for calcium sensors using PD-10 desalting columns.Instrumentation Steady state UV-Vis absorption spectra were recorded using a Cary 300 UV-Vis spectrometer (Agilent Technologies), equipped with a Versa20 Peltier-based temperature-controlled cuvette chamber (Quantum Northwest) and fluorescence data were recorded using a LPS 220 spectrofluorometer (PTI, Monmouth Junction, NJ), equipped with a TLC50TM Legacy/PTI Peltier-based temperature-controlled cuvette chamber (Quantum Northwest). Thermodynamic dissociation constants were determined with a Spark 10M plate reader (Tecan).Thermodynamic analysisThe thermodynamic dissociation constants KD,+ and KD,-were determined in HEPES buffer (HEPES 50 mM, NaCl 259 mM, pH 7.4) containing either 10 mM CaCl2 or 10 mM EGTA, while the pH in solution was kept constant at 7.4. Normalized fluorescence (or bound fraction) were plotted as a function of fluorogen concentration and fitted using one site specific binding equation to provide KD,+ and KD,-. The thermodynamic dissociation constants KD,Ca were determined using buffers with precise free Ca 2+ concentration either purchased commercially (ThermoFisher) or prepared following protocols previously reported by Roger Tsien et al.3 by reciprocal dilution of a Ca 2+ saturated solution (10 mM CaEGTA in HEPES buffer) and a Ca 2+ free solution (10 mM K2EGTA in HEPES buffer) to give a series of solutions with free Ca 2+ concentration ranging from 0 to 0.351 µM at pH 7.2. In these experiments, the fluorogen concentration was fixed at the optimal fluorogen concentration (KD,+KD,-) 1/2 . The normalized

Table 1 . Physico-chemical properties of the circularly permuted FAST (cpFAST) variants bound to HMBR.

 1 Abbreviations are as follows: labs, wavelength of maximal absorption; lem, wavelength of maximal emission; e, molar absorption coefficient at labs; f, fluorescence quantum yield; KD, thermodynamic dissociation constant.

	Variant	Linker size					KD
		(a.a.)	labs	lem	e	f	(µM)
			(nm)	(nm)	(mM -1 cm -1 )
	FAST*	-	481	540	42.5 ± 0.8	0.23 ± 0.03	0.13 ± 0.01
	cpFAST1	0	481	540	~ 35 **	~ 0.2**	5.1 ± 0.70
	cpFAST2	3	481	540	~ 35 **	~ 0.2**	0.99 ± 0.11
	cpFAST3	5	481	540	~ 35 **	~ 0.2**	0.65 ± 0.04
	cpFAST4	8	481	540	36.0 ± 0.5	0.19 ± 0.02	0.30 ± 0.04
	cpFAST5	11	481	542	36.2 ± 0.9	0.28 ± 0.03	0.22 ± 0.03
	cpFAST6	15	481	541	36.9 ± 0.8	0.24 ± 0.03	0.26 ± 0.03
	cpFAST7	18	481	540	33.0 ± 2.9	0.20 ± 0.02	0.16 ± 0.01
	* previously reported 12 .

Table 2 . Physico-chemical properties of the cpFAST-based Ca 2+ biosensors (M13- Reporter-CaM) labeled with HMBR (A) and HBR-3,5-DOM (B).

 2 Abbreviations are as follows: KD,+ and KD,-are the thermodynamic dissociation constants of the sensor:fluorogen complexes in presence and in absence of Ca 2+ ; KD,Ca and nH are the thermodynamic dissociation constant and Hill coefficient associated to Ca 2+ binding (the fluorogen concentration was fixed to (KD,+KD,-) 1/2 ).

	Reporter		KD,+ (µM)	KD,-(µM)	KD,Ca (nM) [nH]
	FAST	A	0.16 ± 0.01	0.36 ± 0.02
	cpFAST1	A	4.0 ± 0.4	18.2 ± 0.8	77 ± 2 [2.8 ± 0.2]
		B	17.4 ± 0.8	37 ±3	63 ± 1 [4.0 ± 0.5]
	cpFAST2	A	1.40 ± 0.04	5.2 ± 0.1	99 ± 4 [3.0 ± 0.3]
		B	3.9 ± 0.1	18.0 ± 0.8	82 ± 1 [3.3 ± 0.2]
	cpFAST3	A	0.80 ± 0.02	2.9 ± 0.1	64 ± 2 [3.3 ± 0.3]
		B	4.2 ± 0.2	14.8 ± 0.9	57 ± 3 [2.3 ± 0.3]
	cpFAST4	A	0.59 ± 0.03	1.39 ± 0.03
		B	2.8 ± 0.1	9.1 ± 0.4
	cpFAST5	A	0.36 ± 0.01	0.82 ± 0.02
		B	2.50 ± 0.09	7.5 ± 0.6

ACKNOWLEDGEMENTS

This work was supported by the European Research Council (ERC-2016-CoG-724705 FLUOSWITCH), the Agence National de la Recherche (ANR-14-CE09-0002-01), France BioImaging (ANR-10-INBS-04), the Equipex Morphoscope 2 (ANR-11-EQPX-0029) and the program «Investissements d'Avenir » launched by the French Government and implemented by ANR (ANR-10-IDEX-0001-02 PSL, project Superline).

Nature 499, 295-300. (18) Berridge, M. J., Bootman, M. D., and Roderick, H. L. (2003) Calcium signalling: dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol 4, 517-529. (19) Porumb, T., Yau, P., Harvey, T. S., and Ikura, M. (1994) A calmodulin-target peptide hybrid molecule with unique calcium-binding properties. Protein Eng. 7, 109-115. (20) Miyawaki, A., Llopis, J., Heim, R., McCaffery, J. M., Adams, J. A., Ikura, M., and Tsien, R. Y. (1997) Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388, 882-887. (21) Bootman, M. D., Cheek, T. R., Moreton, R. B., Bennett, D. L., and Berridge, M. J. (1994) Smoothly graded Ca2+ release from inositol 1,4,5-trisphosphate-sensitive Ca2+ stores. J. Biol. Chem. 269, 24783-24791. (22) Akerboom, J., Rivera, J. D. V., Guilbe, M. M. R., Malavé, E. C. A., Hernandez, H. H., Tian, L., Hires, S. A., Marvin, J. S., Looger, L. L., and Schreiter, E. R. (2009) Crystal structures of the GCaMP calcium sensor reveal the mechanism of fluorescence signal change and aid rational design. J. Biol. Chem. 284, 6455-6464. (23) Shitashima, Y., Shimozawa, T., Asahi, T., and Miyawaki, A. (2018) A dual-ligandmodulable fluorescent protein based on UnaG and calmodulin. Biochem. Biophys. Res. Comm. 496, 872-879. (24) Wu, J., Abdelfattah, A. S., Zhou, H., Ruangkittisakul, A., Qian, Y., Ballanyi, K., and Campbell, R. E. (2018) Genetically Encoded Glutamate Indicators with Altered Color and Topology. ACS Chem. Biol. (25) Qin, Y., Sammond, D. W., Braselmann, E., Carpenter, M. C., and Palmer, A. E. (2016) Development of an Optical Zn 2+Probe Based on a Single Fluorescent Protein. ACS Chem. Biol. 11, 2744-2751. (26) Chen, Z., and Ai, H.-W. (2016) Single Fluorescent Protein-Based Indicators for Zinc Ion (Zn 2+). Anal. Chem. 88, 9029-9036. (27) Sanford, L., and Palmer, A. (2017) Recent Advances in Development of Genetically Encoded Fluorescent Sensors. Meth. Enzymol. 589, 1-49.

SUPPORTING INFORMATION

The supporting information contains Movies S1-S3, SI Text 1, Figures S1-S14 and Materials and Methods.

NOTES

The authors declare the following competing financial interest: A.G. is co-founder and holds equity in Twinkle Bioscience, a company commercializing the FAST technology.

Supporting Information

Circularly permuted fluorogenic proteins for the design of modular biosensors Alison G. Tebo 1,# , Frederico M. Pimenta Mammalian cell culture HeLa cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with phenol red, Glutamax I, and 10% (vol/vol) fetal calf serum (FCS), at 37 °C in a 5% CO2 atmosphere. For imaging, cells were seeded in μDish IBIDI (Biovalley) coated with poly-L-lysine. Cells were transiently transfected using Genejuice (Merck) according to the manufacturer's protocol for 24 hours prior to imaging.

Fluorescence microscopy