Suchergebnisse

The Cre-Lox system is a highly versatile and powerful DNA recombinase mechanism, mainly used in genetic engineering to insert or remove desired DNA sequences. It is widely utilized across multiple fields of biology, with applications ranging from plants, to mammals, to microbes. A key feature of this system is its ability to allow recombination between mutant lox sites. Two of the most commonly used mutant sites are named lox66 and lox71, which recombine to create a functionally inactive double mutant lox72 site. However, a large portion of the published literature has incorrectly annotated these mutant lox sites, which in turn can lead to difficulties in replication of methods, design of proper vectors and confusion over the proper nomenclature. Here, we demonstrate common errors in annotations, the impacts they can have on experimental viability, and a standardized naming convention. We also show an example of how this incorrect annotation can induce toxic effects in bacteria that lack optimal DNA repair systems, exemplified by Mycoplasma pneumoniae. ; This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement 634942 (MycoSynVac) and was also financed by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme, under grant agreement 670216 (MYCOCHASSIS). We also acknowledge support of the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) to the EMBL partnership, the Centro de Excelencia Severo Ochoa and the CERCA Programme/Generalitat de Catalunya

Distinguishing between promoter-like sequences in bacteria that belong to true or abortive promoters, or to those that do not initiate transcription at all, is one of the important challenges in transcriptomics. To address this problem, we have studied the genome-reduced bacterium Mycoplasma pneumoniae, for which the RNAs associated with transcriptional start sites have been recently experimentally identified. We determined the contribution to transcription events of different genomic features: the -10, extended -10 and -35 boxes, the UP element, the bases surrounding the -10 box and the nearest-neighbor free energy of the promoter region. Using a random forest classifier and the aforementioned features transformed into scores, we could distinguish between true, abortive promoters and non-promoters with good -10 box sequences. The methods used in this characterization of promoters can be extended to other bacteria and have important applications for promoter design in bacterial genome engineering. ; European Union Seventh Framework Programme (FP7/2007–2013), through the European Research Council [232913]; Fundación Botín, the Spanish Ministry of Economy and Competitiveness [BIO2007-61762]; National Plan of R + D + i; ISCIII – Subdirección General de Evaluación y Fomento de la Investigación [PI10/01702]; European Regional Development Fund (ERDF) (to the ICREA Research Professor L.S.]; Spanish Ministry of Economy and Competitiveness, 'Centro de Excelencia Severo Ochoa 2013–2017 [SEV-2012-0208]. Funding for open access charge: European Union Seventh Framework Programme (FP7/2007–2013), through the European Research Council [232913]; Fundaci´on Bot´ın, the Spanish Ministry of Economy and Competitiveness [BIO2007-61762]; National Plan of R + D + i; ISCIII – Subdirección General de Evaluación y Fomento de la Investigación [PI10/01702]; European Regional Development Fund (ERDF) (to the ICREA Research Professor L.S.]; Spanish Ministry of Economy and Competitiveness, 'Centro de Excelencia Severo Ochoa 2013–2017 [SEV-2012-0208].

Protein degradation is a crucial cellular process in all-living systems. Here, using Mycoplasma pneumoniae as a model organism, we defined the minimal protein degradation machinery required to maintain proteome homeostasis. Then, we conditionally depleted the two essential ATP-dependent proteases. Whereas depletion of Lon results in increased protein aggregation and decreased heat tolerance, FtsH depletion induces cell membrane damage, suggesting a role in quality control of membrane proteins. An integrative comparative study combining shotgun proteomics and RNA-seq revealed 62 and 34 candidate substrates, respectively. Cellular localization of substrates and epistasis studies supports separate functions for Lon and FtsH. Protein half-life measurements also suggest a role for Lon-modulated protein decay. Lon plays a key role in protein quality control, degrading misfolded proteins and those not assembled into functional complexes. We propose that regulating complex assembly and degradation of isolated proteins is a mechanism that coordinates important cellular processes like cell division. Finally, by considering the entire set of proteases and chaperones, we provide a fully integrated view of how a minimal cell regulates protein folding and degradation. ; This work has been supported by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program, under grant agreement 670216 (MYCOCHASSIS). We also acknowledge the support of the Spanish Ministry of Science and Innovation to the EMBL partnership, the Centro de Excelencia Severo Ochoa, and the CERCA Program from the Generalitat de Catalunya. M.W acknowledges the European Union's Horizon 2020 research and innovation program under grant agreement 634942 (MycoSynVac) and M.L.‐S. The support from FEDER project from Instituto Carlos III (ISCIII, Acción Estratégica en Salud 2016) (reference CP16/00094). The proteomics analyses were performed in the CRG/UPF Proteomics Unit which is part of the Spanish Infrastructure for Omics Technologies (ICTS OmicsTech), and it is a member of the ProteoRed PRB3 consortium which is supported by grant PT17/0019 of the PE I + D+i 2013‐2016 from the Instituto de Salud Carlos III (ISCIII) and ERDF

Transposon sequencing is commonly applied for identifying the minimal set of genes required for cellular life; a major challenge in fields such as evolutionary or synthetic biology. However, the scientific community has no standards at the level of processing, treatment, curation and analysis of this kind data. In addition, we lack knowledge about artifactual signals and the requirements a dataset has to satisfy to allow accurate prediction. Here, we have developed FASTQINS, a pipeline for the detection of transposon insertions, and ANUBIS, a library of functions to evaluate and correct deviating factors known and uncharacterized until now. ANUBIS implements previously defined essentiality estimate models in addition to new approaches with advantages like not requiring a training set of genes to predict general essentiality. To highlight the applicability of these tools, and provide a set of recommendations on how to analyze transposon sequencing data, we performed a comprehensive study on artifacts corrections and essentiality estimation at a 1.5-bp resolution, in the genome-reduced bacterium Mycoplasma pneumoniae. We envision FASTQINS and ANUBIS to aid in the analysis of Tn-seq procedures and lead to the development of accurate genome essentiality estimates to guide applications such as designing live vaccines or growth optimization. ; ERASynBio 2nd Joint Call for Transnational Research Projects: 'Building Synthetic Biology Capacity Through Innovative Translational Projects', with funding from the corresponding ERASynBio National Funding Agencies; European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program [670216] (MYCOCHASSIS); CERCA Programme/Generalitat de Catalunya; Spanish Ministry of Science and Innovation to the EMBL partnership, 'Centro de Excelencia Severo Ochoa 2013–2017'. Funding for open access charge: ERASynBio 2nd Joint Call for Transnational Research Projects: 'Building Synthetic Biology Capacity Through Innovative Translational Projects', with funding from the corresponding ERASynBio National Funding Agencies; European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program [670216] (MYCOCHASSIS); CERCA Programme/Generalitat de Catalunya; Spanish Ministry of Science and Innovation to the EMBL partnership, 'Centro de Excelencia Severo Ochoa 2013–2017'.

Trans-translation is a ubiquitous bacterial mechanism of ribosome rescue mediated by a transfer-messenger RNA (tmRNA) that adds a degradation tag to the truncated nascent polypeptide. Here, we characterize this quality control system in a genome-reduced bacterium, Mycoplasma pneumoniae (MPN), and perform a comparative analysis of protein quality control components in slow and fast-growing prokaryotes. We show in vivo that in MPN the sole quality control cytoplasmic protease (Lon) degrades efficiently tmRNA-tagged proteins. Analysis of tmRNA-mutants encoding a tag resistant to proteolysis reveals extensive tagging activity under normal growth. Unlike knockout strains, these mutants are viable demonstrating the requirement of tmRNA-mediated ribosome recycling. Chaperone and Lon steady-state levels maintain proteostasis in these mutants suggesting a model in which co-evolution of Lon and their substrates offer simple mechanisms of regulation without specialized degradation machineries. Finally, comparative analysis shows relative increase in Lon/Chaperone levels in slow-growing bacteria suggesting physiological adaptation to growth demand. ; This work has been supported by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program under grant agreement 670216 (MYCOCHASSIS), and Conveni La Caixa 2020–2023 (LA CAIXA-EGA, LCF/PR/GN13/10260009). We also acknowledge the support of the Spanish Ministry of Science and Innovation to the EMBL partnership, the Centro de Excelencia Severo Ochoa, and the CERCA Program from the Generalitat de Catalunya. M.W acknowledges the European Union's Horizon 2020 research and innovation program under grant agreement 634942 (MycoSynVac). The proteomics analyses were performed in the CRG/UPF Proteomics Unit which is part of the Proteored, PRB3 and is supported by grant PT17/0019, of the PE I + D + i 2013–2016, funded by ISCIII and ERDF

Mycoplasmas are important model organisms for Systems and Synthetic Biology, and are pathogenic to a wide variety of species. Despite their relevance, many of the tools established for genome editing in other microorganisms are not available for Mycoplasmas. The Tn4001 transposon is the reference tool to work with these bacteria, but the transformation efficiencies (TEs) reported for the different species vary substantially. Here, we explore the mechanisms underlying these differences in four Mycoplasma species, Mycoplasma agalactiae, Mycoplasma feriruminatoris, Mycoplasma gallisepticum and Mycoplasma pneumoniae, selected for being representative members of each cluster of the Mycoplasma genus. We found that regulatory regions (RRs) driving the expression of the transposase and the antibiotic resistance marker have a major impact on the TEs. We then designed a synthetic RR termed SynMyco RR to control the expression of the key transposon vector elements. Using this synthetic RR, we were able to increase the TE for M. gallisepticum, M. feriruminatoris and M. agalactiae by 30-, 980- and 1036-fold, respectively. Finally, to illustrate the potential of this new transposon, we performed the first essentiality study in M. agalactiae, basing our study on more than 199,000 genome insertions. ; This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement 634942 (MycoSynVac) and was also financed by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme, under grant agreement 670216 (MYCOCHASSIS). We also acknowledge support of the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) to the EMBL partnership, the Centro de Excelencia Severo Ochoa and the CERCA Programme/Generalitat de Catalunya.

Here, we propose an approach to identify active metabolic pathways by integrating gene essentiality analysis and protein abundance. We use two bacterial species (Mycoplasma pneumoniae and Mycoplasma agalactiae) that share a high gene content similarity yet show significant metabolic differences. First, we build detailed metabolic maps of their carbon metabolism, the most striking difference being the absence of two key enzymes for glucose metabolism in M. agalactiae. We then determine carbon sources that allow growth in M. agalactiae, and we introduce glucose-dependent growth to show the functionality of its remaining glycolytic enzymes. By analyzing gene essentiality and performing quantitative proteomics, we can predict the active metabolic pathways connected to carbon metabolism and show significant differences in use and direction of key pathways despite sharing the large majority of genes. Gene essentiality combined with quantitative proteomics and metabolic maps can be used to determine activity and directionality of metabolic pathways. ; This project was financed by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program, under grant agreement nos. 634942 (MycoSynVac) and 670216 (MYCOCHASSIS). We acknowledge support of the Spanish Ministry of Science and Innovation to the EMBL partnership, the Centro de Excelencia Severo Ochoa, and the CERCA Programme / Generalitat de Catalunya.

The C-terminal sequence of a protein is involved in processes such as efficiency of translation termination and protein degradation. However, the general relationship between features of this C-terminal sequence and levels of protein expression remains unknown. Here, we identified C-terminal amino acid biases that are ubiquitous across the bacterial taxonomy (1,582 genomes). We showed that the frequency is higher for positively charged amino acids (lysine, arginine), while hydrophobic amino acids and threonine are lower. We then studied the impact of C-terminal composition on protein levels in a library of Mycoplasma pneumoniae mutants, covering all possible combinations of the two last codons. We found that charged and polar residues, in particular lysine, led to higher expression, while hydrophobic and aromatic residues led to lower expression, with a difference in protein levels up to fourfold. We further showed that modulation of protein degradation rate could be one of the main mechanisms driving these differences. Our results demonstrate that the identity of the last amino acids has a strong influence on protein expression levels. ; This project has received funding from the European Union's Horizon 2020 research and innovation program under grant agreement no. 634942 (MycoSynVac) and 670216 (MYCOCHASSIS). ML-S acknowledges the support from FEDER project from Instituto Carlos III (ISCIII, Acción Estratégica en Salud 2016) (reference CP16/00094)

Data de publicació electrònica: 16-12-2020 ; The removal of unwanted genetic material is a key aspect in many synthetic biology efforts and often requires preliminary knowledge of which genomic regions are dispensable. Typically, these efforts are guided by transposon mutagenesis studies, coupled to deepsequencing (TnSeq) to identify insertion points and gene essentiality. However, epistatic interactions can cause unforeseen changes in essentiality after the deletion of a gene, leading to the redundancy of these essentiality maps. Here, we present LoxTnSeq, a new methodology to generate and catalogue libraries of genome reduction mutants. LoxTnSeq combines random integration of lox sites by transposon mutagenesis, and the generation of mutants via Cre recombinase, catalogued via deep sequencing. When LoxTnSeq was applied to the naturally genome reduced bacterium Mycoplasma pneumoniae, we obtained a mutant pool containing 285 unique deletions. These deletions spanned from > 50 bp to 28 Kb, which represents 21% of the total genome. LoxTnSeq also highlighted large regions of non-essential genes that could be removed simultaneously, and other non-essential regions that could not, providing a guide for future genome reductions. ; This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement 634942 (MycoSynVac) and was also financed by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme, under grant agreement 670216 (MYCOCHASSIS). We also acknowledge support of the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) to the EMBL partnership, the Centro de Excelencia Severo Ochoa and the CERCA Programme/Generalitat de Catalunya

Mycoplasma species share a set of features, such as lack of a cell wall, streamlined genomes, simplified metabolism, and the use of a deviant genetic code, that make them attractive approximations of what a chassis strain should ideally be. Among them, Mycoplasma pneumoniae arises as a candidate for synthetic biology projects, as it is one of the most deeply characterized bacteria. However, the historical paucity of tools for editing Mycoplasma genomes has precluded the establishment of M. pneumoniae as a suitable chassis strain. Here, we developed an oligonucleotide recombineering method for this strain based on GP35, a ssDNA recombinase originally encoded by a Bacillus subtilis-associated phage. GP35-mediated oligo recombineering is able to carry out point mutations in the M. pneumoniae genome with an efficiency as high as 2.7 × 10-2, outperforming oligo recombineering protocols developed for other bacteria. Gene deletions of different sizes showed a decreasing power trend between efficiency and the scale of the attempted edition. However, the editing rates for all modifications increased when CRISPR/Cas9 was used to counterselect nonedited cells. This allowed edited clones carrying chromosomal deletions of up to 1.8 kb to be recovered with little to no screening of survivor cells. We envision this technology as a major step toward the use of M. pneumoniae, and possibly other Mycoplasmas, as synthetic biology chassis strains. ; This project has received funding from the European Union's Horizon 2020 research and innovation program under Grant 634942 (MycoSynVac) and was also financed by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program under Grant 670216 (MYCOCHASSIS) and the FEDER project from Instituto Carlos III (ISCIII, Acción Estratégica en Salud 2016) (reference CP16/00094). We also acknowledge support of the Spanish Ministry of Science and Innovation, to the EMBL partnership, the Centro de Excelencia Severo Ochoa, and the CERCA Programme/Generalitat de Catalunya. Finally, we would like to thank Dr. Sarah A. Head for critical manuscript revision.

Aggregates of Pseudomonas aeruginosa form a protective barrier against antibiotics and the immune system. These barriers, known as biofilms, are associated with several infectious diseases. One of the main components of these biofilms is alginate, a homo- and hetero-polysaccharide that consists of β-D-mannuronate (M) and α-L-guluronate (G) units. Alginate lyases degrade this sugar and have been proposed as biotherapeutic agents to dissolve P. aeruginosa biofilms. However, there are contradictory reports in the literature regarding the efficacy of alginate lyases against biofilms and their synergistic effect with antibiotics. We found that most positive reports used a commercial crude extract from Flavobacterium multivorum as the alginate lyase source. By using anion exchange chromatography coupled to nano LC MS/MS, we identified two distinct enzymes in this extract, one has both polyM and polyG (polyM/G) degradation activities and it is similar in sequence to a broad-spectrum alginate lyase from Flavobacterium sp. S20 (Alg2A). The other enzyme has only polyG activity and it is similar in sequence to AlyA1 from Zobellia galactanivorans. By characterizing both of these enzymes together with three recombinant alginate lyases (a polyM, a polyG and a polyM/G), we showed that only enzymes with polyM/G activity such as Alg2A and A1-II' (alginate lyase from Sphingomonas sp.) are effective in dissolving biofilms. Furthermore, both activities are required to have a synergistic effect with antibiotics. ; We acknowledge support of the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program under agreement No 670216 (MYCOCHASSIS), the Spanish Ministry of Economy and Competitiveness and Fondo Europeo de Desarrollo Regional (MINECO-FEDER) (BIO2015-63557-R), 'Centro de Excelencia Severo Ochoa 2013-2017', FEDER project from Instituto Carlos III (ISCIII, Acción Estratégica en Salud 2016) (reference CP16/00094) and "Secretaria d'Universitats i Recerca del Departament d'Economia i Coneixement de la Generalitat de Catalunya / CERCA programme" (2014SGR678 and 2017SGR1079). The CRG/UPF Proteomics Unit is part of the "Plataforma de Recursos Biomoleculares y Bioinformáticos (ProteoRed)" supported by grant PT13/0001 of Instituto de Salud Carlos III from the Spanish Government.

Genome engineering of microorganisms has become a standard in microbial biotechnologies. Several efficient tools are available for the genetic manipulation of model bacteria such as Escherichia coli and Bacillus subtilis, or the yeast Saccharomyces cerevisiae. Difficulties arise when transferring these tools to nonmodel organisms. Synthetic biology strategies relying on genome transplantation (GT) aim at using yeast cells for engineering bacterial genomes cloned as artificial chromosomes. However, these strategies remain unsuccessful for many bacteria, including Mycoplasma pneumoniae (MPN), a human pathogen infecting the respiratory tract that has been extensively studied as a model for systems biology of simple unicellular organisms. Here, we have designed a novel strategy for genome engineering based on the recombinase-assisted genomic engineering (RAGE) technology for editing the MPN genome. Using this strategy, we have introduced a 15 kbp fragment at a specific locus of the MPN genome and replaced 38 kbp from its genome by engineered versions modified either in yeast or in E. coli. A strain harboring a synthetic version of this fragment cleared of 13 nonessential genes could also be built and propagated in vitro. These strains were depleted of known virulence factors aiming at creating an avirulent chassis for SynBio applications. Such a chassis and technology are a step forward to build vaccines or deliver therapeutic compounds in the lungs to prevent or cure respiratory diseases in humans. ; The authors thank Clara Blanchard for English proofreading. We also thank Dr. Don L. Court for providing biological material and advice. We acknowledge the financial support by the European Union's Horizon 2020 research and innovation program under Grant Agreement [No. 634942]; and the European MiniCell project selected by ANR, in the frame of the ERASynBio second Joint Call for Transnational Research Projects [No. ANR-15-SYNB-0001-04].

MyMpn (http://mympn.crg.eu) is an online resource devoted to studying the human pathogen Mycoplasma pneumoniae, a minimal bacterium causing lower respiratory tract infections. Due to its small size, its ability to grow in vitro, and the amount of data produced over the past decades, M. pneumoniae is an interesting model organisms for the development of systems biology approaches for unicellular organisms. Our database hosts a wealth of omics-scale datasets generated by hundreds of experimental and computational analyses. These include data obtained from gene expression profiling experiments, gene essentiality studies, protein abundance profiling, protein complex analysis, metabolic reactions and network modeling, cell growth experiments, comparative genomics and 3D tomography. In addition, the intuitive web interface provides access to several visualization and analysis tools as well as to different data search options. The availability and--even more relevant--the accessibility of properly structured and organized data are of up-most importance when aiming to understand the biology of an organism on a global scale. Therefore, MyMpn constitutes a unique and valuable new resource for the large systems biology and microbiology community. ; European Union through the European Research Council (ERC) ['Celldoctor' GA 232913]; Spanish Ministerio de Economía y Competitividad Plan Nacional [BIO2007-61762]; Fundaci´on Marcelino Botin [to L.S.]. laCaixa-CRG Fellowship, Fundació laCaixa [to J.W.]; Spanish Ministry of Economy and Competitiveness No [PTA2010-4446-I to A.H.; PTA2011-6729-I to L.C.]. Funding for open access charge: ERC [to L.S.].

A common problem encountered when performing large-scale MS proteome analysis is the loss of information due to the high percentage of unassigned spectra. To determine the causes behind this loss we have analyzed the proteome of one of the smallest living bacteria that can be grown axenically, Mycoplasma pneumoniae (729 ORFs). The proteome of M. pneumoniae cells, grown in defined media, was analyzed by MS. An initial search with both Mascot and a species-specific NCBInr database with common contaminants (NCBImpn), resulted in around 79% of the acquired spectra not having an assignment. The percentage of non-assigned spectra was reduced to 27% after re-analysis of the data with the PEAKS software, thereby increasing the proteome coverage of M. pneumoniae from the initial 60% to over 76%. Nonetheless, 33 413 spectra with assigned amino acid sequences could not be mapped to any NCBInr database protein sequence. Approximately, 1% of these unassigned peptides corresponded to PTMs and 4% to M. pneumoniae protein variants (deamidation and translation inaccuracies). The most abundant peptide sequence variants (Phe-Tyr and Ala-Ser) could be explained by alterations in the editing capacity of the corresponding tRNA synthases. About another 1% of the peptides not associated to any protein had repetitions of the same aromatic/hydrophobic amino acid at the N-terminus, or had Arg/Lys at the C-terminus. Thus, in a model system, we have maximized the number of assigned spectra to 73% (51 453 out of the 70 040 initial acquired spectra). ; This work was sup-ported by the European Research Council (ERC), the Fundación Marcelino Botín, the Spanish Ministerio de Economía y Com-petitividad BIO2007-61762 and the ISCIII (PI10/01702). TheCRG/UPF Proteomics Unit is part of the "Plataforma de Re-cursos Biomoleculares y Bioinform´aticos (ProteoRed)" supportedby grant PT13/0001 of Instituto de Salud Carlos III (ISCIII).We acknowledge the support of the Spanish Ministry of Economyand Competitiveness, 'Centro de Excelencia Severo Ochoa 2013–2017', SEV-2012-0208. This project has received funding fromthe European Union's Horizon 2020 research and innovationprogram under grant agreement No 634942

Over the past decade, a new strategy was developed to bypass the difficulties to genetically engineer some microbial species by transferring (or "cloning") their genome into another organism that is amenable to efficient genetic modifications and therefore acts as a living workbench. As such, the yeast Saccharomyces cerevisiae has been used to clone and engineer genomes from viruses, bacteria, and algae. The cloning step requires the insertion of yeast genetic elements in the genome of interest, in order to drive its replication and maintenance as an artificial chromosome in the host cell. Current methods used to introduce these genetic elements are still unsatisfactory, due either to their random nature (transposon) or the requirement for unique restriction sites at specific positions (TAR cloning). Here we describe the CReasPy-cloning, a new method that combines both the ability of Cas9 to cleave DNA at a user-specified locus and the yeast's highly efficient homologous recombination to simultaneously clone and engineer a bacterial chromosome in yeast. Using the 0.816 Mbp genome of Mycoplasma pneumoniae as a proof of concept, we demonstrate that our method can be used to introduce the yeast genetic element at any location in the bacterial chromosome while simultaneously deleting various genes or group of genes. We also show that CReasPy-cloning can be used to edit up to three independent genomic loci at the same time with an efficiency high enough to warrant the screening of a small (<50) number of clones, allowing for significantly shortened genome engineering cycle times. ; This work is part of the European MiniCell project "A model-driven approach to minimal cell engineering for medical therapy" selected by ANR, in the frame of the ERASynBio second Joint Call for Transnational Research Projects (No. ANR-15-SYNB-0001-04). It has also been supported by the National Science Foundation (Grant No. IOS-1110151) and the European Union's Horizon 2020 research and innovation program under Grant Agreement No. 634942.