La production hétérologue des protéines membranaires reste difficile, peut-être parce que l’insertion dans la membrane de la cellule hôte constitue une étape limitante de la production. Afin de tourner cette difficulté, deux modes de synthèse ont été envisagés: la synthèse de protéines dans un système a-cellulaire, en l’absence de membrane mais en présence de détergent, ou l’adressage forcé de la protéine vers les corps d’inclusion dans le cas d’une expression plus classique en bactérie entière. La réalisation des deux stratégies repose sur l’utilisation de protéines de fusion possédant une séquence d’entraînement en amont du gène d’intérêt, soit qu’elles améliorent la traduction du transcrit en limitant le repliement spatial de ce dernier, soit qu’elles favorisent la production de la protéine d’intérêt en corps d’inclusion. La porine OmpX et le peptide T7 ont été choisis en cas d’expression dans les systèmes bactériens. La protéine SUMO est utilisée pour la production dans un lysat eucaryote. Les différentes approches ont été testées sur la production de la pannexine1 humaine (Px1).Si les séquences d’entraînement OmpX et le peptide T7 sont correctement produites in vitro, aucune des deux, en revanche, ne favorise la production de la Px1. Seul l’entraîneur SUMO est efficace. En effet, nous avons observé que cette protéine augmente la production de la Px1 dans un lysat eucaryote de germe de blé. Par ailleurs OmpX, connue pour être largement produite in vivo dans les corps d’inclusion, n’entraîne pas la localisation de la Px1 dans ces structures. Contre toute attente, l’étiquette T7 dirige la Px1 dans les corps d’inclusion. L’étude électrophysiologique de la Px1 a donc été effectuée à partir de la protéine produite in vivo (T7his-Px1) après renaturation, ou produite sous forme soluble in vitro (his6-Px1) dans le lysat eucaryote. Dans le cas de la protéine T7his-Px1 renaturée, une activité canal qui rappelle celle qui est observée après expression dans l’ovocyte de Xénope, a été détectée en patch-clamp, mais dans trois cas seulement. Dans le cas de la protéine his6-Px1, aucune activité canal n’est clairement détectée. Dans une deuxième partie de ce travail on examine le rôle de la boucle périplasmique dans la sensibilité à la pression du MscL, un canal mécanosensible bactérien devenu un système modèle dans l’étude de la mécanosensibilité. Presque toutes les études fonctionnelles sur ce canal ont été réalisées sur le canal de E.coli, alors que la structure a été obtenue à partir de l’homologue de M. tuberculosis. Une étude fonctionnelle a montré que le MscL de M. tuberculosis est difficile à ouvrir : son ouverture requiert l’application d’une pression double de celle qui est nécessaire chez E.coli. Les deux homologues diffèrent principalement par la longueur de leur unique boucle périplasmique. De manière à examiner le rôle de la boucle, on a comparé l’activité du canal MscL de E.coli, celle du canal de M. tuberculosis et celle d’une protéine chimère constituée de la protéine de M. tuberculosis dans laquelle la boucle a été changée pour celle de la protéine de E.coli. De manière inattendue, nous avons constaté que les canaux de E.coli et de M. tuberculosis ont la même sensibilité à la pression. La protéine chimère n’avait pas d’activité canal. Si ce travail ne permet pas de conclure quant au rôle de la boucle, il montre sans ambigüité que contrairement à ce qui a été rapporté les canaux MscL de E.coli et de M. tuberculosis ne diffèrent pas sensiblement sur le plan fonctionnel / The production of heterologous membrane protein is notoriously difficult; this might be due to the fact that insertion of the protein in the membrane host is a limiting step. To by-pass this difficulty, two modes of synthesis were tested: 1) production in a cell-free system devoid of biological membrane but supplemented with detergent or liposomes, 2) production in bacteria, with targeting of the membrane protein to inclusion bodies. Both strategies were tested for the production of the human pannexin 1 channel (Px1). The gene coding the protein was fused with an “enhancer” sequence resulting in the addition of a peptide or short protein at the N terminus of the protein of interest. This enhancer sequence which is well produced in vitro or in vivo is supposed to facilitate the translation of the protein of interest. Three enhancer sequences were chosen: 1) the small porin OmpX of E. coli, which, in addition, should target the protein to inclusion bodies when the protein is expressed in bacteria 2) a peptide of phage T7 for expression in E.coli lysate or E.coli cells 3) the small protein SUMO for production in a wheat germ cell-free system. In a bacterial cell-free system, neither OmpX nor T7 promoted Px1 production. Px1 is only produced when the SUMO enhancer sequence is used in the wheat germ system. In bacteria, OmpX, known to form inclusions bodies did not promote the targeting of the fusion protein to inclusion bodies. Unexpectedly, the peptide T7 was able to do it.Px1 obtained from inclusion bodies (T7his-Px1) was renatured and reconstituted in liposomes. Similarly his6-Px1 produced in wheat germ system was reconstituted in liposomes. Both preparations were used for electrophysiological studies (patch-clamp and planar bilayers). With the refolded T7his-Px1, channel activity reminiscent of that observed with Px1 expressed in Xenope oocyte (Bao et al., 2004) could be detected, but only in three cases. In the case of his6-Px1, no clear channel activity could be observed. The second part of this work deals with the involvement of the periplasmic loop of the bacterial mechanosensitive channel MscL in its sensitivity to pressure. Mscl has become a model system for the investigation of mechanosensisity. Nearly all functional studies have been performed on MscL from E.coli while the structure of the protein has been obtained from the Mycobacterium tuberculosis homologue. In one functional study it was shown that MscL from M. tuberculosis is extremely difficult to open, gating at twice the pressure needed for E.coli MscL The periplasmic loop is the most variable sequence between the two homologues, being longer in E.coli than in M. tuberculosis. In order to assess the role of the periplamic loop in the sensitivity to pressure, we compared the activity of the E.coli and M. tuberculosis MscL and of a chimeric protein made of the M. tuberculosis protein in which the periplasmic loop has been exchanged for that of the E. coli channel. Unexpectedly, M. tuberculosis and E .coli MscL were observed to gate at a similar applied pressure. The chimeric protein had no functional activity. In conclusion, this study does not allow any conclusion as to the role of the loop in the sensitivity to pressure, but it shows clearly that, in contrast to the results of a previous study, there is no functional difference between E. coli and M. tuberculosis MscL.

Mounting concerns about drug-resistant pathogenic bacteria have rekindled the interest in bacteriophages (bacterial viruses). As bacteria’s natural predators, bacteriophages offer a critical advantage over antibiotics, namely that they can be highly specific. This means that phage therapeutics can be designed to destroy only the infectious agent(s), without causing any harm to our microbiota. However, the potential secondary effects on the balance of microbiota through bacteriophage-induced genome evolution remains as one of the critical apprehensions regarding phage therapy. There exists a significant gap in knowledge regarding the direct and indirect effect of phage therapeutics on the microbiota. The aim of this thesis was to: (1) establish an in vivo model for investigation of the evolutionary dynamics and co-evolution of therapeutic phage and its corresponding host bacterium in the gut; (2) determine if phage therapy can affect the composition of the gut microbiota, (3) observe the differences of phage-resistant bacteria mutants evolved in vivo in comparison to those evolved in vitro. We used germ-free mice colonized with a consortium of eight known bacteria, known as the altered Schaedler flora (ASF). The colonizing strain of choice (mock infection) was a non-pathogenic strain E. coli K-12 (JM83) known to co-colonize the ASF model, which was challenged in vivo with T7 phage (strictly lytic). We compared the composition of the gut microbiota with that of mice not subject to phage therapy. Furthermore, the resistant mutants evolved in vivo and in vitro were characterized in terms of growth fitness and motility. / Thesis / Master of Applied Science (MASc) / Bacteriophages are viruses that infect bacteria. After their discovery in 1917, bacteriophages were a primary cure against infectious disease for 25 years, before being completely overshadowed by antibiotics. With the rise of antibiotic resistance, bacteriophages are being explored again for their antibacterial activity. One of the critical apprehensions regarding bacteriophage therapy is the possible perturbations to our microbiota. We set out to explore this concern using a simplified microbiome model, namely germ-free mice inoculated with only 8 bacteria plus a mock infection challenged with bacteriophage. We monitored this model for 9 weeks and isolated a collection of phage-resistant bacterial mutants from the mouse gut that developed post phage challenge, maintaining the community of mock infection inside the gut. A single dose of lytic phage challenge effectively decreased the mock infection without causing any extreme long-term perturbations to the gut microbiota.

The development of shape in multicellular organisms has intrigued human minds for millenia. Empowered by modern genetic techniques, molecular biologists are now striving to not only dissect developmental processes, but to exploit their modularity for the design of custom living systems used in bioproduction, remediation, and regenerative medicine. Currently, our capacity to harness this potential is fundamentally limited by a lack of spatiotemporal control over gene expression in multicellular systems. While several synthetic genetic circuits for control of multicellular patterning have been reported, hierarchical induction of gene expression domains has received little attention from synthetic biologists, despite its fundamental role in biological self-organization. In this thesis, I introduce the first synthetic genetic system implementing population-based AND logic for programmed hierarchical patterning of bacterial populations of Escherichia coli, and address fundamental prerequisites for implementation of an analogous genetic circuit into the emergent multicellular plant model Marchantia polymorpha. In both model systems, I explore the use of bacteriophage T7 RNA polymerase as a gene expression engine to control synthetic patterning across populations of cells. In E. coli, I developed a ratiometric assay of bacteriophage T7 RNA polymerase activity, which I used to systematically characterize different intact and split enzyme variants. I utilized the best-performing variant to build a three-color patterning system responsive to two different homoserine lactones. I validated the AND gate-like behavior of this system both in cell suspension and in surface culture. Then, I used the synthetic circuit in a membrane-based spatial assay to demonstrate programmed hierarchical patterning of gene expression across bacterial populations. To prepare the adaption of bacteriophage T7 RNA polymerase-driven synthetic patterning from the prokaryote E. coli to the eukaryote M. polymorpha, I developed a toolbox of genetic elements for spatial gene expression control in the liverwort: I analyzed codon usage across the transcriptome of M. polymorpha, and used insights gained to design codon-optimized fluorescent reporters successfully expressed from its nuclear and chloroplast genomes. For targeting of bacteriophage T7 RNA polymerase to these cellular compartments, I functionally validated nuclear localization signals and chloroplast transit peptides. For spatiotemporal control of bacteriophage T7 RNA polymerase in M. polymorpha, I characterized spatially restricted and inducible promoters. For facilitated posttranscriptional processing of target transcripts, I functionally validated viral enhancer sequences in M. polymorpha. Taking advantage of this genetic toolbox, I introduced inducible nuclear-targeted bacteriophage T7 RNA polymerase into M. polymorpha. I showed implementation of the bacteriophage T7 RNA polymerase/PT7 expression system accompanied by hypermethylation of its target nuclear transgene. My observations suggest operation of efficient epigenetic gene silencing in M. polymorpha, and guide future efforts in chassis engineering of this multicellular plant model. Furthermore, my results encourage utilization of spatiotemporally controlled bacteriophage T7 RNA polymerase as a targeted silencing system for functional genomic studies and morphogenetic engineering in the liverwort. Taken together, the work presented enhances our capacity for spatiotemporal gene expression control in bacterial populations and plants, facilitating future efforts in synthetic morphogenesis for applications in synthetic biology and metabolic engineering.