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Construction d’un châssis bactérien viable, minimal et non pathogène grâce aux outils de biologie de synthèse

Abstract : A goal of synthetic biology is to create and produce “custom” organisms, for therapeutic and industrial applications. One of the contemplated approaches to achieve this goal is based on synthesis techniques and transplantation of whole genomes, in order to create mutant organisms.The aim of this thesis is to develop synthetic biology tools that will enable the construction of a minimal and non-pathogenic cell based on Mycoplasma pneumoniae. This bacterium is one of the smallest living organisms, with a size smaller than one micron and a genome of 816 kbp. This mycoplasma is one of the most studied, with a large set of genetic and multi- “omics” data available. These characteristics make this naturally “almost minimal” cell an ideal starting point for the construction of a bacterial chassis. Nevertheless, the genetic manipulation of this mycoplasma is difficult, due to the limited number of available tools.A recently developed approach offers the possibility to circumvent these limitations by using the yeast Saccharomyces cerevisiae as a genome engineering platform for M. pneumoniae. The preliminary step to this strategy is to clone the bacterial genome in yeast. To do so, a "yeast elements" cassette is inserted into the genome of M. pneumoniae, to allow its maintenance as an artificial chromosome. The work carried out during this thesis allowed us to insert this cassette through a transposon, and to clone this marked genome in yeast. Then, the stability of the cloned genome was studied, demonstrating that the bacterial chromosome is maintained during ten passages. We then developed a new strategy for the insertion of the "yeast elements", using the CRISPR/Cas9 system to simultaneously clone and edit a mycoplasma genome in yeast: the CReasPy-Cloning. This method was used to remove three different loci containing genes involved in virulence: MPN372 (CARDS toxin), MPN142 (cytoadherence protein) and MPN400 (IgG blocking protein). This method was also used to target two and then three different loci in one step.Once in-yeast cloning and bacterial genome engineering is achieved, it is necessary to transfer the modified chromosome into a recipient cell, to produce a mutant organism. This process, called genome transplantation, is not described for M. pneumoniae, so a significant part of this thesis was dedicated to the development of this tool. We used plasmid transformation as a model mechanism to study the process of DNA entry into M. pneumoniae and to test the use of polyethylene glycol, the key reagent for transplantation. Although we succeeded in developing a plasmid transformation protocol, we have not yet been able to perform genome transplantation.Concurrently, we have developed an alternative strategy for genome editing that does not depend on transplantation. This approach, named "Genomic Transfer - Recombinase-Mediated Cassette Exchange" (GT-RMCE), is used to capture in a vector a section of the edited bacterial genome borne by the yeast. This vector is then transformed into M. pneumoniae, and through to the Cre-lox system the edited section is introduced into the genome. This mechanism allows to carry out large-scale modifications, and is currently used to introduce into M. pneumoniae the ΔMPN372, ΔMPN400 and ΔMPN372-ΔMPN400 deletions produced by CReasPy-cloning. We also used the GT-RMCE to generate a strain of M. pneumoniae carrying two copies of the S10 ribosomal operon.Overall, the M. pneumoniae genome engineering tools developed during this thesis constitute a significant step towards the construction of new bacterial chassis.
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Estelle Ruiz. Construction d’un châssis bactérien viable, minimal et non pathogène grâce aux outils de biologie de synthèse. Génétique. Université de Bordeaux, 2019. Français. ⟨NNT : 2019BORD0132⟩. ⟨tel-02454257⟩

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