Academic literature on the topic 'Ingénierie des Génomes'
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Journal articles on the topic "Ingénierie des Génomes"
DUCOS, A., B. BED'HOM, H. ACLOQUE, and B. PAIN. "Modifications ciblées des génomes : apports et impacts pour les espèces d’élevage." INRA Productions Animales 30, no. 1 (June 14, 2018): 3–18. http://dx.doi.org/10.20870/productions-animales.2017.30.1.2226.
Full textCastagné, Paul, Armelle Guingand, Alexandra Moderc, and Sarah Monard. "Ingénierie du génome bactérien grâce à l’outil CRISPR/Cas12a." médecine/sciences 34, no. 5 (May 2018): 399–400. http://dx.doi.org/10.1051/medsci/20183405009.
Full textDucos, Alain, Bertrand Bed'Hom, Hervé Acloque, and Bertrand Pain. "Modifications ciblées des génomes : apports et impacts potentiels des nouvelles technologies pour les espèces aviaires." Bulletin de l'Académie vétérinaire de France, 2020. http://dx.doi.org/10.3406/bavf.2020.70900.
Full textDissertations / Theses on the topic "Ingénierie des Génomes"
Loubat, Arthur. "Caractérisation fonctionnelle du génome du bactériophage SPP1 de Bacillus subtilis par des approches de Biologie de Synthèse." Electronic Thesis or Diss., université Paris-Saclay, 2024. http://www.theses.fr/2024UPASL006.
Full textA strategy to foster innovation in biotechnology relies on constructing cellular chassis strains with genomes appropriately streamlined for the desired application. Streamlining genomes requires the development of efficient and robust genetic tools for genome engineering. While Gram-negative model bacteria have multiple genetic tools of phage origin, this is not the case for Gram-positive bacteria. This work aims to pioneers methods and techniques for investigating and manipulating bacteriophage genomes, with a particular focus on the SPP1 phage from Bacillus subtilis. SPP1 is one of the best-characterized lytic bacteriophages in the siphovirus family. However, numerous questions persist regarding the function and essentiality of its genes, as well as the processes of SPP1 transcription, replication, and encapsidation.Two complementary libraries of mutants have been constructed. The first one is a library of B. subtilis mutant strains, each carrying one or more phage genes integrated into the bacterial chromosome, with inducible expression. The toxicity of viral proteins to B. subtilis was tested for 82 mutants. Approximately 23% of the mutants displayed altered phenotypes due to the expression of phage genes. For instance, regarding genes of unknown function, the expression of gp29.1 led to a dose-dependent reduction in growth rate, while gp37.1-37.2 expression induced cell filamentation.The second library is composed of semi-synthetic SPP1 phages, each deleted for one or more essential and non-essential genes. A deletion method by in vitro assembly of SPP1 genome fragments followed by host cell transformation was developed. Each phage mutant was built and propagated in the corresponding B. subtilis mutant strain from the first collection to allow for trans-complementation of the phage mutation. The fitness of 36 mutants was characterized during B. subtilis infection, revealing that around 25% of phage genes were found to be essential or nearly essential for phage propagation. For instance, the mutant SPP1 Δgp22, involved in the tail assembly but of unknown function, exhibited significantly reduced capacity for multiplication.Lastly, an in vivo engineering method of genomes of phages from Gram-positive bacteria using CRISPR-Cas9 was developed and validated.These results have helped decipher some interactions between SPP1 and B. subtilis and will ultimately contribute to the design of new genetic engineering tools
Guesdon, Gabrielle. "Développement de méthodes de clonage de génomes entiers chez la levure pour la construction de souches châssis semi-synthétiques de Bacillus subtilis." Thesis, Bordeaux, 2022. http://www.theses.fr/2022BORD0204.
Full textOne of the major challenges in the synthetic biology (BS) field, is to provide new solutions to global issues (therapeutic/sanitary or climatic), in particular through the construction of useful, efficient and environmentally friendly production strains.The well-characterized, non-pathogenic, Gram+ bacterium Bacillus subtilis (Bsu), is widely used in industry as a biotechnological workhorse. Recent studies have established that mutant strains with modified genomes are able to produce larger amounts of recombinant proteins. This suggests that the production of rationally designed Bsu chassis could be an important step in the improvement of valuable strains for industrial purposes.This work was performed within the Bacillus 2.0's ANR project, which aims at applying SB tools for Bsu, and at developing an effective pipeline for the high-throughput construction of versatile Bsu chassis strains. Selected SB technologies for the pipeline include (i) the synthetic genome design, (ii) the in-yeast DNA assembly methods using Saccharomyces cerevisiae, (iii) the from-yeast whole genome isolation and transplantation (GT) to a recipient bacteria cell and, (iv) the characterization of recombinant strains.The objectives of this thesis were to ensure the feasibility of these methods using a Gram+ bacterium, by showing, in particular, that it was possible to clone and maintain in S. cerevisiae the genome of a minimal Bsu strain, MPG192 (2.86 Mbp) and to modify it using the large repertoire of yeast genetic tools. Our first attempts to clone the entire Bsu genome into yeast using already described methods failed. Using a TAR-Cloning approach, we then attempted to clone large DNA fragments obtained by restriction of the Bsu genome. In a first experiment, five out of seven fragments were cloned. Difficulties to clone the largest fragment (1.50 Mbp), are presumably related to its size, and/or the lack of ARS elements. Concerning the other fragment, several factors have been proposed to explain the cloning failure: again, an insufficient number of ARS elements, but also, the presence of many repeated sequences (7 ribosomal operons), and/or the deleterious expression of these genes. Finally with other experiments, the whole 2.86 Mb genome was cloned in 21 pieces ranging from 6 kbp to 515 kbp. As TAR-Cloning imposes constraints in the choice of restriction sites, a new cloning method, called CReasPy-Fusion, was developed. This method allows the simultaneous cloning and engineering of mega-sized genome in yeast using the CRISPR-Cas9 system, after direct bacterial cell to yeast spheroplast cell fusion. As a proof of concept, we demonstrated that the method can be used to capture a piece of genome, or to clone and edit the whole genome from six different Mycoplasma species. This method was then adapted to Bsu, showing for the first-time yeast spheroplast and Gram+ protoplast cell fusion. A fragment of ~150 kb has been successfully cloned in yeast.Even if, the entire Bsu genome has not yet been cloned in yeast, several critical elements have been identified. First of all, this work underlines the importance of the cloning method to be adopted depending on the organism of interest. Then, it emphasizes the existence of both biological and technical factors that explain current difficulties and that will have to be taken into account in subsequent experiments. Finally, it enabled the development of the new in-yeast cloning method called CReasPy-Fusion which expands the catalog of technics already described. Through its versatility, it opens up prospects for the capture of large genome fragments, the suppression of problematic loci, and to support the assembly of synthetic fragments
Ruiz, Estelle. "Construction d’un châssis bactérien viable, minimal et non pathogène grâce aux outils de biologie de synthèse." Thesis, Bordeaux, 2019. http://www.theses.fr/2019BORD0132/document.
Full textA 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
Tsarmpopoulos, Iason. "Ingénierie de génome de bactéries minimales par des outils CRISPR/Cas9." Thesis, Bordeaux, 2017. http://www.theses.fr/2017BORD0787/document.
Full textMycoplasmas are small pathogenic bacteria that are characterized by reduced genomes of about 1 Mbp with a low G+C content. The interest of the scientific community towards these species has been recently renewed by successful synthesis of their genome and transplantation experiments. These new genetic tools opened the way to further applications and developments for large-scale genome engineering programmes. CRISPR/Cas systems are natural systems that provide bacteria and archaea with an adaptive defense mechanism against invading nucleic acids. The CRISPR system from Streptococcus pyogenes includes an endonuclease (SpCas9) and two CRISPR RNAs (crRNA et tracrRNA) which role are to drive Cas9 to a target sequence. Target recognition depends on a specific pairing of the crRNA and the presence of a motif named protospacer adjacent motif (PAM). After recognition, Cas9 cleaves the targeted DNA. From the natural S. pyogenes system, a simplified genetic tool including Cas9 and a guide RNA (gRNA) was developed for many organisms . The first goal of my thesis was to combine the synthetic biology methods of genome cloning in yeast and back transplantation into recipient cells with a CRISPR/Cas9 tool for efficient engineering of mycoplasma genomes cloned in yeast. We succeeded in removing genes and genomic regions in three different species, Mycoplasma mycoides subsp. capri (Mmc), M. capricolum subsp. capricolum and M. pneumoniae. Then, in order to develop a system optimized for mycoplasma genome editing, we characterized a natural CRISPR/Cas9 system derived from Mycoplasma gallisepticum (Mg). Using a combination of in silico and in vivo approaches, MgCas9 PAM sequence was characterized as NNNAAAA. We then started to develop a minimal CRISPR/Cas system from M. gallisepticum for direct genome editing in mollicutes. Thus we introduced MgCas9 encoding gene in Mmc and tried to activate it with a newly designed gRNA, a chimeric molecule between the crRNA and the tracrRNA of M. gallisepticum, without success yet
Zaworski, Julie. "Deinococcus geothermalis genome scale structure study to design and engineer heterologous metabolic pathways." Thesis, Université Paris-Saclay (ComUE), 2017. http://www.theses.fr/2017SACLE031.
Full textDeinococcus geothermalis is a non-model organism of high interest for bio-manufacturing since it shows a extreme resistance and good capacities for fermentation process on different carbon sources. However the engineering tools are limited to finely tuned metabolic pathways for bio-productions. This PhD work aims at contributing to overcome this obstacle through a whole-genome approach to the issue of understanding the genomic organization of D. geothermalis and defined interesting genomic locations. The whole-genome approach is based on the existence of genome-scale patterns that were analyzed in two different ways. A first approach consisted of studying the influence of the genome location on the expression of a reporter cassette. On a library of over 150 strains, the expression is higher near the origin of replication than near the terminus, a common observation. However, other hot spots of expression along the genome additionally appeared with a symmetric distribution about the origin of replication. The second approach consisted of analyzing the genomic patterns under stress through the in-house GREAT:SCAN:patterns software. These patterns interrelate with gene expression regulation and are an interesting key for genome engineering. Testing different stress conditions and considering the matching regulons as described in the literature, it appeared that related stresses share genomic patterns. Moreover these patterns tend to be conserved between distant organisms. These two approaches lead to define interesting genome loci for inserting genes encoding the enzymes of a pathway, with a view to metabolic engineering