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Conception et implémentation in vivo chez la bactérie E. coli de commutateurs ARN synthétiques

Abstract : This thesis work is a contribution to the general field of synthetic biology of RNA and aims at constructing and testing the functioning of RNA switches controlled by small anti-sense RNAs. The corresponding topic of fundamental research addresses the relationship between sequence, folding and function of an RNA molecule.We have devised RNA switches that adopt two alternative conformations. In one conformation, the RNA forms a transcriptional terminator, stopping the expression of a downstream gene. The alternative conformation undoes the terminator structure and allows read-through to the downstream DNA. The choice of which conformation the RNA molecule adopts depends on the presence or absence of a small regulatory anti-sense RNA. We have validated the functioning of our repressible and inducible switches in vitro. In order to validate the functioning of the switches in vivo, we have constructed and optimized a genetic testing system in the bacterium Escherichia coli. The performance of the switch is measured by the expression of a fluorescent reporter gene cloned downstream of the switch. We have thus been able to provide a proof of concept of the design, construction and in vivo functioning of activatable and repressible RNA switches.Our in vitro and in vivo experiments have allowed us to discover new principles of the design of synthetic RNA switches. We have also been able to optimize the expression conditions of the switches and the measurements methods of the in vivo experiments. The dynamic range of our RNA switches needs to be improved and we have discovered several promising avenues to accomplish this goal. More generally, our results form the basis of a more systematic exploration of the relationships between sequence, folding and functioning of RNA molecules.
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Submitted on : Sunday, October 24, 2021 - 1:02:41 AM
Last modification on : Wednesday, November 3, 2021 - 6:15:28 AM


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  • HAL Id : tel-03399389, version 1




Gabriel Joël Espel. Conception et implémentation in vivo chez la bactérie E. coli de commutateurs ARN synthétiques. Biologie structurale [q-bio.BM]. Université Grenoble Alpes [2020-..], 2020. Français. ⟨NNT : 2020GRALV051⟩. ⟨tel-03399389⟩



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