Génie génétique | EPFL Graph Search

Le génie génétique est l'ensemble des outils permettant de modifier la constitution génétique d'un organisme en supprimant, en introduisant ou en remplaçant de l'ADN. Celui-ci peut être introduit directement dans les cellules de l'organisme hôte ou dans des cellules cultivées ex vivo puis réintroduites dans l'organisme. Un prérequis au développement du génie génétique a été la mise au point de techniques recombinantes d'acide nucléique pour former de nouvelles combinaisons de matériel génétique héritable suivies de l'incorporation de ce matériel soit indirectement à travers un système vecteur ou directement par microinjection, macroinjection ou microencapsulation. Il a souvent pour but la modification des génotypes, et donc des phénotypes. Le génie génétique est un champ très actif de la recherche car les applications possibles sont multiples, notamment en santé humaine (correction d’un gène porteur d’une mutation délétère, production de protéines thérapeutiques, élimination de séqu

Identification of genetic interactors of ypa2 in Schizosaccharomyces pombe

Manuela Moraru

In this study, the fission yeast Schizosaccharomyces pombe was used as a model system to study the cellular signaling that underlies cell cycle progression. Phosphorylation plays an important part in the regulation of this process. Kinases catalyze the transfer of a phosphate group to a substrate, which may regulate its activity. Protein phosphatases oppose the activity of protein kinases; the balance of their activities regulates signaling flux in many signaling pathways. The Septation Initiation Network (SIN) is a signaling cascade that regulates cytokinesis in fission yeast. SIN signaling is triggered by a GTPase and is transduced by three protein kinases; phosphorylation plays an important role in controlling SIN signalling. Numerous genetic screens and biochemical analyses have identified phosphatases and kinases that regulate the SIN. The protein phosphatases Calcineurin/Protein Phosphatase 2B (PP2B) and Flp1p promote SIN signaling, while PP2A is a SIN inhibitor. PP2A is conserved and is activated by the chaperone Phosphatase Two A Phosphatase Activator (PTPA). S. pombe has two PTPA-related genes, ypa1 and ypa2; the deletion mutant of the latter rescues SIN mutants which cannot undergo cytokinesis, consistent with PP2A opposing SIN signaling. The phenotype of the deletion mutant of ypa2, indicates that it is involved in the control of cell polarity, cell growth, mitotic commitment and cytokinesis. In this study, we characterized the hypomorphic allele ypa2-S2 which rescues SIN mutants, but is otherwise largely normal. ypa2-s2 was used as bait in a synthetic genetic array screen, to identify genes whose products compensate for reduced function of Ypa2p. A large number of hits were identified, of which approximately ninety percent are novel genetic interactors of ypa2. Validation studies indicated that eighty percent of the interactions seen in the high-throughput screen can be reconstructed. The identified genes regulate several biological processes, including signal transduction and protein phosphorylation. This prompted us to further characterize the roles of Ckb1p and Flp1p, in regulating cytokinesis signaling. Ckb1p is a regulatory subunit for Casein Kinase II (CK II), which was previously implicated in polar growth. The phenotype of the mutant ypa2-S2 ckb1-â indicates that Ckb1p, similar to Ypa2p, contributes to the regulation of mitotic commitment and cytokinesis signaling. Furthermore, ckb1-â showed negative genetic interaction with PP2A and SIN mutants. Flp1p is a protein phosphatase that was reported to regulate mitotic commitment and to promote SIN signaling. The double mutant between ypa2-S2 and flp1-â showed cell separation defects and phenotypic analysis of double mutants between flp1-â and PP2A mutants indicates that these phosphatases cooperate in cell separation. Overall, this work has uncovered novel facets of the regulation of cytokinesis in S. pombe, implicating CK II in controlling SIN signaling, and uncovering cooperative interactions between different phosphatases in controlling cytokinesis.

EPFL2016

Engineering Agrobacterium tumefaciens Adhesion to Target Cells

Zaïnebe Al-Mayyah, Alexandre Louis André Persat, Alix Kim Lan Pham, Xavier Jean-Yves Pierrat, Jeremy Pak Hei Wong

Agrobacterium tumefaciens is a plant pathogen commonly repurposed for genetic modification of crops. Despite its versatility, it remains inefficient at transferring DNA to many hosts, including to animal cells. Like many pathogens, physical contact between A. tumefaciens and host cells promotes infection efficacy. Thus, improving the strength and specificity of A. tumefaciens to target cells has the potential for enhancing DNA transfer for biotechnological and therapeutic purposes. Here, we demonstrate a methodology for engineering genetically encoded exogeneous adhesins at the surface of A. tumefaciens. We identified an autotransporter gene we named Aat that is predicted to show canonical beta-barrel and passenger domains. We engineered the beta-barrel scaffold and linker (Aat(beta)) to display synthetic adhesins susceptible to rewire A. tumefaciens to alternative host targets. As a proof of concept, we leveraged the versatility of a VHH domain to rewire A. tumefaciens adhesion to yeast and mammalian hosts displaying a GFP target receptor. Finally, to demonstrate how synthetic A. tumefaciens adhesion can improve transfer to host cells, we showed improved protein translocation into HeLa cells using a sensitive split luciferase reporter system. Engineering A. tumefaciens adhesion has therefore a strong potential in generating complex heterogeneous cellular assemblies and in improving DNA transfer efficiency against non-natural hosts.

AMER CHEMICAL SOC2022

Probing the growth and mechanical properties of Bacillus subtilis biofilms through genetic mutation strategies

Lei Wang, Chen Zhang

Bacterial communities form biofilms on various surfaces by synthesizing a cohesive and protective extracellular matrix, and these biofilms protect microorganisms against harsh environmental conditions. Bacillus subtilis is a widely used experimental species, and its biofilms are used as representative models of beneficial biofilms. Specifically, B. subtilis biofilms are known to be rich in extracellular polymeric substances (EPS) and other biopolymers such as DNA and proteins like the amyloid protein TasA and the hydrophobic protein BslA. These materials, which form an interconnected, cohesive, three-dimensional polymer network, provide the mechanical stability of biofilms and mediate their adherence to surfaces among other functional contributions. Here, we explored how genetically-encoded components specifically contribute to regulate the growth status, mechanical properties, and antibiotic resistance of B. subtilis biofilms, thereby establishing a solid empirical basis for understanding how various genetic engineering efforts are likely to affect the structure and function of biofilms. We noted discrete contributions to biofilm morphology, mechanical properties, and survival from major biofilm components such as EPS, TasA and BslA. For example, EPS plays an important role in maintaining the stability of the mechanical properties and the antibiotic resistance of biofilms, whereas BslA has a significant impact on the resolution that can be obtained for printing applications. This work provides a deeper understanding of the internal interactions of biofilm components through systematic genetic manipulations. It thus not only broadens the application prospects of beneficial biofilms, but also serves as the basis of future strategies for targeting and effectively removing harmful biofilms.

KEAI PUBLISHING LTD2022

Identification of genetic interactors of ypa2 in Schizosaccharomyces pombe

Manuela Moraru

EPFL2016