local : FR-18008901306731-2021-04-12
Pseudomonas aeruginosa is a Gram-negative Human opportunistic pathogen, responsible for nosocomial infections with high mortality for immunocompromised patients. The pathogenesis is based on the production of many structural or secreted virulence factors. P. aeruginosa is also able to produce several resistance mechanisms to antibiotics, which allows its establishment in hospitals. This bacterial host cannot be used for biotechnological purposes as such, that's why we therefore undertook to remove from the genome of reference strain PAO1, the main genes associated with virulence and antibioresistance mechanisms. Thus, the mutant SM54 obtained, relieved of 37 genes, i.e. 0.8% of the genome, was characterized. Its became hypersusceptible to antibiotics, 53% less cytotoxic (in vitro results) and the mortality of individuals infected with SM54 is decreased in comparison with PAO1 (in vivo results). Then, this new chassis was used as platform to evaluate the recombinant protein production. We developed various expression plasmids carrying the inducible systems XylS/Pm and cmrA/Pcin, and the reporter genes luxCDABE and blaAmpC. The antibiotic susceptibility was determined and demonstrated the production of this homologous protein AmpC, in presence of the respective inducers of systems. With these vectors, we were subsequently able to detect by Western-blot the production of human cytokine IL-1β in P. aeruginosa, thus proving the R&D interest of pyocyanic bacillus for the bioproduction of recombinant proteins.
Data acquisition : from Dec 2017 to Dec 2020
Metadata record :
Creation : 12 Apr 2021
Update : 28 Jun 2021
Additional information :
Data collected as part of the thesis in Biochemistry and Molecular Biology, by Melanie Grosjean (Optimization of genes expression for biotechnological purposes in Pseudomonas aeruginosa), co-supervised by Prof. Patrick Plesiat, Professor at the University of Franche-Comté, and Cedric Muller, PhD, SMALTIS CEO.
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Access details :
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Data acquisition methods :
- Observational data :
Virulence assays, microscopic observations, antibiotic susceptibility tests
- Experimental data :
Determination of bacterial growth and dry weight, determination of antibiotic minimal inhibitory concentration, quantification of gene expression (RT-qPCR), in vivo (mouses, larvae) and in vitro (human cells and murine macrophages) toxicity assays, quantification of protein production (SDS-PAGE and Western blot).