Abstract
Studying bacterial physiology and pathogenesis often requires isolation of targeted mutants. From the early days of bacterial genetics, many genetic tools have been developed to achieve this goal in a lot of bacteria species, and a major key is to be able to manipulate the targeted genome region with a minimum impact on the rest of the genome. Here, we described a two-step protocol relevant in Legionella pneumophila. This efficient two-step protocol uses the natural transformability of L. pneumophila and linear DNA fragments as substrates for recombination without the necessity of intermediate hosts to amplify targeted DNA. Based on a suicide cassette strategy, this genetic toolbox enables to generate clean scar-free deletions, single-nucleotide mutation, transcriptional or translational fusions, as well as insertion at any chosen place in L. pneumophila chromosome, therefore enabling multiple mutations with no need of multiple selection markers.
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Acknowledgments
This work was supported by CNRS (Centre National de la Recherche Scientifique, UMR5308), INSERM (Institut National de la Recherche Medicale; U1111), and University Claude Bernard Lyon1. This work was performed within the framework of the LABEX ECOFECT (ANR-11-LABX-0042) of Université de Lyon, within the program “Investissements d’Avenir” (ANR-11-IDEX-0007) operated by the French National Research Agency (ANR).
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Bailo, N., Kanaan, H., Kay, E., Charpentier, X., Doublet, P., Gilbert, C. (2019). Scar-Free Genome Editing in Legionella pneumophila. In: Buchrieser, C., Hilbi, H. (eds) Legionella. Methods in Molecular Biology, vol 1921. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9048-1_6
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DOI: https://doi.org/10.1007/978-1-4939-9048-1_6
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