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Legionella pp 93-105 | Cite as

Scar-Free Genome Editing in Legionella pneumophila

  • Nathalie Bailo
  • Hussein Kanaan
  • Elisabeth Kay
  • Xavier Charpentier
  • Patricia Doublet
  • Christophe GilbertEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1921)

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.

Key words

Legionella pneumophila Natural transformation Genome editing Recombination Suicide cassette 

Notes

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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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Nathalie Bailo
    • 1
  • Hussein Kanaan
    • 1
  • Elisabeth Kay
    • 1
  • Xavier Charpentier
    • 1
  • Patricia Doublet
    • 1
  • Christophe Gilbert
    • 1
    Email author
  1. 1.CIRI, International Center for Infectiology Research, CNRS, UMR5308, Inserm, U1111, Université Lyon 1Université de LyonLyonFrance

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