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Legionella

Volume 954 of the series Methods in Molecular Biology pp 197-212

Date:

Constructing Unmarked Gene Deletions in Legionella pneumophila

  • Andrew BryanAffiliated withDepartment of Microbiology and Immunology, University of Michigan Medical School
  • , Zachary D. AbbottAffiliated withDepartment of Microbiology and Immunology, University of Michigan Medical School
  • , Michele S. SwansonAffiliated withDepartment of Microbiology and Immunology, University of Michigan Medical School Email author 

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Abstract

The ability to construct recombinant alleles efficiently in strains of interest, particularly unmarked deletions that reduce the potential for polar effects, is essential to studies of both pathogenesis and basic bacterial physiology. Here we describe a three-phase approach for generating unmarked deletions in Legionella pneumophila by constructing a mutant allele in E. coli using λ-Red recombination, so-called recombineering; transferring the allele onto the L. pneumophila chromosome by natural transformation; and then removing the selectable marker by utilizing the Flp site-specific recombinase. This strategy can decrease the amount of clone screening required while also increasing the percentage of the time the desired allele is obtained on the first attempt. The approach is particularly suited for constructing multiple unmarked deletions in a single strain in fewer steps than traditional methods.

Key words

Legionella pneumophila Flp recombinase Recombination Recombineering Unmarked deletions Counter-selection