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Chromosomal Mutagenesis

Volume 435 of the series Methods in Molecular Biology pp 203-215

Mycobacterial Recombineering

  • Julia C. van KesselAffiliated withPittsburgh Bacteriophage Institute and Department of Biological Sciences, University of Pittsburgh
  • , Graham F. HatfullAffiliated withPittsburgh Bacteriophage Institute and Department of Biological Sciences, University of Pittsburgh

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Abstract

Although substantial advances have been made in mycobacterial genetics over the past 15 yr, manipulation of mycobacterial genomes and Mycobacterium tuberculosis in particular, continues to be hindered by problems of relatively poor DNA uptake, slow growth rate, and high levels of illegitimate recombination. In Escherichia coli an effective approach to stimulating recombination frequencies has been developed called “recombineering,” in which phage-encoded recombination functions are transiently expressed to promote efficient homologous recombination. Although homologs of these recombination proteins are rare among mycobacteriophages, we have identified one phage, Che9c, encoding relatives of both RecE and RecT of the E. coli rac prophage. Expression of the Che9c proteins from an inducible expression system in either slow- or fast-growing mycobacteria provides elevated recombination frequencies and facilitates simple allelic exchange using linear DNA substrates. Mycobacterial recombineering, therefore, offers a simple approach for constructing gene replacement mutants in M. smegmatis and M. tuberculosis.

Key Words

Allelic exchange genetic engineering mutagenesis mycobacteriophage Mycobacterium tuberculosis recombineering