Construction of Targeted Mycobacterial Mutants by Homologous Recombination

  • Sharon L. Kendall
  • Rosangela Frita
Part of the Methods in Molecular Biology book series (MIMB, volume 465)


The ability to select genes to knock out of mycobacterial genomes has greatly improved our understanding of mycobacteria. This chapter describes a method for doing this. The gene (including a 1-kb flanking region) is cloned into a pNIL series vector and disrupted by deletion or insertion of a cassette. A selection of marker genes obtained from the pGOAL series of vectors are inserted into the pNIL vector to create a suicide delivery system. This delivery vector is introduced into mycobacteria where the disrupted version of the gene replaces the wild-type version by a two-step homologous recombination process. The method involves selecting for a single crossover event followed by selection of double crossovers. Single crossovers have incorporated plasmid marker genes and are sucroseS, kanamycinR and blue on media containing X-gal. Double crossovers have lost plasmid markers and are sucroseR, kanamycinS and white on media containing X-gal.


allelic replacement gene knock-out homologous recombination mycobacteria mutant 


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Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  1. 1.Department of Pathology and Infectious DiseasesThe Royal Veterinary CollegeLondonUK
  2. 2.Department of Pathology and Infectious DiseasesThe Royal Veterinary CollegeLondonUK

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