Construction of targeted mycobacterial mutants by homologous recombination.
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 sucrose(S), kanamycin(R) and blue on media containing X-gal. Double crossovers have lost plasmid markers and are sucrose(R), kanamycin(S) and white on media containing X-gal.
Kendall
Sharon L
SL
Department of Pathology and Infectious Diseases, The Royal Veterinary College, Royal College Street, London NW1 OTU, UK. skendall@rvc.ac.uk
Frita
Rosangela
R
D055786:Gene Knockout Techniques / Q000379:methods*; D016680:Genome, Bacterial; D009154:Mutation; D009161:Mycobacterium / Q000235:genetics*; D011995:Recombination, Genetic