title: The use of temperature-sensitive plasmids in mycobacteria.
abstract: The construction of allelic exchange mutants is one of the major strategies to decipher the function of a defined gene. In this chapter, protocols are described to perform allelic exchange in the mycobacterial species Mycobacterium smegmatis and Mycobacterium tuberculosis using thermosensitive counterselectable vectors. The antibiotic resistance cassette used makes it possible to efficiently rescue the marker, leaving an unmarked mutation on the chromosome. In addition, a method is provided to construct conditional M. smegmatis mutants and to test whether or not a gene is essential.
authors.affiliation: Mycobacterial Division Research, National Institute for Medical Research, London, UK.
authors.forename: Damien
authors.initials: D
authors.lastname: Portevin
authors.forename: Wladimir
authors.initials: W
authors.lastname: Malaga
authors.forename: Christophe
authors.initials: C
authors.lastname: Guilhot
mesh_terms: D000483:Alleles; D005798:Genes, Bacterial; D005822:Genetic Vectors / Q000235:genetics*; D016296:Mutagenesis; D009154:Mutation; D009161:Mycobacterium / Q000235:genetics*; D020102:Mycobacterium smegmatis / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D013696:Temperature