title: Gene switching and essentiality testing.
abstract: The identification of essential genes is of major importance to mycobacterial research, and a number of essential genes have been identified in mycobacteria, however confirming essentiality is not straightforward, as deletion of essential genes results in a lethal phenotype. In this chapter, protocols are described that can be used to confirm gene essentiality using gene switching, following the construction of a delinquent strain. Because deletion mutants cannot be created for essential genes, a second gene copy is introduced via an integrating vector, which allows the chromosomal gene copy to be deleted. The integrated vector can then be replaced using the gene switching method; where no transformants are obtained, essentiality is confirmed. This technique can also be used to confirm functionality of gene homologues and to easily identify essential operon members.
authors.affiliation: Institute of Cell and Molecular Science, Barts and the London, Queen Mary's School of Medicine and Dentistry, 4 Newark Street, Whitechapel, London E1 2AA, UK. a.c.brown@qmul.ac.uk
authors.forename: Amanda Claire
authors.initials: AC
authors.lastname: Brown
mesh_terms: D005798:Genes, Bacterial; D020043:Genes, Essential; D005822:Genetic Vectors / Q000235:genetics; D009160:Mycobacteriophages / Q000235:genetics; D009161:Mycobacterium / Q000235:genetics*; D009876:Operon; D011995:Recombination, Genetic