Gene switching and essentiality testing.
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.
Brown
Amanda Claire
AC
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
D005798:Genes, Bacterial; D020043:Genes, Essential; D005822:Genetic Vectors / Q000235:genetics; D009160:Mycobacteriophages / Q000235:genetics; D009161:Mycobacterium / Q000235:genetics*; D009876:Operon; D011995:Recombination, Genetic