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 which can be used to confirm gene essentiality using gene switching, following the construction of a strain carrying its only functional copy on an integrated plasmid (Δ'int). Since 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 homologs and to easily identify essential operon members.