Mutational studies aimed at characterizing the function(s) of bacterial genes required for growth or viability are constrained by the inability to generate deletion strains lacking the gene of interest. To circumvent this limitation, it is possible to generate conditional mutants in which a copy of the gene of interest is introduced into the bacteria to compensate for the loss of the native allele. Expression of the non-native copy of the target gene is typically under control of an inducible promoter, which allows for controllable and regulated production of the gene of interest. Conditional mutants are also broadly useful for phenotypic analyses of genes that require a tightly regulated and artificially inducible copy of the target gene. Herein, we describe the methods used to generate and confirm conditional mutant clones in Borrelia burgdorferi utilizing the Borrelia-adapted lac operator/repressor system.
Keywords: Borrelia; Conditional mutants; Essential genes; Genetic manipulation; Inducible expression; LacI; Lyme disease; lac repressor.