Complementation of chromosomal deletions in Mycobacterium tuberculosis
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Tuberculosis (TB) is an infectious disease caused by the bacillus Mycobacterium tuberculosis (Mtb). The World Health Organization estimates that one-third of the world's population is latently infected with Mtb; these individuals have a 10% risk of developing TB. To successfully prevent, diagnose, and treat TB, it is imperative to understand the mechanisms this pathogen uses to survive in a human host. Defining the roles that different Mtb genes play in virulence is typically achieved by characterizing mutants deleted for targeted gene(s). To ensure that the resulting phenotypes are associated with the deleted genes, the wild type gene(s) must be re-introduced in trans. This is typically performed by PCR amplification of the genes and cloning them onto plasmids that can replicate in Mtb. Amplification of large regions of DNA often results in mutations due to proofreading limitations of the DNA polymerase used in vitro. The focus of my research has been to attempt to complement a 16-kb region of the Mtb genome. To help to define functions of genes within this region, a series of plasmids were created containing various portions of the deleted region. Difficulty with amplifying the entire region has led us to pursue an alternate strategy of recovering this region from the chromosome using plasmid-mediated recombination. Efforts are in progress to generate a system to screen for plasmids in which the targeted chromosomal region has been recombined onto it. This work is anticipated to allow the generation of complementation plasmids that have a low risk of PCR-induced mutations. Such a system will aid TB vaccine research by facilitating studies to identify which Mtb genes encode virulence factors.