[Development and application of homologous recombination knockout system in Mycobacterium tuberculosis]

We developed a homologous recombination system to make marker-free mutants in Mtb in an easy screening way.

The plasmid pSL002 harboring recE and recT-like protein gp60 and gp61 was transformed into wt Mtb in order to increase the recombing efficiency. The two homologous arms of target gene were amplified and cloned into pSL001. The purified homologous arms-loxP-gfp-hyg-loxP fragments were further transformed into Mtb (with pSL002 inside) competent cells to obtain the double cross over (DCO) mutants. The plasmid pSL003 was transformed into DCO competent cells to excise the two loxP sites flanked region. The plasmid pSL002 and pSL003 were removed via the counter selection marker sacB.

An efficient homologous recombination system was developed. Three different marker free mutants: Rv1364c phosphatase domain, PstP extracellular domain and PstP transmembrane domain were created by the system developed in this study. The efficiency to obtain DCO was varying from 25% to 62.5%, while the efficiency from DCO to KO was close to 100%. The gfp reporter was used to screen SCO, DCO and KO.

The homologous system shortens the complicated screening process to 3 month in Mtb. This is the fastest allelic exchange system reported so far, providing novel deletion strategy to the repertoire of mycobacterial genetic tools for constructing unmarked mutations.