One step allelic exchange in mycobacteria using in vitro generated conditional transducing phages

Abstract

The present invention provides a method for high frequency of allelic exchange in the slow-growing mycobacteria using in vitro generated specialized transducing mycobacteriophages, as well as the recombinant slow-growing mycobacteria generated using the disclosed method. A transducing mycobacteriophage of the present invention comprises a conditional mycobacteriophage containing an E. coli bacteriophage lambda cosmid inserted into a non-essential region of the mycobacteriophage, said cosmid containing a mutated DNA substrate which is homologous to a wildtype nucleic acid sequence of a slow-growing mycobacterium. When slow-growing mycobacteria infected with the conditional transducing phage are cultured under conditions wherein the conditional transducing phage does not replicate, the mutated DNA substrate is incorporated into the chromosomal DNA of the slow-growing mycobacteria by homologous recombination, thereby generating the recombinant slow-growing mycobacteria of the present invention. The disclosed method may be used to produce mycobacterial auxotrophs, including leucine and lysine auxotrophs.

Dependencies are inferred from the claim text.