Patent ID: 8784842

Claim:
A method for directed allelic exchange mutagenesis of slow-growing mycobacterium (sp), comprising: providing a conditionally replicating transducing mycobacteriophage containing an allelic exchange substrate (AES), the AES comprising a selectable gene flanked by upstream and downstream homologous regions that flank a target locus or gene; culturing a slow-growing mycobacterial strain characterized by clumping during culturing, followed by gravity sedimentation to provide a supernatant fraction and a sediment fraction, low-speed centrifugation of the supernatant fraction to provide a first low-speed mycobacterial pellet, and re-suspension of the low-speed mycobacterial pellet in culture medium suitable for transducing; culturing the re-suspended slow-growing mycobacterial strain in the presence of the transducing mycobacteriophage at a non-permissive temperature to provide a mycobacteriophage-contacted culture; withdrawing an amount of the mycobacteriophage-contacted culture; and selecting, using the withdrawn amount and a suitable selection medium, allelic exchange mutants of the slow-growing mycobacterial strain.