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Volume 146, Issue 8

Use of a flexible cassette method to generate a double unmarked mutant by gene replacement Free

Abstract

Progress in the field of mycobacterial research has been hindered by the inability to readily generate defined mutant strains of the slow-growing mycobacteria to investigate the function of specific genes. An efficient method is described that has been used to generate several mutants, including the first double unmarked deletion strain of . Four mutants were constructed: a marked deletion of the cluster, which encodes three phospholipases C; separate unmarked deletions in and (encoding a haemolysin); and a double unmarked mutant ΔΔ. To accomplish this, two series of vectors were designed, the first of which, named pNIL, allows manipulation of the target gene sequence at a variety of convenient restriction sites. The second series, named pGOAL, contains marker cassettes flanked by I restriction enzyme sites. The final suicide plasmid vectors were then obtained by cloning a marker cassette from a pGOAL vector into the single I site of the pNIL vector with the modified gene of interest. Finally, a two-step strategy was employed whereby single cross-over events were first selected, then screening for the second cross-over was carried out to yield the mutant strains. This technique will now allow the construction of potential vaccine strains without the inclusion of antibiotic resistance markers, the ability to make multiple defined mutations and the possibility of making more subtle defined mutations, such as point mutations.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): haemolysin , homologous recombination , hyg, hygromycin , kan, kanamycin , MCS, multiple cloning site , phospholipase C , rapid cloning system and suc, sucrose
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2000-08-01
2024-04-19
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Use of a flexible cassette method to generate a double unmarked Mycobacterium tuberculosis tlyA plcABC mutant by gene replacement
Microbiology 146, 1969 (2000); https://doi.org/10.1099/00221287-146-8-1969
/content/journal/micro/10.1099/00221287-146-8-1969
/content/journal/micro/10.1099/00221287-146-8-1969
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