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J Microbiol Methods. 2010 Oct;83(1):34-41. doi: 10.1016/j.mimet.2010.07.014. Epub 2010 Jul 27.

Rapid construction of mycobacterial mutagenesis vectors using ligation-independent cloning.

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Department of Pathology and Infectious Diseases, The Royal Veterinary College, Centre for Emerging, Endemic and Exotic Disease, Hawkshead Lane, Hertfordshire, AL9 7TA, United Kingdom.


Targeted mutagenesis is one of the major tools for determining the function of a given gene and its involvement in bacterial pathogenesis. In mycobacteria, gene deletion is often accomplished by using allelic exchange techniques that commonly utilise a suicide delivery vector. We have adapted a widely-used suicide delivery vector (p1NIL) for cloning two flanking regions of a gene using ligation independent cloning (LIC). The pNILRB plasmid series produced allow a faster, more efficient and less laborious cloning procedure. In this paper we describe the making of pNILRB5, a modified version of p1NIL that contains two pairs of LIC sites flanking either a sacB or a lacZ gene. We demonstrate the success of this technique by generating 3 mycobacterial mutant strains. These vectors will contribute to more high-throughput methods of mutagenesis.

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