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

ABSTRACT: 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.

SUBMITTER: Balhana R

PROVIDER: S-EPMC2941038 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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

Balhana Ricardo R Stoker Neil G NG Sikder Mahmudul Hasan MH Chauviac Francois-Xavier FX Kendall Sharon L SL

Journal of microbiological methods 20100727 1

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 ...[more]

PMID: 20650290

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