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Rapid construction of mycobacterial mutagenesis vectors using ligation-independent cloning

Balhana, Ricardo and Stoker, Neil G. and Sikder, Mahmudul Hasan and Chauviac, Francois-Xavier and Kendall, Sharon L. (2010) Rapid construction of mycobacterial mutagenesis vectors using ligation-independent cloning. Journal of Microbiological Methods 83 (1), pp. 34-41. ISSN 0167-7012.

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Official URL: http://dx.doi.org/10.1016/j.mimet.2010.07.014

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.

Item Type: Article
Keyword(s) / Subject(s): Mycobacteria, homologous recombination, ligation independent cloning, gene deletion
School or Research Centre: Birkbeck Schools and Research Centres > School of Science > Biological Sciences
Depositing User: Administrator
Date Deposited: 14 Feb 2011 15:54
Last Modified: 17 Apr 2013 12:33
URI: http://eprints.bbk.ac.uk/id/eprint/3057

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