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

Journal of Microbiological Methods
Ricardo BalhanaSharon L Kendall

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.

References

Oct 25, 1990·Nucleic Acids Research·C Aslanidis, P J de Jong
Jun 1, 1995·Molecular Microbiology·P SanderE C Böttger
Apr 25, 1993·Nucleic Acids Research·Y S YangJ D Capra
Oct 6, 1997·Proceedings of the National Academy of Sciences of the United States of America·V PelicicC Guilhot
Dec 10, 2003·Proceedings of the National Academy of Sciences of the United States of America·Nobuyuki ShimonoLee W Riley
Apr 9, 2004·Proceedings of the National Academy of Sciences of the United States of America·Kenolisa C OnwuemeLuis E N Quadri
Sep 24, 2004·The International Journal of Biochemistry & Cell Biology·Edith E MachowskiValerie Mizrahi
Apr 27, 2005·Tuberculosis·Tanya ParishKen Duncan
Feb 18, 2006·Journal of Bacteriology·Ashwini ChauhanMalini Rajagopalan
Feb 13, 2007·Nature Methods·Mamie Z Li, Stephen J Elledge

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Citations

Jul 19, 2012·Yi chuan = Hereditas·Qin-Lin DuJian-Ping Xie
Sep 24, 2013·Biochemical and Biophysical Research Communications·Zhongshi lyuQin Zhou
Jul 6, 2014·International Journal of Gynecological Cancer : Official Journal of the International Gynecological Cancer Society·Xiaolong LiangZhu Yang
Aug 27, 2017·In Vitro Cellular & Developmental Biology. Animal·Hua XiaYajun Xie

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Methods Mentioned

BETA
PCR

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