Rapid construction of mycobacterial mutagenesis vectors using ligation-independent cloning
| Autor: | Francois-Xavier Chauviac, Neil G. Stoker, Mahmudul Hasan Sikder, Sharon L. Kendall, Ricardo Balhana |
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| Jazyk: | angličtina |
| Předmět: |
Microbiology (medical)
Genetic Vectors Mutant Mutagenesis (molecular biology technique) Biology bcs Microbiology Article Mycobacterium 03 medical and health sciences Plasmid Vector (molecular biology) Cloning Molecular Homologous recombination Molecular Biology Gene Ligation independent cloning 030304 developmental biology Genetics Cloning 0303 health sciences Gene deletion 030306 microbiology Ligation-independent cloning Mycobacteria Mutagenesis |
| Zdroj: | Journal of Microbiological Methods |
| ISSN: | 0167-7012 |
| DOI: | 10.1016/j.mimet.2010.07.014 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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