title: 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.
authors.affiliation: Department of Pathology and Infectious Diseases, The Royal Veterinary College, Centre for Emerging, Endemic and Exotic Disease, Hawkshead Lane, Hertfordshire, AL9 7TA, United Kingdom.
authors.forename: Ricardo
authors.initials: R
authors.lastname: Balhana
authors.forename: Neil G
authors.initials: NG
authors.lastname: Stoker
authors.forename: Mahmudul Hasan
authors.initials: MH
authors.lastname: Sikder
authors.forename: Francois-Xavier
authors.initials: FX
authors.lastname: Chauviac
authors.forename: Sharon L
authors.initials: SL
authors.lastname: Kendall
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D016296:Mutagenesis; D009161:Mycobacterium / Q000235:genetics*