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