title: Development of a new generation of vectors for gene expression, gene replacement, and protein-protein interaction studies in mycobacteria.
abstract: Escherichia coli-mycobacterium shuttle vectors are important tools for gene expression and gene replacement in mycobacteria. However, most of the currently available vectors are limited in their use because of the lack of extended multiple cloning sites (MCSs) and convenience of appending an epitope tag(s) to the cloned open reading frames (ORFs). Here we report a new series of vectors that allow for the constitutive and regulatable expression of proteins, appended with peptide tag sequences at their N and C termini, respectively. The applicability of these vectors is demonstrated by the constitutive and induced expression of the Mycobacterium tuberculosis pknK gene, coding for protein kinase K, a serine-threonine protein kinase. Furthermore, a suicide plasmid with expanded MCS for creating gene replacements, a plasmid for chromosomal integrations at the commonly used L5 attB site, and a hypoxia-responsive vector, for expression of a gene(s) under hypoxic conditions that mimic latency, have also been created. Additionally, we have created a vector for the coexpression of two proteins controlled by two independent promoters, with each protein being in fusion with a different tag. The shuttle vectors developed in the present study are excellent tools for the analysis of gene function in mycobacteria and are a valuable addition to the existing repertoire of vectors for mycobacterial research.
authors: National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India.
authors: Amit
authors: A
authors: Parikh
authors: Devanand
authors: D
authors: Kumar
authors: Yogesh
authors: Y
authors: Chawla
authors: Krishna
authors: K
authors: Kurthkoti
authors: Shazia
authors: S
authors: Khan
authors: Umesh
authors: U
authors: Varshney
authors: Vinay K
authors: VK
authors: Nandicoori
mesh_terms: D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D018390:Gene Targeting; D005822:Genetic Vectors; D008967:Molecular Biology / Q000379:methods*; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D025941:Protein Interaction Mapping; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA