Highly fluorescent GFPm 2+ -based genome integration-proficient promoter probe vector to study Mycobacterium tuberculosis promoters in infected macrophages.
Bottom Line: Study of activity of cloned promoters in slow-growing Mycobacterium tuberculosis during long-term growth conditions in vitro or inside macrophages, requires a genome-integration proficient promoter probe vector, which can be stably maintained even without antibiotics, carrying a substrate-independent, easily scorable and highly sensitive reporter gene.In order to meet this requirement, we constructed pAKMN2, which contains mycobacterial codon-optimized gfp(m) (2+) gene, coding for GFP(m) (2+) of highest fluorescence reported till date, mycobacteriophage L5 attP-int sequence for genome integration, and a multiple cloning site. pAKMN2 showed stable integration and expression of GFP(m) (2+) from M. tuberculosis and M. smegmatis genome.Stable expression from genome-integrated format even without antibiotic, and high sensitivity of detection by flow cytometry and fluorescence imaging, in spite of single copy integration, make pAKMN2 useful for the study of cloned promoters of any mycobacterial species under long-term in vitro growth or stress conditions, or inside macrophages.
Affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore - 560012, Karnataka, India.Show MeSH
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Mentions: The construction of pAKMN2, from the source vectors, pMN406 (Roy et al., 2004) and pDK20 (DasGupta et al., 1998), through the intermediate episomal pAKMN1, is given in the self‐explanatory Fig. 1. In order to verify the stability of genome‐integrated pAKMN2‐promoter constructs, M. tuberculosis/pAKMN2‐PQ1K1 (Mt) and M. smegmatis/pAKMN2‐PQ1K1 (Ms) integrants, carrying total promoter region, Q1‐K1, of M. tuberculosis cell division gene, ftsZ, MtftsZ (Fig. 2A; Roy and Ajitkumar, 2005), were grown to mid‐log phase without hygromycin and plated on hygromycin‐containing and hygromycin‐free plates. The colony‐forming units (cfu) for both the integrants were comparable in the presence and absence of hygromycin (checked up to 30 and 60 generations for Mt and Ms integrants respectively) (Fig. 2B), with statistically insignificant values (two‐sided P‐values obtained by unpaired t‐test: 0.3078 for Mt and 0.1374 for Ms). On the contrary, statistically significant reduction (two‐sided P‐values: 0.0008 for Mt and 0.0035 for Ms) in cfu was found in the absence of hygromycin for the episomal pMN406‐PQ1K1 transformants of M. tuberculosis and M. smegmatis, grown under same conditions (Fig. 2B).
Affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore - 560012, Karnataka, India.