Present addresses: Cardiovascular Research Institute, University of California San Francisco, San Francisco, CA 94158, USA;
Highly fluorescent GFPm2+-based genome integration-proficient promoter probe vector to study Mycobacterium tuberculosis promoters in infected macrophages
Article first published online: 29 SEP 2011
© 2011 The Authors. Microbial Biotechnology © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd
Volume 5, Issue 1, pages 98–105, January 2012
How to Cite
Roy, S., Narayana, Y., Narayanaswamy Balaji, K. and Ajitkumar, P. (2012), Highly fluorescent GFPm2+-based genome integration-proficient promoter probe vector to study Mycobacterium tuberculosis promoters in infected macrophages. Microbial Biotechnology, 5: 98–105. doi: 10.1111/j.1751-7915.2011.00305.x
- Issue published online: 14 DEC 2011
- Article first published online: 29 SEP 2011
- Received 5 June, 2011; accepted 26 August, 2011.
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 gfpm2+ gene, coding for GFPm2+ 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 GFPm2+ from M. tuberculosis and M. smegmatis genome. Expression of GFPm2+, driven by the cloned minimal promoters of M. tuberculosis cell division gene, ftsZ (MtftsZ), could be detected in the M. tuberculosis/pAKMN2-promoter integrants, growing at exponential phase in defined medium in vitro and inside macrophages. 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.