An Accurate Method for the Qualitative Detection and Quantification of Mycobacterial Promoter Activity



Saurabh Mishra, Deepak Anand, Namperumalsamy Vijayarangan, Parthasarathi Ajitkumar*
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore – 560012, Karnataka, India


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© Mishra et al.; Licensee Bentham Open.

open-access license: This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.

* Address correspondence to this author at the Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore-560012, India; Tel: 91-80-2293-2344; Fax: 91-80-2360-2697; E-mail: ajit@mcbl.iisc.ernet.in


Abstract

The present study was designed to determine the half-life of gfpm2+ mRNA, which encodes mycobacterial codon-optimised highly fluorescent GFPm2+ protein, and to find out whether mycobacterial promoter activity can be quantitated more accurately using the mRNA levels of the reporter gene, gfpm2+, than the fluorescence intensity of the GFPm2+ protein. Quantitative PCR of gfpm2+ mRNA in the pulse-chased samples of the rifampicin-treated Mycobacterium smeg-matis/gfpm2+ transformant showed the half-life of gfpm2+ mRNA to be 4.081 min. The levels of the gfpm2+ mRNA and the fluorescence intensity of the GFPm2+ protein, which were expressed by the promoters of Mycobacterium tuberculosis cell division gene, ftsZ (MtftsZ), were determined using quantitative PCR and fluorescence spectrophotometry, respectively. The data revealed that quantification of mycobacterial promoter activity by determining the gfpm2+ mRNA levels is more accurate and statistically significant than the measurement of GFPm2+ fluorescence intensity, especially for weak promoters.

Keywords: gfpm2+, half-life, promoter activity, mycobacteria, quantitative PCR, fluorescence intensity.