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

Abstract

The present study was designed to determine the half-life of gfp_m²⁺ mRNA, which encodes mycobacterial codon-optimised highly fluorescent GFP_m²⁺ protein, and to find out whether mycobacterial promoter activity can be quantitated more accurately using the mRNA levels of the reporter gene, gfp_m²⁺, than the fluorescence intensity of the GFP_m²⁺ protein. Quantitative PCR of gfp_m²⁺ mRNA in the pulse-chased samples of the rifampicin-treated Mycobacterium smeg-matis/gfpm²⁺ transformant showed the half-life of gfp_m²⁺ mRNA to be 4.081 min. The levels of the gfp_m²⁺ mRNA and the fluorescence intensity of the GFP_m²⁺ 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 gfp_m²⁺ mRNA levels is more accurate and statistically significant than the measurement of GFP_m²⁺ fluorescence intensity, especially for weak promoters.