Mishra Saurabh, Anand Deepak, Vijayarangan Namperumalsamy, Ajitkumar Parthasarathi
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore - 560012, Karnataka, India.
Open Microbiol J. 2013;7:1-5. doi: 10.2174/1874285801307010001. Epub 2013 Jan 21.
The present study was designed to determine the half-life of gfp(m) (2+) mRNA, which encodes mycobacterial codon-optimised highly fluorescent GFP(m) (2+) protein, and to find out whether mycobacterial promoter activity can be quantitated more accurately using the mRNA levels of the reporter gene, gfp(m) (2+), than the fluorescence intensity of the GFP(m) (2+) protein. Quantitative PCR of gfp(m) (2+) mRNA in the pulse-chased samples of the rifampicin-treated Mycobacterium smeg-matis/gfpm(2+) transformant showed the half-life of gfp(m) (2+) mRNA to be 4.081 min. The levels of the gfp(m) (2+) mRNA and the fluorescence intensity of the GFP(m) (2+) 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(m) (2+) mRNA levels is more accurate and statistically significant than the measurement of GFP(m) (2+) fluorescence intensity, especially for weak promoters.
本研究旨在确定编码经分枝杆菌密码子优化的高荧光GFP(m)(2+)蛋白的gfp(m)(2+)mRNA的半衰期，并探究与通过GFP(m)(2+)蛋白的荧光强度相比，使用报告基因gfp(m)(2+)的mRNA水平是否能更准确地定量分枝杆菌启动子活性。对利福平处理的耻垢分枝杆菌/gfpm(2+)转化体的脉冲追踪样品中的gfp(m)(2+)mRNA进行定量PCR，结果显示gfp(m)(2+)mRNA的半衰期为4.081分钟。分别使用定量PCR和荧光分光光度法测定了由结核分枝杆菌细胞分裂基因ftsZ（MtftsZ）的启动子表达的gfp(m)(2+)mRNA水平和GFP(m)(2+)蛋白的荧光强度。数据显示，通过测定gfp(m)(2+)mRNA水平来定量分枝杆菌启动子活性比测量GFP(m)(2+)荧光强度更准确且具有统计学意义，尤其是对于弱启动子。
