Recombinant Reporter Assay Using Transcriptional Machinery of Mycobacterium tuberculosis
Recombinant Reporter Assay Using Transcriptional Machinery of Mycobacterium tuberculosis
复制标题
DOI：
10.1128/jb.02445-14
复制
发表时间：
2015-02-01
影响因子：
3.2
通讯作者：
Mukhopadhyay, Jayanta
中科院分区：
文献类型：
--
作者：
Banerjee, Rajdeep;Rudra, Paulami;Mukhopadhyay, Jayanta
由于生长极其缓慢的细菌突变株的产生非常繁琐，开发体内基因报告测定法来评估结核分枝杆菌转录机制组件之间的相互作用仍然是科学家面临的挑战。我们开发了一种重组 mCherry 报告基因测定法，使我们能够监测大肠杆菌体内结核分枝杆菌转录调节因子与其启动子的相互作用。该测定涉及大肠杆菌中的三质粒表达系统，其中两个质粒负责结核分枝杆菌 RNA 聚合酶 (RNAP) 的产生，第三个质粒包含受 sigma 因子或转录调节因子依赖性启动子控制的 mCherry 报告基因表达盒。我们观察到内源性大肠杆菌 RNAP 和 sigma 因子不会干扰测定。通过使用报告基因检测，我们发现结核分枝杆菌环AMP受体蛋白（CRP）的功能相互作用是与其自身的RNA聚合酶发生的，而不是与大肠杆菌聚合酶发生的。在大肠杆菌中进行重组报告基因检测比在结核分枝杆菌中进行重组报告检测要快得多，并且避免了处理致病菌的危险。该方法可以扩展到开发其他致病性和生长缓慢的细菌系统的报告分析。
Development of an in vivo gene reporter assay to assess interactions among the components of the transcription machinery in Mycobacterium tuberculosis remains a challenge to scientists due to the tediousness of generation of mutant strains of the extremely slow-growing bacterium. We have developed a recombinant mCherry reporter assay that enables us to monitor the interactions of Mycobacterium tuberculosis transcriptional regulators with its promoters in vivo in Escherichia coli. The assay involves a three-plasmid expression system in E. coli wherein two plasmids are responsible for M. tuberculosis RNA polymerase (RNAP) production and the third plasmid harbors the mCherry reporter gene expression cassette under the control of either a sigma factor or a transcriptional regulator-dependent promoter. We observed that the endogenous E. coli RNAP and sigma factor do not interfere with the assay. By using the reporter assay, we found that the functional interaction of M. tuberculosis cyclic AMP receptor protein (CRP) occurs with its own RNA polymerase, not with the E. coli polymerase. Performing the recombinant reporter assay in E. coli is much faster than if performed in M. tuberculosis and avoids the hazard of handling the pathogenic bacterium. The approach could be expanded to develop reporter assays for other pathogenic and slow-growing bacterial systems.