Biochemical elucidation of the novel ECP bacterial stress response

本博士项目将专注于详细阐明细菌中新发现的基因调控网络的生化机制。在之前的一项研究中，我们证明了在渗透和翻译胁迫的条件下，大肠杆菌细胞会发生排泄细胞质蛋白（ECP）现象，从而将大量的细胞成分排出到细胞外环境中。本研究的动机是了解细菌应激反应对细菌生理学、宿主-病原体相互作用以及利用细菌细胞作为生物生产宿主的生物技术应用具有重要意义。本博士项目将重点阐述arfA和mscL转录后调控的生化机制。这两个基因的mRNA将通过体外转录制备，并接受RNase III介导的加工，或单独或相互复合。所得的sRNA产物将通过变性尿素- page进行分析，并在cDNA合成后进行测序。在细胞内，这两种转录本的丰度和半衰期，以及它们对细胞周期和ECP发病的依赖性将使用qRT-PCR进行研究。

This PhD project will focus on the detailed elucidation of the biochemical mechanism of a recently discovered gene regulatory network in bacteria. In a previous study we demonstrated that under conditions of both osmotic and translation stress E. coli cells undergo an excretion cytoplasmic proteins (ECP) phenomenon, whereby abundant cellular components are expelled into the extracellular environment. The motivation of this current study is that understanding bacterial stress responses has important implications for bacterial physiology, host-pathogen interactions, and for the biotechnological application of using bacterial cells as bio-production hosts.This PhD project will focus on the detailed elucidation of the biochemical mechanism of post-transcriptional regulation of arfA and mscL. The mRNA of the both these genes will be prepared by in vitro transcription, and subjected to RNase III mediated processing either or in isolation or in complex with each other. The resultant sRNA products will be analyzed by denaturing urea-PAGE and sequenced following cDNA synthesis. Inside the cells the abundance and half-life of the two transcripts, and the dependency of these upon the cell cycle and the onset of ECP will be studied using qRT-PCR.