Biochemical elucidation of the novel ECP bacterial stress response

这个博士项目将专注于详细阐明最近发现的细菌基因调控网络的生化机制。在以前的研究中，我们证明了在渗透胁迫和翻译胁迫条件下，E。大肠杆菌细胞经历分泌胞质蛋白（ECP）现象，由此丰富的细胞组分被排出到细胞外环境中。本研究的动机是了解细菌的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。本博士项目将专注于详细阐明arfA和mscL转录后调控的生化机制。这两种基因的mRNA将通过体外转录制备，并进行RNA酶III介导的加工，或者单独进行，或者彼此复合进行。将通过变性尿素-PAGE分析所得sRNA产物，并在cDNA合成后测序。在细胞内，将使用qRT-PCR研究两种转录物的丰度和半衰期，以及它们对细胞周期和ECP发作的依赖性。

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.