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Date:
2020-10-05
[Abstract] Investigation of bacterial gene regulation upon environmental changes is still a challenging task. For example, Vibrio cholerae, a pathogen of the human gastrointestinal tract, faces diverse transient conditions in different compartments upon oral ingestion. Genetic reporter systems have been demonstrated to be extremely powerful tools to unravel gene regulation events in complex conditions, but so far focused mainly on gene induction. Herein, we describe the TetR-controlled recombination-based in vivo expression technology TRIVET, which allows detection of gene silencing events. TRIVET resembles a modified variant of the in vivo expression technology (IVET) as well as recombination-based in vivo expression technology (RIVET), which were used to ...
[摘要] [摘要]研究细菌基因对环境变化的调控仍然是一项艰巨的任务。例如,人胃肠道的病原体霍乱弧菌在口服后会在不同的隔室中遇到各种短暂的状况。事实证明,遗传报告系统是揭示复杂条件下基因调控事件的极有力工具,但到目前为止,它主要集中在基因诱导上。在本文中,我们描述了基于TetR控制的重组的体内表达技术TRIVET,该技术可检测基因沉默事件。TRIVET类似于体内表达技术(IVET)以及基于重组的体内变异体 表达技术(RIVET),用于鉴定宿主定殖过程中几种细菌的条件基因诱导。像它的前辈一样,TRIVET是一个基于单细胞的报告系统,可以通过耐药谱的表型变化以时空方式分析细菌基因的阻遏。简而言之,无启动子的tetR (编码转录阻遏物TetR)可通过转座子诱变随机地整合到细菌基因组中,或通过同源重组在目标启动子的下游特异性整合到细菌基因组中。的TetR导致的去阻遏的转录表达的减少的TetR控制解离TNPR,这反过来又导致切除ö F A Ñ抗生素抗性盒(也称为RES-盒)和改变的电阻曲线可观察到的通过划线上氨苄青霉素和卡那霉素板。然后可以将这种改变量化为抗性和非抗性分离株之间的比例。此外,新引入的第二报道基因,promot erless ...
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