| TetR Regulated in vivo Repression Technology to Identify Conditional Gene Silencing in Genetically Engineerable Bacteria Using Vibrio cholerae Murine Infections as Model System
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Author:
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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| Phenol-based Extraction of RNA from Chlamydomonas reinhardtii
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Author:
Date:
2018-07-05
[Abstract] RNA extraction is a basic procedure in molecular biology and a wide variety of commercial kits are available. Some of these kits have been successfully used in Chlamydomonas. However, in some cases RNA quality and quantity may be dramatically reduced depending on the strains and/or the conditions where cells were grown or treated. Phenol-based protocols are the most robust methods to obtain both high quantity and quality RNA from any strain of this alga grown in any condition. Here, we describe an easy and cheap protocol using phenol but avoiding the acute toxicity of guanidine isothiocyanate present in the commercial phenol-based mixtures.
[摘要] RNA提取是分子生物学中的基本程序，并且可获得各种商业试剂盒。 其中一些试剂盒已成功用于 Chlamydomonas 。 然而，在一些情况下，取决于菌株和/或细胞生长或处理的条件，RNA质量和数量可以显着降低。 基于苯酚的方案是从任何条件下生长的这种藻类的任何菌株获得高数量和高质量RNA的最稳健的方法。 在这里，我们描述了使用苯酚的简单且廉价的方案，但避免了商业苯酚基混合物中存在的异硫氰酸胍的急性毒性。
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| A Protocol of Using White/Red Color Assay to Measure Amyloid-induced Oxidative Stress in Saccharomyces cerevisiae
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Author:
Date:
2017-08-05
[Abstract] The yeast Saccharomyces cerevisiae (S. cerevisiae) harboring ade1 or ade2 mutations manifest red colony color phenotype on rich yeast medium YPD. In these mutants, intermediate metabolites of adenine biosynthesis pathway are accumulated. Accumulated intermediates, in the presence of reduced glutathione, are transported to the vacuoles, whereupon the development of the red color phenotype occurs. Here, we describe a method to score for presence of oxidative stress upon expression of amyloid-like proteins that would convert the red phenotype of ade1/ade2 mutant yeast to white. This assay could be a useful tool for screening for drugs with anti-amyloid aggregation or anti-oxidative stress potency.
[摘要] 携带 ade1 或 ade2 突变体的酵母酿酒酵母（ S。cerevisiae ）在富酵母上显示红色菌落色表型 中等YPD。 在这些突变体中，积累了腺嘌呤生物合成途径的中间代谢物。 在还原型谷胱甘肽存在下，累积的中间体被转移到空泡中，由此发生红色表型的发生。 在这里，我们描述了一种通过淀粉样样蛋白的表达来评估氧化应激存在的方法，其将将ade1 / ade2突变体酵母的红色表型转化为白色。 该测定可能是用于筛选具有抗淀粉样蛋白聚集或抗氧化应激效力的药物的有用工具。
【背景】ADE1或ADE2基因的酵母（Saccharomyces cerevisiae）突变体（例如，，ade1Δ，ade2Δ ade1-14 ade2-），当在YPD（酵母蛋白胨葡萄糖）培养基上生长时，作为腺嘌呤生物合成途径的中间代谢物的液泡（Sharma等人，2003）。包含早熟终止密码子的可抑制等位基因ade1-14 已被广泛用于评价翻译的朊病毒状态终止因子Sup35蛋白。在[ psi - ]酵母中，Sup35p保持可溶性和功能性，因此翻译在有效地终止于ade1-14的早熟终止密码子>等位基因导致截短和非功能性Ade1蛋白的合成。因此，腺嘌呤生物合成级联保持不完整，导致中间体代谢产物的积累，产生酵母的红色表型。相比之下，在[PSI + ...
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