| 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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| Whole-genome Identification of Transcriptional Start Sites by Differential RNA-seq in Bacteria
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Author:
Date:
2020-09-20
[Abstract] Gene transcription in bacteria often starts some nucleotides upstream of the start codon. Identifying the specific Transcriptional Start Site (TSS) is essential for genetic manipulation, as in many cases upstream of the start codon there are sequence elements that are involved in gene expression regulation. Taken into account the classical gene structure, we are able to identify two kinds of transcriptional start site: primary and secondary. A primary transcriptional start site is located some nucleotides upstream of the translational start site, while a secondary transcriptional start site is located within the gene encoding sequence.
Here, we present a step by step protocol for genome-wide transcriptional start sites determination by differential RNA-sequencing (dRNA-seq) ...
[摘要] [摘要] 细菌中的基因转录通常起始于起始密码子上游的一些核苷酸。识别SPE cific Ť ranscriptional 小号挞小号ITE （TSS）为遗传操作必需的，因为在许多情况下，起始密码子上游有中涉及的基因表达调控序列元件。考虑到经典的基因结构，我们能够鉴定出两种转录起始位点：一级和二级。主要转录起始位点位于翻译起始位点上游的一些核苷酸上，而次要转录起始位点位于基因编码序列内。
这里，我们提出一步步协议全基因组吨ranscriptional 小号馅饼小号ITES d etermination通过差RNA测序（DRNA 使用肠道病原体-SEQ）福氏痢疾杆菌血清型菌株5A作为M90T模型。但是，该方法可以用于选择的任何其他细菌物种。第一步，使用热酚法从细菌培养物中纯化总RNA。核糖体RNA（rRNA）是使用商业试剂盒通过杂交探针特异性去除的。然后准备一个富含5'- 一磷酸依赖性核酸外切酶（TEX）处理的，富含初级转录本的RNA文库，用于与未进行TEX处理的文库进行比较，然后连接已知序列的RNA接头衔接子，从而确定具有单核苷酸精度的TSS。最后，对RNA进行处理以制备Illumina测序文库，并按购买的服务进行测序。通过内部生物信息学分析鉴定TSS。
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| In vitro Crosslinking Reactions and Substrate Incorporation Assays for The Identification of Transglutaminase-2 Protein Substrates
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Author:
Date:
2020-06-20
[Abstract] Transglutaminase (TG2) catalyzes protein crosslinking between glutamyl and lysyl residues. Catalytic activity occurs via a transamidation mechanism resulting in the formation of isopeptide bonds. Since TG2-mediated transamidation is of mechanistic importance for a number of biological processes, assays that enable rapid and efficient identification and characterization of candidate substrates are an important first-step to uncovering the function of crosslinked proteins. Herein we describe an optimized and flexible protocol for in vitro TG2 crosslink reactions and substrate incorporation assays. We have previously employed these techniques in the identification of the protein high mobility group box 1 (HMGB1) as a TG2 substrate. However, the protocol can be adapted for ...
[摘要] [摘要]转谷氨酰胺酶(TG2)催化谷氨酰和赖氨酰残基之间的蛋白质交联。催化活性通过转酰胺机制发生，导致异肽键的形成。由于TG2介导的transamidation是一些生物过程的机械重要性，检测，使快速和有效的识别和表征的候选底物是一个重要的第一步，以揭示交联蛋白的功能。在这里，我们描述了一个优化和灵活的协议，用于体外TG2交联反应和底物结合测定。我们以前曾采用这些技术在鉴定蛋白质高流动性基团盒1（HMGB1）作为TG2底物。然而，该协议可以适应任何候选转酰胺化底物的鉴定。
[背景]转谷氨酰胺酶(TG2)是转谷氨酰胺酶家族的成员，催化钙依赖性转酰胺化反应。转谷氨酰胺酶家族包括TG1-7和FXIIIa，以及带4.2的非催化活性成员，在蛋白质支架中发挥作用(Satchwell等，2009；Gundemir等，2012)。TG2在该家族中的独特之处在于，它是无处不在的表达和多向性功能；除了蛋白交联外，TG2还具有蛋白二硫异构酶(Hasegawa等，2003；Mastroberardino等，2006)、G蛋白(Nakaoka等，1994；Baek等，1996；Vezza等，1999)和激酶(Mishra和Murphy，2004；Mishra等，2006和2007)的功能。
蛋白质交联发生在与肽结合的谷氨酰残基(谷氨酰胺供体底物)的γ-羧酰胺基与赖氨酰残基(赖氨酸受体底物)的ϵ-氨基基之间，从而形成ϵ-(γ-谷氨酰)赖氨酸异肽键(Folk和Finlayson，1977)。交联反应的示意图如图1所示。 ...
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