| FRET-based Microscopy Assay to Measure Activity of Membrane Amino Acid Transporters with Single-transporter Resolution
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Author:
Date:
2021-04-05
[Abstract] Secondary active transporters reside in cell membranes transporting polar solutes like amino acids against steep concentration gradients, using electrochemical gradients of ions as energy sources. Commonly, ensemble-based measurements of radiolabeled substrate uptakes or transport currents inform on kinetic parameters of transporters. Here we describe a fluorescence-based functional assay for glutamate and aspartate transporters that provides single-transporter, single-transport cycle resolution using an archaeal elevator-type sodium and aspartate symporter GltPh as a model system. We prepare proteo-liposomes containing reconstituted purified GltPh transporters and an encapsulated periplasmic glutamate/aspartate-binding protein, PEB1a, labeled with donor and acceptor fluorophores. We then ...
[摘要] [摘要]次级活性转运蛋白驻留在细胞膜中，利用离子的电化学梯度作为能量源，可针对陡峭的浓度梯度转运极性氨基酸（如氨基酸）。通常，基于集合的放射性标记底物摄取或转运电流的测量可确定转运蛋白的动力学参数。在这里，我们描述了一种基于荧光的谷氨酸和天冬氨酸转运蛋白功能测定方法，该方法使用古细菌升降剂型钠和天冬氨酸共转运蛋白Glt Ph作为模型系统，提供了单转运蛋白，单转运周期的分辨率。我们准备包含重组的纯化的Glt Ph转运蛋白和封装的周质谷氨酸/天冬氨酸结合蛋白，PEB1a，用供体和受体荧光团标记的蛋白脂质体。然后，我们将蛋白脂质体表面固定化，并使用单分子全内反射荧光（TIRF）显微镜测量随时间变化的运输依赖性荧光共振能量转移（FRET）效率变化。与放射性配体摄取测定法相比，该测定法在时间分辨率上提高了10-100倍。它还可以对不同转运周期步骤进行动力学表征，并识别转运蛋白种群内的动力学异质性。
[背景]膜驻留的二级主动转运蛋白或溶质载体（SLC）介导氨基酸，激素，神经递质，维生素和药物等溶质的细胞摄取。他们将集中的底物摄取与主要通过Na + / K + ATPases的作用维持的离子电化学梯度的能量上有利的耗散结合在一起（Lingrel and Kuntzweiler ...
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| Detecting Spaciotemporal Transcript Accumulation in Maize by RNA In Situ Hybridization
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Author:
Date:
2021-02-20
[Abstract] RNA in situ hybridization is a method for visualizing spatiotemporal transcript accumulation in cells and tissues. The method provides clear resolution, is highly sensitive and specific, and can uncover gradients of transcript accumulation within a histologically-intact tissue, which is not possible currently with other methods for transcript detection. RNA in situ hybridization, however, is not a quantitative approach for gene expression. Protocols for RNA in situ hybridization have numerous steps that can span several days of work, complicating troubleshooting procedures. Here, we build on previously published RNA in situ hybridization protocols optimized for paraffin-embedded and sectioned maize tissue (Jackson, 1991; Long et al., 1996 ; ...
[摘要] [摘要] RNA原位杂交是一种可视化时空转录本在细胞和组织中积累的方法。该方法提供清晰的分辨率，高度灵敏和特异，并且可以揭示组织完整的组织内转录物积累的梯度，这是当前其他方法无法检测到转录物的方法。然而，RNA原位杂交不是用于基因表达的定量方法。RNA原位杂交的协议有许多步骤，可能需要花费数天的时间，从而使故障排除过程变得更加复杂。在这里，我们基于先前发布的RNA原位构建 杂交方案针对石蜡包埋和切片的玉米组织进行了优化（Jackson，1991； Long等，1996；Javelle等，2011），它提供了优化转录本检测的其他措施。
图形摘要：
RNA原位杂交的工作流程
[背景技术]在时间和空间差异基因表达允许具有相同的遗传物质的细胞呈现不同的身份。确实，基因表达的这种变化通常负责驱动整个生物体中模式或形态的进化变化（Carroll，2005）。因此，通过观察组织内天然背景下的基因表达，可以增强对发育过程的见识。诸如RT-qPCR和RNA- ...
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| 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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