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Agarose

琼脂糖

Company: Sigma-Aldrich
Catalog#: A9539
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FRAP: A Powerful Method to Evaluate Membrane Fluidity in Caenorhabditis elegans
Author:
Date:
2018-07-05
[Abstract]  FRAP (Fluorescence Recovery After Photobleaching) is probably the most direct method to investigate the dynamics of molecules in living cells. Here, we describe FRAP to quantify membrane fluidity in C. elegans. Using FRAP, we have shown that cold, glucose and exogenous saturated fatty acids can decrease the fluidity of cellular membranes in certain mutants. [摘要]  FRAP(光漂白后的荧光恢复)可能是研究活细胞中分子动力学的最直接方法。 在这里,我们描述FRAP来量化 C中的膜流动性。线虫。 使用FRAP,我们已经表明冷,葡萄糖和外源饱和脂肪酸可以降低某些突变体中细胞膜的流动性。

【背景】生物膜,定义所有细胞的特征,主要由磷脂组成(Van Meer 等人,,2008)。存在于磷脂双层中的脂肪酸物质极大地影响其性质。例如,高饱和脂肪酸含量增加了膜的刚性,而高不饱和脂肪酸含量促进了流动性(Pilon,2016)。细胞膜的脂肪酸组成通常与膳食脂肪的组成明显相关,膳食脂肪可直接掺入磷脂中(Abbott et al。,2012; Dancy et al。,2015)。然而,考虑到饮食中存在广泛的变化,细胞必须具有调节机制,监测和调节膜组成并获得所需的膜性质,例如流动性,曲率,厚度,等等。使用FRAP对在肠道细胞中表达prenylated GFP的蠕虫,我们先前已经证明,缺乏PAQR-2(哺乳动物脂联素受体的同源物)的突变体在葡萄糖或富含饱和脂肪的饮食中培养时降低了膜流动性。酸(Svensk et al。,2016; Devkota et ...

In vivo Analysis of Cyclic di-GMP Cyclase and Phosphodiesterase Activity in Escherichia coli Using a Vc2 Riboswitch-based Assay
Author:
Date:
2018-03-05
[Abstract]  Cyclic di-guanosine monophosphate (c-di-GMP) is a ubiquitous second messenger that regulates distinct aspects of bacterial physiology. It is synthesized by diguanylate cyclases (DGCs) and hydrolyzed by phosphodiesterases (PDEs). To date, the activities of DGC and PDE are commonly assessed by phenotypic assays, mass spectrometry analysis of intracellular c-di-GMP concentration, or riboswitch-based fluorescent biosensors. However, some of these methods require cutting-edge equipment, which might not be available in every laboratory. Here, we report a new simple, convenient and cost-effective system to assess the function of DGCs and PDEs in E. coli. This system utilizes the high specificity of a riboswitch to c-di-GMP and its ability to regulate the expression of a downstream ... [摘要]  环状二磷酸鸟苷(c-di-GMP)是一种无处不在的第二信使,它调节细菌生理学的不同方面。 它由diguanylate环化酶(DGC)合成并被磷酸二酯酶(PDE)水解。 迄今为止,通常通过表型分析,细胞内c-di-GMP浓度的质谱分析或基于核糖开关的荧光生物传感器来评估DGC和PDE的活性。 但是,其中一些方法需要尖端设备,而这些设备可能不适用于每个实验室。 在这里,我们报告了一个新的简单,方便和具有成本效益的系统,用于评估E中DGC和PDE的功能。大肠杆菌。 该系统利用核糖开关对c-di-GMP的高特异性及其响应于c-di-GMP浓度而调节下游β-半乳糖苷酶报道基因的表达的能力。 在该协议中,我们描述了该系统的构建及其用于评估DGC和PDE酶的活性。

【背景】Cyclic-di-GMP是细菌中重要且无处不在的第二信使,其调节各种过程,例如运动到衰退转变,生物膜形成,毒力和细胞周期进展(Römling等人, ,2013)。 GG(D / ...

Identification of Insertion Site by RESDA-PCR in Chlamydomonas Mutants Generated by AphVIII Random Insertional Mutagenesis
Author:
Date:
2018-02-05
[Abstract]  Chlamydomonas reinhardtii is frequently used as a model organism to study fundamental processes in photosynthesis, metabolism, and flagellar biology. Versatile tool boxes have been developed for this alga (Fuhrmann et al., 1999; Schroda et al., 2000; Schroda, 2006). Among them, forward genetic approach has been intensively used, mostly because of the high efficiency in the generation of hundreds of thousands of mutants by random insertional mutagenesis and the haploid nature therefore phenotypic analysis can be done in the first generation (Cagnon et al., 2013; Tunçay et al., 2013). A major bottleneck in the application of high throughput methods in a forward genetic approach is the identification of the genetic lesion(s) responsible for the ... [摘要]  莱茵衣藻(Chlamydomonas reinhardtii)是光合作用,代谢和鞭毛生物学的基础研究者。已经为这种藻类开发了多功能的工具箱(Fuhrmann等人,1999; Schroda等人,2000; Schroda,2006)。其中,正向遗传方法已经被广泛使用,主要是因为通过随机插入诱变产生了数十万个突变体的高效率,并且可以在第一代进行单倍体性质表型分析(Cagnon等人 2013,Tunçay et。 2013)。在正向遗传方法中应用高通量方法的主要瓶颈是鉴定与观察到的表型相关的遗传损伤。在该协议中,我们详细描述了最初在(González-Ballester等人,2005)中报道的限制性定点扩增PCR(RESDA-PCR)的改进版本。优化包括引物组合的优化,DNA聚合酶的选择,PCR循环参数的优化以及PCR产物直接测序的应用。这些修改使得获得特定的PCR产物变得更加容易,并且加速了亚克隆步骤以更快地获得测序数据。


【背景】除了限制酶位点定向扩增PCR(RESDA-PCR)(González-Ballester等人,2005)之外,还发现了其它几种分子技术。 包括Genome ...

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