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Ammonium sulfate for molecular biology, ≥99.0%

硫酸铵

Company: Sigma-Aldrich
Catalog#: A4418
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Laminarin Quantification in Microalgae with Enzymes from Marine Microbes
Author:
Date:
2018-04-20
[Abstract]  The marine beta-glucan laminarin is an abundant storage polysaccharide in microalgae. High production rates and rapid digestion by heterotrophic bacteria turn laminarin into an ideal carbon and energy source, and it is therefore a key player in the marine carbon cycle. As a main storage glucan laminarin also plays a central role in the energy metabolism of the microalgae (Percival and Ross, 1951; Myklestad, 1974; Painter, 1983). We take advantage of enzymes that digest laminarin selectively and can thereby quantify only this polysaccharide in environmental samples. These enzymes hydrolyze laminarin into glucose and oligosaccharides, which are measured with a standard reducing sugar assay to obtain the laminarin concentration. Prior to this assay, the three enzymes need to be produced via ... [摘要]  海洋β-葡聚糖昆布多糖是微藻中丰富的储存多糖。高生产率和异养细菌的快速消化将昆布多糖转化为理想的碳源和能源,因此它是海洋碳循环的关键参与者。作为主要的储存葡聚糖昆布多糖也在微藻的能量代谢中发挥核心作用(Percival and Ross,1951; Myklestad,1974; Painter,1983)。我们利用可以选择性消化昆布多糖的酶,从而可以对环境样品中的这种多糖进行定量。这些酶将昆布多糖水解成葡萄糖和寡糖,用标准的还原糖测定法测定得到昆布多糖浓度。在此测定之前,需要通过异源表达和纯化产生三种酶。该测定可用于监测环境微藻中的昆布多糖浓度,其通过过滤从海水中浓缩,或用来自藻类实验室培养物的样品中浓缩。

【背景】海洋多糖在海洋碳循环中起着重要作用,是浮游植物生理学的重要组成部分,但受到严重影响。几十年来,农业食品工业一直使用基于酶分析的即用试剂盒来分析各种不同的多糖(Whitaker,1974)。这些快速,稳健和特异性的基于酶的方法评估源自陆地植物即淀粉的多糖,因为它们广泛用于食品,饲料和其他工业应用中(Brunt等人, ,1998)。然而,海洋多糖的类似测定仍然缺乏。受到使用酶在藻类中进行多糖定量的想法的启发,我们开发了一种基于酶的方法来量化在硅藻和其他微藻中生态相关的β-葡聚糖昆布氨酸,也称为菊科金刚烷。

这种应用的三种糖苷水解酶(GH)来自福尔摩沙(Formosa)。并且它们的特征如下:FbGH30是GH30家族的外切型β-1,6-葡聚糖酶,特别是水解与昆布多糖骨架连接的β-1,6-连接的葡萄糖单体分支;并且FaGH17A和FbGH17A是GH家族17的两种内作用β-1,3-葡聚糖酶,其特异性地作用于β-1,3-连接的昆布多糖主链上(Becker等人,2017年, ...

Single-step Precision Genome Editing in Yeast Using CRISPR-Cas9
Author:
Date:
2018-03-20
[Abstract]  Genome modification in budding yeast has been extremely successful largely due to its highly efficient homology-directed DNA repair machinery. Several methods for modifying the yeast genome have previously been described, many of them involving at least two-steps: insertion of a selectable marker and substitution of that marker for the intended modification. Here, we describe a CRISPR-Cas9 mediated genome editing protocol for modifying any yeast gene of interest (either essential or nonessential) in a single-step transformation without any selectable marker. In this system, the Cas9 nuclease creates a double-stranded break at the locus of choice, which is typically lethal in yeast cells regardless of the essentiality of the targeted locus due to inefficient non-homologous end-joining ... [摘要]  芽殖酵母中的基因组修饰已经非常成功,主要归功于其高度同源性的DNA修复机制。之前已经描述了几种用于修饰酵母基因组的方法,其中许多方法涉及至少两个步骤:插入选择标记并用该标记取代预期的修饰。在这里,我们描述了CRISPR-Cas9介导的基因组编辑方案,用于在没有任何选择标记的情况下在单步转化中修饰任何感兴趣的酵母基因(基本或非必需)。在该系统中,Cas9核酸酶在选择的基因座处产生双链断裂,这在酵母细胞中通常是致死的,而不管由于无效的非同源末端连接修复导致的靶基因座的重要性。该致死性通过使用源自PCR的修复模板的同源重组导致有效的修复。在涉及必需基因的情况下,用功能性等位基因编辑基因组病变的必要性作为额外的选择层。作为一个激励性的例子,我们描述了使用这种策略替代HEM2,一种必需的酵母基因,以及相应的人类直向同源物ALAD。

【背景】酿酒酵母(Baccharomyces cerevisiae,Baker's酵母)作为一种遗传易处理的生物体具有悠久的历史,并且有许多操作酵母基因组的方法。然而,直到最近,有必要应用选择以分离具有所需遗传改变的克隆(Kearse等人,2012; DiCarlo等人,2013; Lee等人,等,2015; ...

RNA Cap Methyltransferase Activity Assay
Author:
Date:
2018-03-20
[Abstract]  Methyltransferases that methylate the guanine-N7 position of the mRNA 5’ cap structure are ubiquitous among eukaryotes and commonly encoded by viruses. Here we provide a detailed protocol for the biochemical analysis of RNA cap methyltransferase activity of biological samples. This assay involves incubation of cap-methyltransferase-containing samples with a [32P]G-capped RNA substrate and S-adenosylmethionine (SAM) to produce RNAs with N7-methylated caps. The extent of cap methylation is then determined by P1 nuclease digestion, thin-layer chromatography (TLC), and phosphorimaging. The protocol described here includes additional steps for generating the [32P]G-capped RNA substrate and for preparing nuclear and cytoplasmic extracts from mammalian cells. This assay is ... [摘要]  甲基化mRNA 5'帽结构的鸟嘌呤-N7位置的甲基转移酶在真核生物中普遍存在并且通常由病毒编码。这里我们提供生物样品的RNA帽甲基转移酶活性的生化分析的详细方案。该测定包括将含有帽 - 甲基转移酶的样品与[32 P] G-加帽的RNA底物和S-腺苷甲硫氨酸(SAM)温育以产生具有N7-甲基化帽的RNA。然后通过P1核酸酶消化,薄层色谱(TLC)和磷成像确定帽甲基化的程度。此处描述的方案包括用于产生[32 P] G-加帽的RNA底物和用于从哺乳动物细胞制备核和细胞质提取物的附加步骤。该分析也适用于分析其他生物样品(包括重组蛋白制剂和来自分析分离和免疫沉淀/下拉实验的级分)的帽甲基转移酶活性。

【背景】mRNA的5'端的N7-甲基鸟苷帽是适当的真核mRNA加工,定位和翻译所必需的修饰。 ...

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