| DNA-free Genome Editing of Chlamydomonas reinhardtii Using CRISPR and Subsequent Mutant Analysis
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Author:
Date:
2017-06-05
[Abstract] We successfully introduced targeted knock-out of gene of interest in Chlamydomonas reinhardtii by using DNA-free CRISPR. In this protocol, the detailed procedures of an entire workflow cover from the initial target selection of CRISPR to the mutant analysis using next generation sequencing (NGS) technology. Furthermore, we introduce a web-based set of tools, named CRISPR RGEN tools (http://www.rgenome.net/), which provides all required tools from CRISPR target design to NGS data analysis.
[摘要] 我们通过使用无DNA的CRISPR成功地引入了目标克隆在赖氨酸衣藻中的目标基因敲除。在该协议中，整个工作流程的详细过程涵盖从初始目标选择CRISPR到使用下一代测序（NGS）技术的突变体分析。此外，我们介绍一种基于Web的工具集，名为CRISPR RGEN工具（ http：// www.rgenome.net/ ），其中提供了CRISPR目标设计到NGS数据分析的所有必需工具。
背景 我们最近报道（Baek等人，2016），使用预先组装的Cas9蛋白质指导RNA核糖核蛋白，模型绿色微藻（Chlamydomonas ...
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| Assay of Arabinofuranosidase Activity in Maize Roots
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Author:
Date:
2016-03-20
[Abstract] Root is a perfect model for studying the mechanisms of plant cell growth. Along the root length, several zones where cells are at different stages of development can be visualized (Figure 1). The dissection of the root on these zones allows the investigation of biochemical and genetic aspects of different growth steps. Maize primary root is much more massive than the root of other Monocots and thus more convenient for such type of research. Plant cell wall, mainly consisting of polysaccharides, plays an important role in plant life. Therefore, measurement of plant carbohydrate content and glycoside-modifying enzyme activity in plant cells has become an important aspect in plant physiology. One of the well-documented changes of hemicelluloses molecules during elongation growth of monocots ...
[摘要] 根是研究植物细胞生长机制的完美模型。沿着根长度，可以可视化细胞处于不同发育阶段的几个区（图1）。在这些区域上的根的解剖允许调查不同生长步骤的生化和遗传方面。玉米主根比其他单根的根更大，因此这种类型的研究更方便。植物细胞壁，主要由多糖组成，在植物生活中发挥重要作用。因此，植物碳水化合物含量和糖苷修饰酶活性在植物细胞中的测量已经成为植物生理学中的重要方面。在单子叶植物细胞的延长生长期间半纤维素分子的充分证明的变化之一是葡糖醛酸阿拉伯木聚糖的阿拉伯糖取代的减少。这可能是由于该多糖的合成的变化或阿拉伯呋喃糖苷酶的作用引起的。在这里，我们描述的分光光度测量阿拉伯呋喃糖苷酶活性在玉米根中的水解发色底物（4-硝基苯基α-L-阿拉伯呋喃糖苷）的速率的协议。
图1. 四日龄黑暗生长的玉米幼苗面板）。根据Kozlova等人（2012）（右图），初级玉米根的不同区和细胞发育的相应阶段。
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| Determination of Keto-deoxy-d-manno-8-octanoic acid (KDO) from Lipopolysaccharide of Escherichia coli
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Author:
Date:
2015-12-20
[Abstract] 2-Keto-3-deoxy-octonate (KDO) is an essential constituent of lipopolysaccharide (LPS) that forms the outermost leaflet of Gram-negative bacterial outer membrane. LPS is mainly composed of lipid A, O-antigen and a core oligosaccharide. Two molecules of KDO are present per one molecule of LPS. A proper level of LPS is required to maintain the outer membrane integrity and either high or low levels of LPS are toxic to the cell. Various methods are available for quantification of LPS; of these, determination of KDO is a simple and accurate method and it can be estimated either directly from crude bacterial cell lysates or from purified LPS by a simple colorimetric assay. Although this procedure can be theoretically used for any Gram-negative bacterium, we used it routinely to measure KDO from ...
[摘要] 2-酮-3-脱氧 - 辛酸（KDO）是形成革兰氏阴性细菌外膜的最外侧叶的脂多糖（LPS）的必需成分。 LPS主要由脂质A，O-抗原和核心寡糖组成。每一分子LPS存在两个KDO分子。需要适当水平的LPS以维持外膜完整性，并且高或低水平的LPS对细胞有毒性。各种方法可用于定量LPS;其中，KDO的测定是简单和准确的方法，并且可以通过简单的比色测定直接从粗制细菌细胞裂解物或从纯化的LPS估计。尽管该方法可以理论上用于任何革兰氏阴性菌，我们常规地使用它来测量来自大肠杆菌（大肠杆菌）K12菌株的细胞裂解物的KDO。 br /> 方法：协议取自Karkhanis等人（1978）。它是一种测量KDO的简单，灵敏和可靠的方法。该测定在细胞裂解物或LPS完全酸水解后释放LPS的各种组分进行。此外，与高碘酸盐，亚砷酸盐和硫代巴比妥酸的反应产生粉红色至红色的发色团，其在用DMSO稳定后在548nm测量。
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