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Yeast Extract

酵母抽提物

Company: BD
Catalog#: 212750
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Bacterial Microcolonies in Gel Beads for High-throughput Screening
Author:
Date:
2018-07-05
[Abstract]  High-throughput screening of a DNA library expressed in a bacterial population for identifying potentially rare members displaying a property of interest is a crucial step for success in many experiments such as directed evolution of proteins and synthetic circuits and deep mutational scanning to identify gain- or loss-of-function mutants.

Here, I describe a protocol for high-throughput screening of bacterial (E. coli) microcolonies in gel beads. Single cells are encapsulated into monodisperse water-in-oil emulsion droplets produced with a microfluidic device. The aqueous solution also contains agarose that gelates upon cooling on ice, so that solid gel beads form inside the droplets. During incubation of the emulsion, the cells grow into monoclonal microcolonies ...
[摘要]  在细菌群体中表达的DNA文库的高通量筛选用于鉴定显示感兴趣性质的潜在稀有成员是在许多实验中成功的关键步骤,例如蛋白质和合成回路的定向进化以及用于鉴定增益的深度突变扫描 - 或功能丧失的突变体。

在这里,我描述了一种用于高通量筛选凝胶珠中细菌(大肠杆菌)微菌落的方案。将单细胞包封成用微流体装置产生的单分散油包水乳液液滴。水溶液还含有琼脂糖,其在冰上冷却时凝胶化,从而在液滴内部形成固体凝胶珠。在乳液温育期间,细胞在珠内生长成单克隆微菌落。在从乳液中分离凝胶珠并通过荧光激活细胞分选(FACS)分选后,从凝胶珠中回收细菌,然后准备进行进一步的分选,诱变或分析。为了通过FACS分类,该方案需要荧光读数,例如荧光报告蛋白的表达。测量微小菌落的平均荧光信号降低了高表型细胞间变异性的影响,并且与单细胞分选相比提高了灵敏度。我们应用这种方法在ON和OFF状态下对pBAD启动子文库进行分类(Duarte et al。,2017)。

【背景】荧光激活细胞分选(FACS)具有> 10 7 事件/ h的无与伦比的筛选通量(Davies,2012)。然而,通过FACS根据其荧光分选单个细胞以筛选合成回路的文库(Schaerli和Isalan,2013)经常受到高表型细胞间变异性的阻碍。或者,可以对水凝胶珠中所含的小细胞集落(微集落)进行分类(Weaver ...

Method for CRISPR/Cas9 Mutagenesis in Candida albicans
Author:
Date:
2018-04-20
[Abstract]  Candida albicans is the most prevalent and important human fungal pathogen. The advent of CRISPR as a means of gene editing has greatly facilitated genetic analysis in C. albicans. Here, we describe a detailed step-by-step procedure to construct and analyze C. albicans deletion mutants. This protocol uses plasmids that allow simple ligation of synthetic duplex 23mer guide oligodeoxynucleotides for high copy gRNA expression in C. albicans strains that express codon-optimized Cas9. This protocol allows isolation and characterization of deletion strains within nine days. [摘要]  白色念珠菌是最普遍和最重要的人类真菌病原体。 CRISPR作为基因编辑手段的出现极大地促进了 C中的遗传分析。白色假丝酵母。 在这里,我们描述一个详细的分步过程来构建和分析 C。 白色念珠菌缺失突变体。 该协议使用质粒,允许合成的双链体23mer引导寡聚脱氧核苷酸在高拷贝gRNA表达的简单连接。 表达密码子优化的Cas9的白色念珠菌菌株。 该协议允许在9天内分离和鉴定缺失菌株。

【背景】℃。白色念珠菌是一种难以处理遗传的有机体。由于它通常作为不容易进行有性生殖的二倍体存在,所以纯合隐性突变需要对每个基因座进行连续修饰。克隆间规则间隔短回文重复(CRISPR)突变的发展和应用。白色念珠菌促进遗传操作,因为它允许两个等位基因同时突变(Vyas et al。,2015; Min et al。,2016; Ng and Dean,2017 )。 CRISPR基因编辑涉及将RNA引导的核酸酶募集至邻近NGG原型间隔子邻接基序(PAM)的互补靶位点(Jinek等人,2012; Cong等人, 2013年;马里等人,2013年)。 CRISPR相关(Cas)核酸酶通过与结合Cas9的激活CRISPR RNA(tracrRNA)相关的指导RNA之间的互补碱基配对以高特异性进行靶向(Gasiunas等人, ...

Metal-tagging Transmission Electron Microscopy for Localisation of Tombusvirus Replication Compartments in Yeast
Author:
Date:
2018-04-20
[Abstract]  Positive-stranded (+) RNA viruses are intracellular pathogens in humans, animals and plants. To build viral replicase complexes (VRCs) viruses manipulate lipid flows and reorganize subcellular membranes. Redesigned membranes concentrate viral and host factors and create an environment that facilitates the formation of VRCs within replication organelles. Therefore, efficient virus replication depends on the assembly of specialized membranes where viral macromolecular complexes are turned on and hold a variety of functions. Detailed characterization of viral replication platforms in cells requires sophisticated imaging approaches. Here we present a protocol to visualize the three-dimensional organization of the tombusvirus replicase complex in yeast with MEtal-Tagging Transmission Electron ... [摘要]  正链(+)RNA病毒是人,动物和植物中的细胞内病原体。构建病毒复制酶复合物(VRC)病毒操纵脂质流动和重组亚细胞膜。重新设计的膜集中了病毒和宿主因子,并创造了促进复制细胞器内VRC形成的环境。因此,有效的病毒复制取决于病毒大分子复合物开启并具有各种功能的特殊膜的组装。细胞中病毒复制平台的详细特征需要复杂的成像方法。在这里我们提出一个协议,用肉眼标记透射电子显微镜(METTEM)可视化酵母中的tombusvirus复制酶复合物的三维组织。该协议使我们能够用METTEM和电子断层扫描成像三维病毒复制酶分子的细胞内分布。我们的研究显示病毒复制酶分子如何在特化细胞膜内构建复制复合物。

【背景】正链RNA病毒的复制取决于细胞膜的重塑。细胞内膜作为VRC装配的结构支架,提供调节病毒复制酶活性和保护病毒RNA免受宿主抗病毒防御的必需脂质和辅因子(Miller和Krijnse-Locker,2008; den Boon <等,2010; nagy和pogany,2011;="" nagy,2016)。电子显微镜观察到具有活性vrc的复制细胞器的结构。="" vrc以单个膜囊或'小球',管状球形立方体膜,双膜囊泡(dmv)或平面寡聚体阵列装配(de="" castro等人,2013)。通常在rna病毒感染的细胞中观察到小球。它们通过在各种细胞器中内陷而形成,并具有对胞质溶胶的狭窄开口(den="" boon="" et=""> ...

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