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Dextrose (D-Glucose) Anhydrous

Company: Fisher Scientific
Catalog#: D16
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Quantification of Hydrogen Sulfide and Cysteine Excreted by Bacterial Cells
Author:
Date:
2018-05-20
[Abstract]  Bacteria release cysteine to moderate the size of their intracellular pools. They can also evolve hydrogen sulfide, either through dissimilatory reduction of oxidized forms of sulfur or through the deliberate or inadvertent degradation of intracellular cysteine. These processes can have important consequences upon microbial communities, because excreted cysteine autoxidizes to generate hydrogen peroxide, and hydrogen sulfide is a potentially toxic species that can block aerobic respiration by inhibiting cytochrome oxidases. Lead acetate strips can be used to obtain semiquantitative data of sulfide evolution (Oguri et al., 2012). Here we describe methods that allow more-quantitative and discriminatory measures of cysteine and hydrogen sulfide release from bacterial cells. An ... [摘要]  细菌释放半胱氨酸以调节细胞内池的大小。它们也可以通过硫的氧化形式的异化还原或通过细胞内半胱氨酸的故意或无意降解来释放硫化氢。这些过程会对微生物群落产生重要影响,因为排泄的半胱氨酸会自动氧化生成过氧化氢,而硫化氢是一种潜在的毒性物种,可通过抑制细胞色素氧化酶来阻断有氧呼吸。醋酸铅条可用于获得硫化物演化的半定量数据(Oguri et al。,2012)。在这里,我们描述的方法,允许更多的定量和歧视措施半胱氨酸和硫化氢释放细菌细胞。提供了一个说明性实例,其中当暴露于外源性胱氨酸时,大肠杆菌迅速产生半胱氨酸和硫化物(Chonoles Imlay等人,2015; Korshunov等人, ,2016)。

【背景】微生物通过几种途径产生了减少的硫物质。硫酸盐还原菌利用还原过程作为能量生成的组成部分。其他细菌释放硫化物,作为硫物质(包括半胱氨酸)的蓄意或偶然降解的副产物。我们观察到半胱氨酸本身是在细胞内水平异常高时排泄的,这种情况可能通过不受控制的氨基酸输入或半胱氨酸合成失调发生。这些硫物质具有非同寻常的反应性,因为它们以高亲和力与金属结合,也是与分子氧发生化学反应的少数生物分子之一。结果是减少的硫化合物可以对细胞产生重要影响。因此,跟踪各种情况下含硫化合物的动态变化是非常重要的。

硫醇试剂 - 特别是5,5-二硫代双(2-硝基苯甲酸)(DTNB) ...

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, ...

Method for Multiplexing CRISPR/Cas9 in Saccharomyces cerevisiae Using Artificial Target DNA Sequences
Author:
Date:
2017-09-20
[Abstract]  Genome manipulation has become more accessible given the advent of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) editing technology. The Cas9 endonuclease binds a single stranded (single guide) RNA (sgRNA) fragment that recruits the complex to a corresponding genomic target sequence where it induces a double stranded break. Eukaryotic repair systems allow for the introduction of exogenous DNA, repair of existing mutations, or deletion of endogenous gene products. Targeting of Cas9 to multiple genomic positions (termed ‘multiplexing’) is achieved by the expression of multiple sgRNAs within the same nucleus. However, an ongoing concern of the CRISPR field has been the accidental targeting of Cas9 to alternative (‘off-target’) DNA locations within a genome. We ... [摘要]  鉴于CRISPR(集群定期间隔短回归重复)编辑技术的出现,基因组操纵变得更加易于使用。 Cas9核酸内切酶将募集复合物的单链(单向导)RNA(sgRNA)片段结合到相应的基因组靶序列,引发双链断裂。真核修复系统允许引入外源DNA,修复现有突变或内源基因产物的缺失。通过在同一核内表达多个sgRNA来实现Cas9对多个基因组位置的定位(称为“多重”)。然而,CRISPR领域的持续关注是将Cas9意外地定位到基因组内的替代(“脱靶”)DNA位置。我们将安装的人造Cas9靶序列的使用(称为人造基因座上的Cas9复制)描述为允许(i)与单个sgRNA复用的酵母基因组中的用途; (ii)减少/消除可能的脱靶效应,以及(iii)精确控制预定目标序列的放置。
【背景】CRISPR(集群定期间隔回归重复)机制已经在原核生物中演变为具有很高精度编辑任何基因组的能力的原始适应性免疫系统(Jinek等,2012; Sorek等,2013)。这种生物技术需要使用来自化脓性链球菌(或othologous物种)的内切核酸酶(Cas9),单个RNA'引导'序列和外源供体DNA(如果需要)。仅在短短几年内,CRISPR / ...

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