| Genomic Edition of Ashbya gossypii Using One-vector CRISPR/Cas9
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Author:
Date:
2020-06-20
[Abstract] The CRISPR/Cas9 system is a novel genetic tool which allows the precise manipulation of virtually any genomic sequence. In this protocol, we use a specific CRISPR/Cas9 system for the manipulation of Ashbya gossypii. The filamentous fungus A. gossypii is currently used for the industrial production of riboflavin (vitamina B2). In addition, A. gossypii produces other high-value compounds such as folic acid, nucleosides and biolipids. A large molecular toolbox is available for the genomic manipulation of this fungus including gene targeting methods, rapid assembly of heterologous expression modules and, recently, a one-vector CRISPR/Cas9 editing system adapted for A. gossypii that allows marker-free engineering strategies to be implemented. The CRISPR/Cas9 ...
[摘要] [摘要] CRISPR/Cas9系统是一种新的基因工具，可以精确地操作几乎所有的基因组序列。在这个协议中，我们使用一个特定的CRISPR/Cas9系统来操纵棉蚜。丝状真菌A.gossypii目前用于核黄素（vitamina B2）的工业生产。此外，棉蚜还产生其他高价值化合物，如叶酸、核苷和生物脂类。一个大型的分子工具箱可用于该真菌的基因组操作，包括基因靶向方法、异源表达模块的快速组装，以及最近一个适用于棉铃虫的单载体CRISPR/Cas9编辑系统，该系统允许实施无标记工程策略。CRISPR/Cas9系统包括RNA引导的DNA内切酶（Cas9）和与基因组靶区互补的引导RNA（gRNA）。Cas9核酸酶需要5′-NGG-3′三核苷酸，称为原间隔基序（PAM），在基因组靶区产生一个双链断裂（DSB），该断裂可以通过同源重组（HR）由合成的致突变供体DNA（dDNA）修复，从而引入一个特定的设计突变。适用于棉铃虫的CRISPR/Cas9系统极大地促进了这种工业真菌的基因组编辑。
[背景] ...
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| Preparation of Golgi Membranes from Rat Liver
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Author:
Date:
2014-11-20
[Abstract] This protocol details the isolation of enriched Golgi membranes from rat liver, using discontinuous density gradient centrifugation. This high-yield extraction method is useful for several applications, including immunoprecipitation of solubilised Golgi membrane proteins (preparation included) and electron microscopy. Protocol adapted from Leelavathi et al. (1970).
[摘要] 该协议详述了使用不连续密度梯度离心从大鼠肝脏分离富集的高尔基体膜。 这种高产率提取方法可用于几种应用，包括溶解的高尔基膜蛋白（包括制备）和电子显微镜的免疫沉淀。 从Leelavathi等人改编的方案（1970）。
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