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使用CRISPR干扰 (CRISPRi) 技术评估基因沉默的实用实验室课程,用于考古团Haloferax volcanii中的基因沉默

R A Paggi¹, M C Ferrari¹, M Cerletti¹

¹Instituto de Investigaciones Biológicas (IIB-CONICET-UNMDP), Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata, Mar del Plata, Argentina.

Biochemistry and molecular biology education : a bimonthly publication of the International Union of Biochemistry and Molecular Biology

|December 19, 2024

概括

这项研究介绍了一种使用CRISPR干扰 (CRISPRi) 沉默Haloferax volcanii中的lonB基因的实验室类. 学生们学习了基因沉默及其对胡卜素生产和生长的影响.

科学领域:

微生物学微生物学
分子生物学分子生物学
生物化学生物化学

背景情况:

使用RNA干扰 (RNAi) 和CRISPR干扰 (CRISPRi) 的基因沉默有助于理解基本的基因功能.
在Haloferax volcanii中,CRISPR-Cas系统已被适应用于CRISPRi介导的基因沉默.
在H. volcanii中的LonB蛋白酶基因调节着胡卜素生物合成.

研究的目的:

为本科生开发一个实验室课程,以学习使用CRISPRi的基因沉默.
研究H. volcanii lonB基因作为胡卜素生物合成的负调节器的功能.
评估lonB基因沉默对色素和生长速度的影响.

主要方法:

一个实验室课程的开发,重点是对H. volcanii.中的CRISPRi基因沉默.
通过CRISPRi通过减少lonB基因表达的H. volcanii突变菌株的生成.
分析细胞颜色,生长速度和lonB基因抑制,使用西方涂抹 (WB).

主要成果:

在H. volcanii.中成功生成和选CRISPRi沉默的lonB突变菌株.
观察到lonB基因沉默对细胞色素和生长速度的影响.
通过西式涂抹证明了lonB基因表达的抑制.

结论:

开发的实验室课程有效地教导使用CRISPRi在非致病性archaeon中的基因沉默技术.
学生在微生物学,生物化学和分子生物学协议方面获得实践经验.
该课程促进协作工作,数据解释和科学沟通技巧.

关键词:

这项技术是CRISPRi技术.一个LonB蛋白质酶基因基因.考古学是指古物质 (archaea) 是指古物质.胡卜素是一种胡卜素.实验室类是实验室类的转录压制压制

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