相关实验视频

Updated: May 10, 2025

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

Published on: February 5, 2015

在大肠杆菌中增强功能CRISPR-AsCas12a蛋白的产生

Orlando S Goméz-Quintero¹, Melissa D Morales-Moreno¹, Erick G Valdés-Galindo¹

¹Laboratory of Biomolecular Engineering and Bionanotechnology, Departamento de Biomacromoléculas, Instituto de Química, Universidad Nacional Autónoma de México, Ciudad de México, Mexico.

Protein expression and purification

|April 27, 2025

概括

这项研究优化了大肠杆菌中CRISPR-Cas12a蛋白质的产生,实现了产量增加4.5倍. 该增强方法为基因组编辑和SARS-CoV-2检测提供了功能AsCas12a.

科学领域:

生物技术是生物技术.
分子生物学分子生物学
基因组学就是基因组学.

背景情况:

在基因组编辑和诊断方面,CRISPR-Cas12a是至关重要的.
实验室规模优化Cas12a蛋白质的生产是有限的.
有效的生产方法是广泛研究应用所需的.

研究的目的:

为了增强实验室规模的重组生产的Acidaminococcus sp Cas12a (AsCas12a) 在大肠杆菌.
确定简单,有效的参数来增加AsCas12a产量.
为了验证优化的AsCas12a.a的功能活动.

主要方法:

使用大肠杆菌BL21(DE3) 进行重组蛋白质表达.
优化的生长介质 (TB + 1% 葡萄糖) 和诱导条件 (0.3 mM IPTG, 6-9h, 30°C).
采用染色学净化方法来分离功能AsCas12a.

主要成果:

在每毫升细菌溶解酸 (0.95至3.73毫克/毫升) 中,AsCas12a的增加达到3倍.
净化后的最终蛋白质产量增加了大约4.5倍 (5.2至23.4毫克/升).
保持了DNA cis-和 trans-cleavage 的完整功能活动.

结论:

开发了一种高效,高产量的协议,用于实验室规模的AsCas12a生产.
优化的方法可供许多研究实验室使用.
功能性AsCas12a成功检测到了SARS-CoV-2 N基因,证明了诊断效用.

关键词:

这就是CRISPR-Cas12a.实验室规模的生产生产.优化优化优化优化蛋白质的生物合成.再组合产品的生产.

更多相关视频

Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins

Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins

Published on: October 18, 2022

CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art

CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art

Published on: May 28, 2019

相关概念视频

CRISPR and crRNAs

CRISPR and crRNAs

Bacteria and archaea are susceptible to viral infections just like eukaryotes; therefore, they have developed a unique adaptive immune system to protect themselves. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) are present in more than 45% of known bacteria and 90% of known archaea.
The CRISPR-Cas system stores a copy of foreign DNA in the host genome and uses it to identify the foreign DNA upon reinfection. CRISPR-Cas has three different...

CRISPR

CRISPR

Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced...