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在Bacillus Subtilis中对重组蛋白质表达的关键基因进行全基因组的CRISPRi查
Xuyang Zhu1, Hui Luo1, Xinrui Yu1
1School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of Education, State Key Laboratory of Food Science and Resources, International Joint Laboratory on Food Safety, Jiangnan University, Wuxi, 214122, China.
概括
这项研究引入了一种新的CRISPRi和CRISPRa选策略,以确定用于增强Bacillus subtilis (B. subtilis) 微生物细胞工厂中复合蛋白质生产的关键基因. 该方法成功识别了关键的基因并改善了蛋白质表达,为先进的细胞工厂设计铺平了道路.
科学领域:
- 微生物学 微生物学
- 合成生物学 合成生物学
- 生物技术是生物技术.
背景情况:
- 细菌细菌是重组蛋白质生产的关键微生物细胞工厂,因其安全性和分泌能力而受到重视.
- 对功能基因的有限知识阻碍了B. subtilis进一步优化,以增强蛋白质表达.
- 开发高效的遗传工具对于推进工业微生物应用至关重要.
研究的目的:
- 开发和验证一个结合的全基因组CRISPR干扰 (CRISPRi) 查和CRISPR激活 (CRISPRa) 策略.
- 确定调节B. subtilis.重组蛋白质生产的新基因.
- 提高B. subtilis作为工业蛋白质合成的细胞工厂的效率.
主要方法:
- 构建一个全基因组的CRISPRi库,针对B. subtilis.中的4225个编码基因.
- 高通量查以确定影响重组蛋白表达的基因.
- 应用CRISPRa来调节目标基因转录以提高蛋白质的生产.
- 转录组分析以了解通路表达关系.
- 高密度发酵和通用性验证.
主要成果:
- 鉴定了12个关键基因,这些基因对于重组蛋白质表达至关重要,包括以前未被描述的基因.
- 使用CRISPRa显著增强复合蛋白基因转录.
- 通过转录组分析阐明工程菌株中差异性通路作用.
- 通过高密度发酵和验证来证明战略的可靠性.
结论:
- 结合的CRISPRi和CRISPRa方法是功能性基因发现和B. subtilis.代谢工程的一个强大的工具.
- 这一策略有效地提高了重组蛋白质的生产,并促进了改进的微生物细胞工厂的设计.
- 该方法适用于其他工业微生物宿主,有助于基因注释和新型细胞工厂的开发.
相关概念视频
01:59CRISPR
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 Short...
02:53CRISPR 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...
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...
01:59CRISPR
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 Short...