生物学杂志

生物学杂志 ›› 2019, Vol. 36 ›› Issue (6): 17-.doi: 10.3969/j.issn.2095-1736.2019.06.017

• 研究报告 • 上一篇    下一篇

利用CRISPR/Cas9系统敲除大肠杆菌tnaA基因

  

  1. 复旦大学 生命科学学院， 上海 200433
  • 出版日期:2019-12-18 发布日期:2019-12-12
  • 通讯作者: 乐科易，讲师，研究方向为微生物菌种基因工程改造及微生物产物的发酵研究，E-mail：Kyle@fudan.edu.cn
  • 作者简介:何京桦，硕士研究生，研究方向为微生物菌种基因工程改造，E-mail：fudan2001@163.com

CRISPR/Cas9-mediated tnaA knockout in Escherichia coli 

  1. School of Life Sciences, Fudan University, Shanghai 200433, China
  • Online:2019-12-18 Published:2019-12-12

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摘要：

大肠杆菌的tnaA基因编码色氨酸酶，该基因的失活有利于大肠杆菌积累更多的L-色氨酸。利用CRISPR/Cas9系统介导的基因编辑技术对大肠杆菌tnaA基因进行敲除。首先针对tnaA基因设计CRISPR/Cas9作用靶点，构建gRNA表达质粒；随后人工设计同源修复供体基因序列，构建成完整的CRISPR/Cas9基因敲除系统；将该系统应用到大肠杆菌w3110-Δ中，经PCR和测序鉴定，证实目的基因tnaA敲除成功，敲除效率75%。成功构建了大肠杆菌tnaA基因敲除菌，为大肠杆菌L-色氨酸高产菌株的构建奠定了基础。


关键词: CRISPR/Cas9系统, 敲除, 大肠杆菌, tnaA基因

Abstract:

In Escherichia coli， tnaA gene encodes tryptophanase and it′s inactivation contributes to the L-tryptophan accumulation in E.coli. In the present study, CRISPR/Cas9-mediated gene editing technology was used to knockout the tnaA gene of E.coli. Sequence targeted by CRISPR/Cas9 was designed and gRNA expression plasmid was constructed. Donor DNA for homologous repair was constructed. Donor DNA, gRNA expression plasmid and pCas plasmid co-constituted the CRISPR/Csa9 system. This system was performed on E.coli w3110-Δ. PCR and sequencing results confirmed the successful knockout of tnaA gene with the knockout efficiency of 75%. In conclusion, CRISPR/Cas9-mediated knockout system for E.coli tnaA gene was successfully established, which would provide an effective tool for strain construction for L-tryptophan production.


Key words: CRISPR/Cas9 system； knockout, Escherichia coli； tnaA gene

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