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菌根菌株的有针对性的突变通过同源重组

Shikui Song¹, Zhengding Su²

¹Laboratory of Protein Engineering and Biopharmaceutical Sciences, Key Laboratory of Industrial Fermentation and Cooperative Innovation Center of Industrial Fermentation, Hubei University of Technology, Wuhan, Hubei, China.

Methods in molecular biology (Clifton, N.J.)

|August 29, 2023

概括

这项研究引入了通过同源重组 (TMHR) 进行向突变发生的方法,以实现高效的细菌基因组工程. 这种方法可以在两周内在Mycobacterium neoaurum HGMS2中实现无基因淘汰和淘汰.

科学领域:

合成生物学合成生物学
细菌遗传学细菌遗传学
分子生物学分子生物学

背景情况:

通过同源重组 (TMHR) 进行向突变发生提供了一种高效的基交换方法,用于细菌基因组工程.
与其他方法不同,TMHR不需要异构重组酶来进行可选标记物插入或切除.
选择是通过使用自杀载体编码的蛋白质和宿主细胞蛋白质实现的.

研究的目的:

描述使用TMHR的基因淘汰和淘汰的简要协议.
通过准3 - 固醇-1,2 - 脱酶基因 (kstd) 来设计Mycobacterium neoaurum HGMS2.

主要方法:

通过PCR通过kstd基因侧面的同样性臂的放大.
将同源臂的亚克隆化变成同源重组载体.
载体的电穿孔进入Mycobacterium neoaurum HGMS2有能力的细胞.
利用抗生素耐药性的单交叉重组和无标记的双交叉突变的糖糖介导的反选择.

主要成果:

在Mycobacterium neoaurum HGMS2.2.中成功淘汰和淘汰kstd基因.
无突变的产生,直到一个单一的DNA基对.
在不到两周的时间内完成了整个过程.

结论:

TMHR提供了一种高效,快速的细菌基因组工程方法.
这一协议促进了Mycobacterium neoaurum中精确的基因操纵.
无标记突变能力扩大了合成生物学和遗传学研究中的应用.

关键词:

3 - 奥克斯类1-脱酶.基因淘汰赛基因淘汰赛同类的重组组合.在Knockin中敲打.变异发生的突变.这是一种Mycobacterium细菌.这是一个PCRPCR.

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