用于提高大肠杆菌中操作效率的苯丙氨酸-tRNA合成酶反选择标记的分子工程。
Molecular engineering of a PheS counterselection marker for improved operating efficiency in Escherichia coli.
作者信息
Miyazaki Kentaro
机构信息
Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Sapporo, Hokkaido, Japan.
Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Sapporo, Hokkaido, Japan.
出版信息
Biotechniques. 2015 Feb 1;58(2):86-8. doi: 10.2144/000114257. eCollection 2015 Feb.
Escherichia coli phenylalanyl-tRNA synthetase, a-subunit (ePheS) can be useful as a counterselection marker since its A294G variant misincorporates 4-chloro-phenylalanine (4CP) into cellular proteins during translation, thereby causing cell death. The drawback of this method is that selection must be performed in minimal or semisynthetic medium to avoid interference from phenylalanine in the medium. Here, I reengineered ePheS for improved 4CP incorporation efficiency, obtaining variants (T251A/ A294G and T251S/A294G) that exhibited high lethality in Luria-Bertani medium (LB) containing 4CP. These new variants were superior to the A294G variant when used as a counterselection marker in vector curing experiments.
大肠杆菌苯丙氨酰 - tRNA合成酶α亚基（ePheS）可作为反向选择标记，因为其A294G变体在翻译过程中将4 - 氯苯丙氨酸（4CP）错误掺入细胞蛋白质中，从而导致细胞死亡。该方法的缺点是必须在基本培养基或半合成培养基中进行选择，以避免培养基中苯丙氨酸的干扰。在此，我对ePheS进行了改造以提高4CP掺入效率，获得了在含有4CP的LB培养基中表现出高致死率的变体（T251A / A294G和T251S / A294G）。在载体清除实验中用作反向选择标记时，这些新变体优于A294G变体。
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