Xie Zhoujie, Zhang Zhao, Cao Zhenju, Chen Meng, Li Pengwei, Liu Weifeng, Qin Hua, Zhao Xuejin, Tao Yong, Chen Yihua
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, People's Republic of China.
University of Chinese Academy of Sciences, Beijing, 100049, China.
Appl Microbiol Biotechnol. 2017 May;101(9):3811-3820. doi: 10.1007/s00253-017-8252-2. Epub 2017 Mar 28.
Since the lacZα-based blue/white screening system was introduced to molecular biology, several different visual reporter systems were developed and used for various purposes in Escherichia coli. A common limit to the existent visual reporter systems is that an extracellular chromogenic substrate has to be added for the visible pigment production. In this study, we developed a new blue/white screening system based on a non-ribosomal peptide synthetase encoded by idgS from Streptomyces and a phosphopantetheinyl transferase encoded by sfp from Bacillus. When IdgS is activated from an apo-form to a holo-form via a posttranslational modification catalyzed by Sfp, it can synthesize a blue pigment indigoidine using L-glutamine, the amino acid abundant in cells, as a substrate. The new blue/white screening system contains a recipient E. coli strain with an optimized idgS gene cassette and a cloning vector harboring an sfp gene with an in-frame insertion of a multiple cloning site close to its N-terminal. We demonstrated that the IdgS/Sfp-based blue/white screening system is a powerful alternative to the lacZα-based screening system, which does not require any external substrate addition.
自从基于lacZα的蓝/白筛选系统被引入分子生物学以来，已经开发了几种不同的视觉报告系统，并在大肠杆菌中用于各种目的。现有视觉报告系统的一个共同局限是，必须添加细胞外显色底物才能产生可见色素。在本研究中，我们基于链霉菌idgS编码的非核糖体肽合成酶和芽孢杆菌sfp编码的磷酸泛酰巯基乙胺基转移酶，开发了一种新的蓝/白筛选系统。当IdgS通过Sfp催化的翻译后修饰从脱辅基形式激活为全酶形式时，它可以使用细胞中丰富的氨基酸L-谷氨酰胺作为底物合成蓝色色素靛蓝。新的蓝/白筛选系统包含一个带有优化的idgS基因盒的受体大肠杆菌菌株和一个克隆载体，该载体带有一个sfp基因，在其N端附近有一个读码框内插入的多克隆位点。我们证明，基于IdgS/Sfp的蓝/白筛选系统是基于lacZα的筛选系统的有力替代方案，该系统不需要添加任何外部底物。
