title: An external substrate-free blue/white screening system in Escherichia coli.
abstract: 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.
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, People's Republic of China.
authors.forename: Zhoujie
authors.initials: Z
authors.lastname: Xie
authors.affiliation: 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.
authors.forename: Zhao
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: 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.
authors.forename: Zhenju
authors.initials: Z
authors.lastname: Cao
authors.affiliation: 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.
authors.forename: Meng
authors.initials: M
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, People's Republic of China.
authors.forename: Pengwei
authors.initials: P
authors.lastname: Li
authors.affiliation: CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China.
authors.forename: Weifeng
authors.initials: W
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, People's Republic of China.
authors.forename: Hua
authors.initials: H
authors.lastname: Qin
authors.affiliation: CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China.
authors.forename: Xuejin
authors.initials: X
authors.lastname: Zhao
authors.affiliation: University of Chinese Academy of Sciences, Beijing, 100049, China.|CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Tao
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, People's Republic of China. chenyihua@im.ac.cn.|University of Chinese Academy of Sciences, Beijing, 100049, China. chenyihua@im.ac.cn.
authors.forename: Yihua
authors.initials: Y
authors.lastname: Chen
mesh_terms: D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D003116:Color; D004926:Escherichia coli / Q000235:genetics*; D005820:Genetic Testing / Q000379:methods*; D005822:Genetic Vectors; D005827:Genetics, Microbial / Q000379:methods*; D008967:Molecular Biology / Q000379:methods*; D010453:Peptide Synthases / Q000235:genetics / Q000378:metabolism; D010860:Pigments, Biological / Q000032:analysis*; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics; D017855:Transferases (Other Substituted Phosphate Groups) / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D010860:Pigments, Biological; C082662:phosphopantetheinyl transferase; D017855:Transferases (Other Substituted Phosphate Groups); D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase
keywords: Blue/white screening; External substrate-free; Indigoidine synthetase; Sfp