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.
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Acknowledgements
The study was funded in part by the Ministry of Science and Technology of China (2015CB150600 and 2013CB734000) and the National Natural Science Foundation of China (31400048 and 31522001). Y.C. is an awardee for the “Hundred Talents Program” of the Chinese Academy of Sciences.
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Yihua Chen and Zhoujie Xie have filed a patent application related to this work.
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Xie, Z., Zhang, Z., Cao, Z. et al. An external substrate-free blue/white screening system in Escherichia coli . Appl Microbiol Biotechnol 101, 3811–3820 (2017). https://doi.org/10.1007/s00253-017-8252-2
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DOI: https://doi.org/10.1007/s00253-017-8252-2