An external substrate-free blue/white screening system in Escherichia coli

Appl Microbiol Biotechnol. 2017 May;101(9):3811-3820. doi: 10.1007/s00253-017-8252-2. Epub 2017 Mar 28.

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.

Keywords: Blue/white screening; External substrate-free; Indigoidine synthetase; Sfp.

MeSH terms

  • Bacillus / enzymology
  • Bacillus / genetics
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Cloning, Molecular*
  • Color
  • Escherichia coli / genetics*
  • Genetic Testing / methods*
  • Genetic Vectors*
  • Genetics, Microbial / methods*
  • Molecular Biology / methods*
  • Peptide Synthases / genetics
  • Peptide Synthases / metabolism
  • Pigments, Biological / analysis*
  • Streptomyces / enzymology
  • Streptomyces / genetics
  • Transferases (Other Substituted Phosphate Groups) / genetics
  • Transferases (Other Substituted Phosphate Groups) / metabolism

Substances

  • Bacterial Proteins
  • Pigments, Biological
  • phosphopantetheinyl transferase
  • Transferases (Other Substituted Phosphate Groups)
  • Peptide Synthases
  • non-ribosomal peptide synthase