title: Molecular engineering of a PheS counterselection marker for improved operating efficiency in Escherichia coli.
abstract: 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.
authors: 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.
authors: Kentaro
authors: K
authors: Miyazaki
mesh_terms: D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D005453:Fluorescence; D005798:Genes, Bacterial; D005818:Genetic Engineering / Q000379:methods*; D005819:Genetic Markers / Q000235:genetics; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D010652:Phenylalanine-tRNA Ligase / Q000235:genetics* / Q000378:metabolism*; D012641:Selection, Genetic
chemical_list: D005819:Genetic Markers; D050505:Mutant Proteins; D010652:Phenylalanine-tRNA Ligase
keywords: 4-chloro-phenylalanine; Escherichia coli; PheS; counterselection; phenylalanyl-tRNA synthetase