RRC ID 58474
Author Miyazaki K.
Title Molecular engineering of a PheS counterselection marker for improved operating efficiency in Escherichia coli.
Journal Biotechniques
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.
Volume 58(2)
Pages 86-8
Published 2015-2-1
DOI 10.2144/000114257
PII 000114257
PMID 25652032
MeSH Escherichia coli / enzymology* Escherichia coli / genetics* Escherichia coli / growth & development Fluorescence Genes, Bacterial Genetic Engineering / methods* Genetic Markers / genetics Mutant Proteins / genetics Mutant Proteins / metabolism Phenylalanine-tRNA Ligase / genetics* Phenylalanine-tRNA Ligase / metabolism* Selection, Genetic*
IF 1.541
Times Cited 22
DNA material pUC18K_ePAG2 (RDB17947) pUC18Z_ePAG2 (RDB17948)