Molecular Engineering of a PheS Counterselection Marker for Improved Operating Efficiency in Escherichia Coli

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.