Journal
BIOTECHNIQUES
Volume 58, Issue 2, Pages 86-88Publisher
FUTURE SCI LTD
DOI: 10.2144/000114257
Keywords
counterselection; Escherichia coli; PheS; phenylalanyl-tRNA synthetase; 4-chloro-phenylalanine
Funding
- Japan Society for the Promotion of Science (JSPS) [26292048, 26670219]
- Grants-in-Aid for Scientific Research [26292048, 26670219] Funding Source: KAKEN
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Escherichia coli phenylalanyl-tRNA synthetase, alpha-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.
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