pAcFRS.2.t1

This RS/tRNA pair was evolved in the C321.A E. coli strain from the AcetylPhe RS (pAcF-RS) originally reported in in 2002 foundational paper, used as a model RS in the 2010 foundational paper and characterized for permissivity in the 2011 foundational paper. In 2015 further residues were allowed to evolve to optimize p-acetyl-Phe and tRNA interactions and improve expression of multiple ncAA containing proteins using low RS levels. This RS showed a 15-fold increased expression of GFP(3UAG) compared to pAcFRS using a chromosome incorporated RS. Figure 4a of foundational paper shows that when included as multicopy plasmid it still highly outperforms the starting RS for incorporating 30 ncAAs into a single protein construct, but does not outperform the original unevolved RS when expressing GFP with 3 ncAAs, and it also has a lower fidelity (higher protein production in the absence of ncAA). This RS was also shown to be highly promiscuous, incorporating Phe variants pAcetyl, pIodo, pBromo, pChloro, pMethyl, ptrifluoromethyl along with methylTyr, 2-napthylAla and styrylAla. Was selected to discrimate against pAzf and did when chromosome incorporated, but not when provided as a multicopy plasmid. Also especially effective at incorporating O-methyl-Tyr, p-IodoPhe, p-trifluormethylPhe, p-tert-butyl-Phe, O-tert-Butyl and p-phenylPhe.