pAzFRS.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-azido-Phe and tRNA interactions and improve expression of multiple ncAA containing proteins using low RS levels. This evolved RS showed 25 fold higher expression of pAzF in GFP(3) compared to the progenitor enzyme (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 in a construct, but that only outperforms the original unevolved RS form by <2-fold when expressing GFP with 3 ncAAs, and that along with the improved expression is a much lower fidelity (higher protein production in the absence of ncAA). Note that the "pAzRS" in this figure is the pCNFRS shown in the 2011 foundational paper to work very well for pAzF. The pAzFRS.2.t1 RS was also shown to be highly promiscuous, incorporating Phe variants pAcetyl, pIodo, pBromo, pChloro, pMethyl, ptrifluoromethyl along with methylTyr, 2-napthylAla and styrylAla. This is the highest efficiency RS for pAzPhe incorporation among those evolved in this paper.