During the past decade over eighty orthogonal tRNA/aminoacyl tRNA synthetase pairs have been evolved in order to incorporate unnatural amino acids into specific locations within a protein structure in vivo.  The directed evolution method of creating these pairs focuses on negative and positive screens that remove tRNA/aaRS pairs which either do not incorporate a specific unnatural amino acid or incorporate an unnatural amino acid and natural amino acids in response to an amber codon.  However, these screening methods do not de-select pairs that could potentially incorporate unnatural amino acids other than the particular one the pair was evolved for.  Recently, a method has been developed that does allow for this screening of promiscuous unnatural amino acid incorporation with orthogonal tRNA/aaRS pairs using a green fluorescent protein (GFP) that contains an amber codon at a central, but non-essential position.  In this study, we expanded on this previously developed GFP screening method by screening additional unnatural amino acids and orthogonal tRNA/aaRS pairs that were not included in the original work.  Additionally, we worked on developing a mCherry (a red fluorescent protein) variant of the screening method that could be used to screen for incorporation of fluorescent unnatural amino acids whose emission color overlaps that of GFP.

Screening the permissivity of unnatural amino acid tRNA/aminoacyl tRNA pairs in <i>E. coli</i>

248th National Meeting (2014)

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Screening the permissivity of unnatural amino acid tRNA/aminoacyl tRNA pairs in E. coli