Using E. Coli to Incorporate Fluorescent Unnatural Amino Acids into Proteins
Dr. Thomas Goyne
Arts and Sciences
0000-0003-4189-5018 (Alyson Kneusel), 0000-0001-8882-1169 (Claire Mammoser), 0000-0002-7189-0704 (Dr. Thomas Goyne), 0000-0003-3900-8379 (Dr. Laura Rowe), 0000-0003-3002-2967 (Amy Gunter)
The goal of this work is to identify unnatural amino acids (UAAs) that an be incorporated into proteins by E.coli using four orthogonal aminoacyl tRNA synthetase/suppressor tRNA (aaRS/tRNA) pairs. A fluorescence screening assay was used to test the incorporation of ten UAAs into proteins using four aaRS/tRNA pairs that had been previously shown to possess varying degrees of promiscuity. The screen utilized a mutant green fluorescent protein (GFP) that included the amber stop codon within the open reading frame. Multiple results demonstrated that aaRS/tRNA pairs are able to bind other UAAs to a level greater than the UAA designed to match that synthetase, yet all UAA mutants had significantly lower incorporation than the wild type GPF control (UAA absent). Correlation of the relative incorporation levels versus the UAA structures provides a basis for understanding the shape and flexibility of the synthetase binding pocket and thus provides experimental data that can be used to evaluate molecular modeling results.
Kneusel, Alyson; Brown, Bayland; Gunter, Amy; Mammoser, Claire C.; Goyne, Thomas; and Rowe, Laura, "Using E. Coli to Incorporate Fluorescent Unnatural Amino Acids into Proteins" (2016). Symposium on Undergraduate Research and Creative Expression (SOURCE). 581.
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