Title

Using E. Coli to Incorporate Fluorescent Unnatural Amino Acids into Proteins

Faculty Sponsor

Dr. Thomas Goyne

College

Arts and Sciences

Department/Program

Chemistry Department

ORCID Identifier(s)

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)

Document Type

Poster Presentation

Celebration Date

Summer 7-28-2016

Abstract

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.

Biographical Information about Author(s)

Alyson Kneusel is a Chemistry and Biology double major at Valparaiso University. She has interests in the biomedical field and plans to go to graduate school and pursue a career in biomedical research following her graduation from Valparaiso University in 2018. This project was of particular interest to her due to its biochemical subject area and potential biomedical applications.

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