Title

Screening for Unnatural Amino Acid Incorporation in Cellular Machinery

Faculty Sponsor

Dr. Laura Rowe

College

Arts and Sciences

Department/Program

Chemistry

ORCID Identifier(s)

0000-0001-8882-1169, 0000-0003-3002-2967

Document Type

Poster Presentation

Celebration Date

Summer 7-28-2016

Abstract

Optical imaging using fluorescence is limited by fluorophore size and unsuitability for in vivo analysis. Unnatural fluorescent amino acids (UFAAs) provide an alternative to bulkier fluorophores for cellular and protein imaging. Additional small, red-shifted, nontoxic, and cell-permeable fluorophores that can be incorporated into proteins in vivo would be a significant advancement in the field (1-4). In order to find these fluorophores, the viability of pairings of UFAAs and the aminoacyl tRNA synthetases (tRNA-aaRS) which attach them to tRNA molecules were tested. Our work has screened seven UFAAs with four tRNA-aaRS developed for specific UFAAs, looking for incorporation into green fluorescent protein (GFP) plasmids using the amber suppression method. A stop (TAG) codon in a GFP plasmid inserted into the host E. Coli causes incomplete formation of the barrel-shaped protein, which then does not fluoresce. If the stop codon, however, is suppressed by the incorporation of a UFAA, GFP fluorescence is expressed from the complete protein, and this fluorescence can be measured and is proportional to the amount of incorporation. Overall, positive and negative controls behave as expected, and other promising hits will be further examined. Miniaturization of the screening has allowed the process to be performed in a microtiter plate, reducing screening time from 3 days to 1.5 days and enabled analyzation by a single instrument.

1.Lang, Chin. Chemical Reviews 2014.

2.Young. Biochemistry 2011.

3.Miyake-Stoner. Biochemistry 2010.

4.Speight. JACS 2013.

Biographical Information about Author(s)

Claire Mammoser is a junior chemistry major at Valparaiso University. She plans to attend graduate school in chemistry following completion of her degree.

Amy Gunter is a junior biochemistry major at Valparaiso University. She plans to attend graduate school in biochemistry following completion of her degree.

Bayland Brown is a junior nursing major at Ivy Tech Community College.

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