Screening Unnatural Fluorescent Amino Acids for Incorporation into E. Coli Cellular Machinery
Arts and Sciences
Fluorescence can be used in optical imaging to view cell activity in vivo. Fluorescent proteins and organic dyes are the primary method of visually tracking biomolecules in vivo. However, several research groups have also incorporated unnatural fluorescent amino acids (UFAAs) into proteins for in vivo analysis of said proteins. However, only a handful of UFAAs have been successfully incorporated into proteins using this method (sometimes called the amber suppression method)1. To expand the library of UFAAs viable for cell imaging, several UFAAs were tested in a Green Fluorescent Protein (GFP) screening system to determine if these UFAAs could be incorporated into GFP in response to an amber stop codon using previously developed ‘promiscuous’ tRNA-aaRS (aminoacyl tRNA synthetase) pairs2. This incorporation was quantified by fluorescence readings of mutated green fluorescent protein (GFP). The GFP was mutated to have a TAG (stop) codon deactivating its fluorescence. This deactivation could be overcome by the incorporation of a UFAA in response to the TAG codon, achieved by insertion of the UFAA by the tRNA specific to this codon. Several different UFAAs and several different amber suppression based tRNA/aaRS pairs were analyzed with this screening system, and the viability of these UFAAs to be incorporated into proteins using available amber suppression systems assessed.
1. Lampkowski, Jessica S.; Uthappa, Diya M.; Young, Douglas D.. Site-specific incorporation of a fluorescent terphenyl unnatural amino acid. Bioorg. Med. Chem. Lett. 2015, Available online 25 September 2015, accessed 10 October 2015.
2. Wang, Feng; Niu, Wei; Guo, Jianto; Schultz, Peter G.. Unnatural Amino Acid Mutagenesis of Fluorescent Proteins. Angew. Chem. Int. Ed. 2012, 51, 10132 –10135.
Dhar, Shilpa and Mammoser, Claire C., "Screening Unnatural Fluorescent Amino Acids for Incorporation into E. Coli Cellular Machinery" (2016). Symposium on Undergraduate Research and Creative Expression (SOURCE). 538.