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Optimizing a Screen for Incorporating Unnatural Amino Acids into Bacterial Proteins
Author(s) -
Dhar Shilpa,
Mammoser Claire,
Gunter Amy,
Kneusel Alyson,
Burkhart Grace,
Brown Bayland,
Rowe Laura
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.922.6
Subject(s) - green fluorescent protein , amino acid , fluorescence , fluorescent protein , transfer rna , biomolecule , biochemistry , protein biosynthesis , in vivo , chemistry , computational biology , genetic code , aminoacyl trna synthetase , biology , genetics , rna , gene , physics , quantum mechanics
Several unnatural amino acids (UAAs), including some fluorescent variants, were tested in a Green Fluorescent Protein (GFP) –based screening system that assesses the ability of the UAA's to be incorporated into proteins in vivo within E. coli bacteria. The fluorescent UAA variants were tested due to their ability to be utilized as tags or labels. Fluorescent UAAs also provide an alternative to bulkier fluorophores for applications such as more efficient cellular and protein imaging to visually track biomolecules in vivo. A number of research groups have incorporated UAAs, including fluorescent ones, into proteins using the amber suppression in vivo method also applied in this work1. However, in this work, UAA's that had never been incorporated into proteins using this method before were tested. The screening system was miniaturized and optimized so that it can be easily, and quickly used by researchers not expert in the field. Overall, over a dozen UAA's were tested with six different orthogonal tRNA/aaRS pairs2 using the screen, with several UAA's incorporating into the protein using a tRNA/aaRS that was not specific to that UAA. Moreover, the initial screen was miniaturized from 250 mL flasks to 96 well plates, and volumes, incubation times, and UAA solubility parameters were optimized in order to increase the efficiency of the screen.3.