Synthesizing Green Fluorescent Protein with Unnatural Amino Acids

The first task of the summer was synthesizing and purifying green fluorescent protein (GFP) with various unnatural amino acids. The unnatural amino acids (UAAs) that were of interest were 3-fluorotyrosine (3FY), ortho-nitrobenzyl tyrosine (ONBY), and p-propargyloxyphenylalanine (pPrF). The first step in the synthesis of GFP with the UAAs is inserting the genes that code for GFP (either TAG-66 or Waldo) and the genes for the enzyme that inserts the UAA into the protein (a synthetase) into E. coli by electroporation. The transformation of the bacteria with the plasmids was verified by plating the cells onto agar containing the antibiotics ampicillin and chloramphenicol. Since the plasmids contain antibiotic resistance genes, only the cells that have been successfully transformed will grow on the plates. After this, colonies are transferred from the plates into culture vials and induced with IPTG/arabinose and the desired UAA. Once the cells have grown and expressed the protein overnight, the protein is extracted from the cells and purified. To verify that the cells have expressed the protein of interest and that it has been successfully purified, the protein sample is run on a gel by gel electrophoresis.This process has led to some unexpected challenges as often times you are unable to visualize the protein on the gel.  Some issues faced throughout this process include unsuccessful transformation of the bacteria, phage infections in some of the culture media and plates, insufficient expression of the protein, and failure to synthesize the UAA. After 5 weeks of trial and error, I was finally able to obtain GFP with the UAAs of interest.