Team:ULaVerne Collab/Improve
As an improvement to Sydney Australia's project, we submitted our main composite part which is the LacP + RFP + single-chain long lasting insulin. The alterations that this part has are that we added Lac promoter which is an inducible promoter that will yield higher amounts of protein. Also, we decided to use a medium RBS in between the LacP and RFP. In addition, we added some GSS linkers next to the His Tag to add more flexibility to the circuit and avoid steric hindrance. The TEV tag has a different sequence which is ENLYFQ/S. In addition, we built another linker chain to hold together the A chain and B chain of insulin. Changing the sequence of the linker (GGYLGGGGGGGR) allows us to decrease the isoelectric point of the Long Lasting single chain insulin. If the isoelectric point is closer to physiological pH (0.73-0.75), then when patients inject themselves, insulin stays in the tissues for longer time and it eventually diffuses through the bloodstream. Once it diffuses to the bloodstream it changes its conformation from a hexamer to a monomer which is the form that binds to the insulin receptor. In addition, we made a mutation in the A chain of the insulin since we changed amino acid 21 Aspartic acid to Alanine. We based this mutation on Glargine Insulin that is currently sold on the market. However, their mutation is from Aspartic acid to Glycine. Since this mutation has a patent, we decided to make a mutation based on the conformation of glycine. Therefore, alanine was chosen for our mutation because it was the most similar amino acid to glycine.
Figure 1. Image of overnight expressing gene reporter RFP