Team:ULaVerne Collab/Construct-two
Design Iterations
For our second construct we decided to use a Lac Promoter since it could behave as a constitutive promoter or an inducible promoter depending on the conditions in where the circuit is located. In addition, we decided to stay with the medium RBS from construct # 1. RFP is still our reporter gene. Ecotin tag is present to send proteins to the periplasmic membrane to form disulfide bonds. In this circuit we incorporated a GSS linker because in the previous construct we noticed that our nickel purification was not giving us positive results. Therefore, by incorporating a flexible linker in between the Ecotin tag and His tag and another in between the His Tag and the TEV tag, the small his tag protein would not be hided in between such big proteins. His tag was still used in this construct because the purification process was still the same as construct in which nickel beads will be used to isolate our construct from any other E. Coli protein. TEV tag is also use in this construct as cleavage location in order to isolate the target protein which could be proinsulin or our single chain insulins. In this construct, our negative control does have a TEV tag as well.
For our second construct, we decided to create the following circuits:
Experimental Circuits
Figure 1. Design of our experimental circuit containing different basic parts. The lac promoter, medium RBS (BBa_B0032) and RFP were all basic parts looked in the registry. The circuit is also composed of an Ecotin tag, GSS linkers next to the His tag, a His tag, a TEV tag. Since this is our experimental circuit, the C peptide is not present. Therefore, we included different linkers in the single chain insulin. At the end of the circuit we included a double terminator.
We created three experimental circuits. The modified insulin in the first experimental circuit has a pI of 5.50, native B & A chains and a 12 amino acid linker chain GGYLPGGGVGR . Our second experimental circuit is meant to be a fast acting insulin. Therefore, it has the same pI of 5.50 and the same 12 amino acid linker chain GGYLPGGGVGR . Also, we incorporated some mutations in the B chain since we flipped the amino acid B28 & B29. This mutations yield a change known as a Lispro change which has be done in one of the insulin that is already in the market. Our third experimental circuit is meant to be a long lasting insulin. The modified insulin has a linker that contains 12 amino acids GGYLGGGGGGGR and overall it has a pI of 6.46. This design was inspired on the Glargine insulin that is already manufactured. Thus, we mutated the 21st amino acid on the A chain from a Aparagine acid to Alanine
Figure 2. Experimental insulin proteins with different linkers and pI
Control Circuit
Figure 3. Design of our positive control circuit containing different basic parts. The lac promoter, medium RBS (BBa_B0032) and RFP were all basic parts looked in the registry. The circuit is also composed of an Ecotin tag, GSS linkers next to the His tag, a His tag, a TEV tag. Since this is our positive control circuit, the C peptide is present. At the end of the circuit we included a double terminator.
You can see more about the parts we used for our construct, you can see them in more detail on our Parts Overview page.