Team:ULaVerne Collab/Construct-one
Design Iterations
This circuit design is influenced by the 2017 Sydney Australia iGEM team. Based off of their results sections, we chose to continue their periplasmic circuit of the secretion of insulin due to their lack of results for this construct. Therefore, we used a K608019 part that contains a strong promoter, medium RBS and RFP gene. RFP is a reporter gene that will turn the cells read if our construct has been successfully transformed. Our circuit also contains an Ecotin Tag which is placed in the N terminus of the protein. The purpose of the Ecotin tag is to translocate our proinsulin proteins to the periplasmic membrane of E.coli. In periplasmic membrane of E.Coli, there is a higher chance of lowering the misfolding of our protein and successfully form the disulfide bonds of proinsulin and the single chain insulins. Following thee Ecotin tag, we placed a His Tag to be able to isolate our protein form the rest of the proteins of E.Coli. Therefore, when we lyse the cell, it would be easy to locate our desire protein. A TEV tag was incorporated to cleave of the tags off the experimental insulins. However, our positive control circuit which contains the proinsulin protein has an arginine tag instead of a TEV tag. The reason why we chose to have an R tag instead of TEV is because when there is time to cleave off the C- peptide of proinsulin, we would only need a trypsin to cleave off the C- peptide and the R tag as well.
For our first construct, we decided to create the following circuits:
Experimental Circuits
Figure 1. Design of our experimental circuit containing different basic and composite parts. The constitutive promoter, RBS and RFP was a composite part (BBa_K608014). The following RBS is a medium RBS (BBa_B0032). The circuit is also composed of an Ecotin tag, a His tag, a TEV tag. Since this is our experimental circuit, the C peptide is not present. Therefore, we included different linkers.
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 been done in one 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 different isoelectric points
Control Circuit
Figure 3. Design of our control circuit containing different basic and composite parts. The constitutive promoter, RBS and RFP was a composite part (BBa_K608014). The following RBS is a medium RBS (BBa_B0032). The circuit is also composed of an Ecotin tag, a His tag, an R tag. Since this is our control circuit, we included a native human proinsulin sequence.
We used a constitutive promoter found in the composite part K608014 as well as RFP, as a reporter gene. Our ecotin tag would send our insulin protein to the periplasm, where it would form disulfide bonds. In order to retrieve our insulin from within the periplasm , we decided to lyse our cells with cell lytic B. We then used Ni-NTA purification to retrieve our insulin using the HIS tag. After Ni-NTA purification we ran a gel with all of our samples and saw that in our cell lysis we are expecting a band that is around 30 kDa, which was the expected size of the protein with attached tags. However, in our Ni-NTA gel we did not have an expected band. This led us to believe that our HIS tag was not able to bind to our nickel beads due to having two large proteins directly adjacent to the HIS tag. Due to this we decided to make some changes to our circuits. Therefore, we designed another circuit which could be seen in our Design Phase Two page.