We are making strides at Counter Culture Labs with the generous volunteering of many driven biohackers that are eager to be part of a wave of change!
In an attempt to draw inspiration for the coming phase of insulin expression we have delved into research done by other scientific groups.
This blog post is a reflection of our adventures in conjunction with insights on the paper titled: Synthesis of the Human Insulin Gene:Protein Expression, Scaling Up and Bioactivity by Redwan et al.
Our proinsulin gene
Our proinsulin construct is part of a plasmid; its production was outsourced to one of our backers, Arcturus Bio-Cloud. As of recently, we successfully expressed our plasmid. Our proxy was the red fluorescent protein (RFP). However, we have had issues with parts of the protocol. As a result, we have decided to take a step back and get our plasmid sequenced from a third party. This a step that will enable us to make a better protocol or choose an altogether better plasmid sequence for the sake of better yield.
So how do Redwan et al. make a gene?
A popular method is via assembling PCR - a proven process by which we can make many copies of a specific DNA sequence for cheap. One generates overlapping nucleotides, which are assembled and form parts of the template of the gene of interest. Finally, extend these parts via PCR and thus create the gene by its parts, by having a polymerase such Taq DNA polymerase (one of Mother Nature's many offspring) do the job for us.
How did we assess expression?
After induction of our genes we sonicated the cells, so as to break them up. Subsequently we separated the proteins from the other parts of the cells via an elution buffer and ran them through a gel via a process called SDS - PAGE. This process yielded a significant band at 20kDa, which we attributed to a dimer version of proinsulin.
Growing the bacteria
In house fermentation of the cell culture would be a step not too distant from now! As a measure of scale, the average patient needs between 0.5-1 g of insulin/year according to Redwan et al. Using a continuous fed batch process and a 3L bench-top Bioflo III bioreactor they were able to produce 59.8 mg of active insulin/L.
- Optimize the protocol for the proinsulin generation.
- Test an additional proinsulin construct.
- Keep researching the methods necessary to maximize our yield of insulin out of proinsulin.