Proinsulin Gene in E. coli and Construction of Microalgae Vector (Update)

It has been an incredibly busy few weeks, which is why this overdue update is a bit late. The algal DNA construct is already done but we do not know if it works, so we needed to test in bacteria first. We had a few setbacks that required us to troubleshoot again. Fortunately, we are pretty resourceful.

Initially, everything seemed like smooth sailing, we synthesized the proinsulin gene and had it delivered to Sebastian's laboratory. He successfully inserted the gene into an E. coli DNA plasmid and we got transformed bacterial colonies.

 

To verify whether the colonies did indeed have the proinsulin gene in them, we then ran a colony PCR. This is where we ran into problems. We weren't getting any bands to confirm that the gene was inserted properly.

 

We tried several times and ended up getting the same results. We theorized that perhaps there was a secondary structure (i.e. hairpin loop structure) in the 5' or 3' ends of the gene that was preventing the primers from annealing and producing a PCR product.

We then decided to try a different plasmid, lifting out only the proinsulin coding DNA sequence. Fortunately, that process was straight-forward, and we were able to incorporate the coding DNA into another plasmid that has a built-in T7 bacteriophage promoter. We have transformed colonies and we will run colony PCR on them next week to confirm that the insulin coding DNA sequence is integrated. We are feeling more confident about this latest batch since the plasmid we're using now has worked pretty well in the past for Sebastian. We will post another update next week with more details about it. .

Also, the proinsulin gene was successfully integrated into the microalgae DNA plasmid, so it can be tested soon, but we have no idea if it works yet. We are waiting to characterize the expression in E. coli first, so we can work more efficiently. Transforming microalgae is a much more arduous task than using bacteria first. We'll get there soon though. We'll have another update next week.