3 min read
Things are getting really exciting here! We just finished designing our new proinsulin construct, and have successfully been growing cultures that contain the new construct. We are now moving on to expressing the proinsulin. If this succeeds, we’ll have accomplished our first milestone of expressing and purifying proinsulin.
For background, a “construct” is the piece of DNA we insert synthetically into an existing microbe. Once inserted properly, we can trigger the microbe to produce the protein (in our case, proinsulin) that the construct encodes.
Sounds easy right? We wish, but things always get veeeery hairy when tinkering with living systems. Read on to hear about all the work that’s gotten us to where we are now…
For the past few months we were using another insulin construct to produce proinsulin, but were having trouble determining what exactly was being produced. So, we re-designed the construct to allow us to better troubleshoot whether or not we’re actually producing the right proteins.
The new construct codes for proinsulin, but also green fluorescent protein (GFP). If we manage to get the microbes to make insulin, they will also make the GFP. This turns out to be very useful because it’s now easy to visually detect that we’re expressing proinsulin. It will also help with purification by keeping the protein soluble and out of inclusion bodies (which require more work to get protein out of).
Once we received the construct in late April, we discovered there was an errant methionine in the plasmid. We used a technique called directed mutagenesis to fix 1) the error and 2) add a stop codon after the GFP. This is so we have a GFP-only control to see if the two proteins are interfering with each other or harming the e. coli when joined. The directed mutagenesis succeeded for both the GFP-only control version and the full fusion protein version of our construct, and in the past few days we’ve grown cultures on selective media to verify this.
Unfortunately, right after we began work on the new construct, our centrifuge began to fail, and we had to devote attention to looking into repairing it. Our equipment is mostly old, donated equipment from labs so this type of thing happens fairly often. Fixing it took over a month, but we’re almost done now. Stephen Treder and David Digor helped us with troubleshooting, and determined that the bearings were worn and were causing excess vibration and needed to be replaced. We had to find a machine shop to remove the old bearings from the shaft and press new ones onto it. We also purchased a small temporary replacement to fill in for it in the meantime.
Noel Carrascal continues to lead work on computational modeling of insulin which we may eventually use to vet new ideas on how to fold the protein properly.
He remains engaged in investigating the possibility of using leucine zippers to bring the A and B chains of insulin together. (Recall that the final active form of insulin is composed of two parts of the proinsulin protein that are bonded to each other in a certain configuration. Leucine zippers are extra bits of protein with high affinity for each other that might be useful in bringing the two pieces together.) The next step in his work is to finish modeling the linking between insulin and the leucine zipper, and find the energy minimum of the complex.
The amount of computational power the project needs has led to discussing if we might be able to make good use of a homebrew computing cluster, and if our team wants to build one. We are still undecided if this is the best use of our time and funds, but the discussion continues. Using cloud computing resources is also a possibility, and might prove most economical, if a bit less educational.
We are ramping up the lab work again and continue to keep the momentum up and attract new volunteers to work with us. Thank you again for your support!
From the PDB molecule of the month, on Insulin: http://pdb101.rcsb.org/motm/14
Here's a brief introductory excerpt:
Our cells communicate using a molecular postal system: the blood is the postal service and hormones are the letters. Insulin is one of the most important hormones, carrying messages that describe the amount of sugar that is available from moment to moment in the blood. Insulin is made in the pancreas and added to the blood after meals when sugar levels are high. This signal then spreads throughout the body, binding to insulin receptors on the surface of liver, muscle and fat cells. Insulin tells these organs to take glucose out of the blood and store it, in the form of glycogen or fat.
Insulin is a tiny protein. It moves quickly through the blood and is easily captured by receptors on cell surfaces, delivering its message. Small proteins pose a challenge to cells: it is difficult to make a small protein that will fold into a stable structure. Our cells solve this problem by synthesizing a longer protein chain, which folds into the proper structure. Then, the extra piece is clipped away, leaving two small chains in the mature form.
Read more in the PDB article!