Increasing the Scale

Welcome back everyone! This week taught me about just how much waiting there can be for experiments. We decided that it was time to start increasing the amount of bacteria we were producing by ten fold, going from 100mL to 1L of bacteria. Much of my time was spent waiting for the bacteria to grow (about 2 hours after having grown it overnight), then waiting for the bacteria to induce (4 hours), and a bunch more waiting from processes like centrifugation or running a gel or waiting for something to heat/cool. There is just so much waiting! Although, all of this waiting did allow me to finally straighten up my notes and label my SDS-PAGEs on PowerPoint, which I had to learn how to use.

Was finally able to label this and make it look all pretty! All Sizes are given in kDa.

From some prior research that I had received from my on-site mentor Dr. Gustin, I found that the size of pfu is about 95kDa. But, the research that I had obtained that number from also used a different type of pfu; a type that has a histidine tag. Histidine is an amino acid that has an attraction to metals like Ni, nickel. This means that if a protein has a bunch of histidine attached to its end, it will stick to that metal, and thus purify itself because it will be the only protein sticking to the metal. The pfu that I am making does not have a tag, so we are planning on purifying it through simple heat treatment, aka making it so hot that all other proteins fall apart leaving pfu by itself as it is thermostable. As of right now, I have made a protein soup by sonicating the 1L culture of bacteria after centrifuging it down into a pellet and re-suspending it. We could use the pfu in PCR right now, but it is probably way too concentrated plus we do not know the proper dilutions, and it still has DNA from the E. Coli in it. Next week, I will remove the DNA by treating it with an enzyme called DNase and hopefully further purify my pfu. Thank you!