Check your cells before you wreck your cells

Amazing that six weeks have already passed! While research has been continuing at a rapid pace, the results, unfortunately, have not. Last time I mentioned that we haven’t found the right conditions to synthesize the proteins that we want to study. After much repetition of experiments and confused looks at the results (for instance, the protein that we work with is called Green Fluorescent Protein…it was not green) we have finally (hopefully) found the source of the hold-up: bad cells. That is right. The cells that we used to synthesize the protein that we then study and manipulate had somehow become contaminated such that the cells were not properly taking up the plasmid (DNA material) that they needed to obtain the ability to manufacture the protein of interest. New stock cells have been made and research can continue with more promising results.

Once we get past that little jam (and lesson to never trust the reagents that you use), my five projects have been proceeding rather well. To review, all of my projects deal in some way, shape or form with unnatural amino acids that we incorporate into protein. There are two main proteins that I have been working with: PRMT1 methyltransferase (important in the development of heart disease) and GFP (green fluorescent protein from deep sea jellyfish). A couple other proteins (such as myoglobin and T4 lysozyme are used but mainly as a base for the chemical reaction called a click to occur). I have finally synthesized PRMT1 which means that as soon as it comes in, I will be able to perform an activity assay to see if the unnatural amino acids that I have incorporated actually makes a difference in the enzyme activity! I am very excited to see this transfer from what I made to what it actually does! The GFP projects are still being problematic…

On the other side of the lab, I have been working on a method to enhance a very common biological technique called polymerase chain reaction (PCR). This technique is used to amplify DNA sequences, but is currently limited to a volume of only 50 microliters. By using the microwave, we can move the scale of the reaction to the milliliters! That is a lot more DNA! The results of this project have so far been promising but we need to perform more trials to really ensure that it is working.

In summary, research, while it does have its brief bumps and set-backs, has been providing some really fantastic results in the Young Lab this summer!

Comments

  1. mrstern says:

    What stock cells are you using (HeLa, DH5, etc.)? Will you be performing any assays to ascertain the epigenetic ramifications of adding the exogenous mutated methyltransferase?

  2. emparrish says:

    It’s so strange to think that issues in the lab can be chalked up to things like bad cells. I personally can attest to the “Don’t trust your reagents” comment, except in my case it is protein samples I have been receiving. Troubleshooting is incredibly frustrating, but I have definitely seen it as an opportunity to really understand my project better and it looks like you have discovered that as well. Proteins are finicky, but it looks like everything you’ve been accomplishing is coming together. The project seems very interesting and I’m glad you’ve gotten past the bumps in the road to make some headway!