Today marks the end of my first week as a student in the START REU program. The first couple days did not involve much hands-on lab experience as it was primarily introductions, basic instructions, and safety training. Although I did not work directly in the lab for the initial portion of this week, it was great getting to meet new people who are just as dedicated to their work as I am and just as excited to have this opportunity. Moreover, I also learned a lot more in-depth details about lab safety, relating to both chemicals and biological materials, that I have not come across in my high school or community college labs. This is, of course, because these institutions do not have abundant resources like Rice does, especially high schools. The second half of the training, relating to hazardous biological materials, was particularly interesting to me as it opened my eyes to how dangerous seemingly safe biological materials can be, such as bacteria and viruses. I will be working with viruses, specifically to determine the structure of an unknown protein within dinoRNAVs, so this lab safety training was both helpful for me and relevant to the project I will be working on for these next nine weeks.

I had the chance to meet my mentor, Clark Hamor, for the second time, with the first time being in early February, and he refreshed my memory on how the Tao lab is organized and lab-specific safety guidelines. Moreover, he has also made these past three days both fascinating through the learning experience and comfortable through the smooth adjustment process, being understanding of schedule-related changes. On Wednesday, which was the first day I got to actually start work in the lab, he taught me how to do certain basic, every-day lab procedures, such as loading and running an SDS-PAGE gel. I had only worked with basic agarose gels and DNA molecules prior to this experience, so it was all new to me. What I understand about SDS-PAGE is that it is a method of electrophoresis that allows protein separation by mass. The medium used in this method is a polyacrylamide-based discontinuous gel. While running, this thin, fragile gel is placed between two glass plates in a slab gel. The concept behind this method is that, when an electrical current is run through the gel, smaller proteins migrate faster due to less resistance from the gel matrix whereas larger proteins move slower, leading to separation of the proteins based on their varying sizes. In the SDS-PAGE method, the use of SDS, which is sodium dodecyl sulfate, and polyacrylamide gel removes the influence of the structure and charge of the proteins, thus they are separated based on their size. After watching him load a few wells, I tried doing the same, and it is a really difficult process when you’re new to it, especially given how thin the pipette tips are. Once we were done with the SDS-PAGE gel and it was de-staining, we moved on to practicing streaking plates with E. coli (but unfortunately left those in the incubator too long as we forgot about them) and also making liquid colonies.

Yesterday, which was a, Thursday was a full day in the lab, and I was there from about 8:30 AM to 5:10 PM. Yesterday, we started off by making four one-liter flasks of LB broth because a large amount of growth medium is needed to grow cells for protein expression. The procedure involved mixing 25 g of Luria Bertani powder and 1 L of purified water into each of the four Erlenmeyer flasks and mixing them (for evenness) using a magnetic stirrer. Following this, we autoclaved each flask to ensure sterility. Then, we followed some steps Clark outlined for protein purification that are specific to his protein (as these steps are very subjective), which involved taking the frozen E. coli cells and putting certain buffers into them, putting the tube into an ice bath, and sonicating it. Following all of this, Clark showed me an ultracentrifuge and taught me how to use it, then told me to go back to the office (for safety purposes) while he ran it. This was pretty much all of day 2, it also involved a lot of reading and concept review/understanding.

^ Me with one flask of LB broth.

^ Me pouring antibiotics into the LB broth as a way to check if the protein has been expressed.

Today, a Friday, I got into the lab around 8:30 AM and Clark went over important, relevant concepts with me, such as lac operons and various protein-related concepts. Then, at around 10 AM, we removed the test tubes from the ultracentrifuge (it was done running around then) and noticed the thin bands the proteins separated into. Clark then showed me how to remove those bands using a syringe, but since they were too close together, it didn’t work out as well for two of them (there were three total). Then, once the liquid removed from the test tubes were put into microcentrifuge tubes of 1.5 mL each, I loaded the wells of an SDS gel with the ladder, loading dye, and the protein samples. One thing I learned from this was that it is important to fill all the wells of the gel (even if it is with loading dye) so that the gel does not run crooked. Then, we ran the gel and went back into the office to read and eat lunch and just discuss things related to the project. After, when the gel was done running, we could tell based on how far the molecules have traveled (they should have come down all the way). After we were done with the gel, we stained and de-stained it and placed it into a pipette holder (box) for sufficient de-staining. Since we left the plates from Wednesday in the incubator for too long, we had to re-streak new plates.

^ The thin bands.

^ Me pipetting loading dye into the wells of the SDS-PAGE gel.

^ The loaded wells, ready to run.

^ Leftover (taken off) polyacrylamide gel named “Blob”

Coming from a background with limited knowledge of advanced topics like virology, proteomics, and general biochemistry, I initially felt a little overwhelmed and uncertain about my ability to understand such complex subjects without prior education. However, Clark has been a great mentor who has been incredibly helpful and made the transition into the program easy and accessible, which helped ease my concerns. I am highly motivated to excel during this internship and am excited to continue learning and enhancing my research skills.