These last few weeks in the lab have been both really exciting and challenging. After completing the first main step of the transformation, inserting a galactose sequence into the virus DNA, we began attempting the second phase. The process is very similar but has a much lower efficiency rate, so we had to be very precise in our technique. We prepared the sample and repeated the transformation procedure, this time, seeking to replace the galactose sequence with a DHFR sequence. Having already performed the procedure twice, we were a little more comfortable with the equipment; the process went a lot smoother. Unfortunately, we missed an important step in the protocol, and recovered our samples for only an hour (as we did in the first step) not for four hours as it was necessary, here. We decided to continue with the process - with the hope that even though the samples did not recover for the desired amount – maybe, we would get lucky and get a colony from transformed bacteria. However, after growing the samples on plates and sequencing the DNA, we determined that the colonies did not contain the desired sequences. A few days later, we repeated the entire procedure; this time, we had no major mistakes. Although after sequencing the DNA we discovered that not only was there no DHFR - and we noticed that the GalK had been removed by the negative selection from the plates - our viruses were back to how we started.
This was a little discouraging but our mentor ensured us that this was completely normal and that it usually takes a few tries. Thus, we began the process again. This time, we got some growth on our control plates, so we were not so optimistic. However, we continued with the DNA purification. The gel results came out very unclear, but we weren’t sure what the problem was. We tried many different things to figure out what the problem was by using different ladders, agarose percentages, and voltages. After 5 rounds on the Gel, we were able to get clear bands, and much to our surprise, they looked very promising. We sent the DNA to sequencing; they confirmed that the transformation was successful! Our mentor told us that he was very proud and that many professional researchers give up on this process because the procedure is so long. This was extremely gratifying and exciting that our work could be used in future experiments.
Another project we have been helping with is a ligation experiment to create viruses that each expresses a specific guide RNA. To do so, a bacterium is cut with restriction enzymes at specific locations, and then, ligation is done with a pool of guide RNA that, then, attach to the bacteria. This process is done many times until every represented guide RNA is incorporated into at least one bacteria colony, and then, each one is transfected into a virus. I have really enjoyed working on this experiment and hearing about its applications and guiding principles.
Additionally, over the last few weeks, I have been thinking a lot about how science is discovered, presented, and shared. Every Friday, the Gladstone institute hosts a seminar for all the summer interns in the building to discuss relevant topics in science and research. This past Friday, the chief editor of all Gladstone research publications came to talk to us about what makes good scientific writing. Reading scientific journals has always been something I have struggled with because - between the dense language and obscure jargon - I often have a difficult time just figuring out what concept or idea is being explored. During the workshop, we spent time discussing and rewriting sentences from scientific papers, in order to ensure that the text clearly presented the ideas. She emphasized that while there is often pressure to use complicated language and sentence structure in the scientific field; it is often detrimental to clearly convey the research. I found this really interesting and appreciated how her focus was on the best way to present research, so that it can be understood and built off on.