Ben Tan pipettes a solution into centrifuge tubes as he performs an RNA cleanup.
Author: Benjamin Tan | Major: Biology | Semester: Fall 2022
My name is Benjamin Tan and I am a senior majoring in Biology in the Fulbright College of Arts and Sciences. During the fall of 2022, I worked in Dr. Mack Ivey’s lab culturing and analyzing certain bacterial species for my thesis project. The main idea of my thesis is to investigate the minimal carbon requirements of certain species of bacteria that could colonize other planets. This project was selected due to its relation with the work that my mentor does in lab regarding organisms of a similar nature. This information holds particular relevance today as interest in astrobiology reaches new heights and technology allows us to explore other planets in ways unavailable before. There is a risk of contamination from Earth-originating bacteria to other celestial bodies, and knowing what environments potential colonizers could grow in is crucial in preventing misidentification when true otherworldly bacteria are encountered.
The two species my advisor and I collectively decided on testing were Desulfotalea psychrophila and Desulfovibrio arcticus. Both species are anaerobic sulfate reducers and cannot grow at room temperature, with D. psychrophila growing at around -5° C and D. arcticus growing around 42° C. Over the course of last semester, I spent most of my time in the lab growing the bacteria and cleaning up the RNA from each trial. During the summer of 2022, I was able to finally complete the synthesis of cDNA and subsequent qPCR for D. arcticus. I spent the weeks leading up to the start of school in August obtaining the data by running several rounds of qPCR. Then in the fall, my focus shifted to D. psychrophila. As a famous Daft Punk song goes, my next step was doing every procedure one more time.
As I wrote previously, culturing these bacteria proved to be exceptionally difficult. However, after the former graduate student (Sergio) and I successfully got a culture going, I let it grow for six months. By that point, the bacteria had grown to a concentration that was acceptable to continue with the experiment. In August, I began to process all the bacteria that I had cultured. After that, it was relatively smooth sailing. Although Sergio had graduated and was no longer available to help me along the way, my thorough training and Dr. Ivey’s advice along the way was more than enough to suffice. Since everything that needed to be done was something that I had done before with the other species of bacteria, the whole process was going through the motions again. Moreover, because I was now familiar with all the techniques and what needed to be done, I could do it much faster and with less mistakes.
In the summer, I was trained on qPCR technique. I synthesized cDNA for all of the RNA that was isolated and created a dilution standard that would help me determine the concentration of the cDNA during the actual qPCR process. Then I subjected the cDNA to qPCR, using triplicates of each sample. The result collection and processing was simple on the other side, consisting of some excel spreadsheet work.
In the fall, I began to process the D. psychrophila samples. I started with processing the mature cells and lysing them. This was a complicated three-step process that was very time sensitive and required three consecutive days to complete. However, the procedures themselves were relatively simple and repetitive. The next step was then to cleanup the RNA. This was accomplished using a kit and was also a long and repetitive process. Between the extraction and cleanup, I was held up in lab for 2 months. I then began to isolate DNA, which took another few weeks to accomplish.