Dean Thompson
Extending Life by Deleting FUN
August 2022
I have always wondered how things work. One of my favorite question words is why? I have had several jobs in my life, but my first career was an Associate Scientist working in biology research labs. While working actively in research I always enjoyed working with new technicians, showing them the ropes and teaching them new techniques to make them successful. This eventually led me to the Fred Hutchinson Cancer Center’s Science Education Partnership (SEP). In 2004, while working at a biotech company called Amgen, I was a mentor to a SEP science teacher for a week during the summer. I realized I really liked teaching people science and wanted to share my enthusiasm for science and research with others on a more professional level. When the opportunity presented itself, I changed careers and became a full time science educator. I have been teaching Introduction to Biology and Biotechnology courses to high school students in the Bellevue School District in Washington State ever since. Now that I was a teacher, I realized I could participate in the SEP program from the teacher’s perspective, and participated as a member of the 2021 SEP teacher cohort. It never ceases to amaze me how events can come full circle. The next year, I joined the 2022 cohort of the Hutch Teacher Fellowship so I could continue to improve my lab skills and think about developing a curriculum related to real world experiments.
Pipetting in the Bedalov lab.
Photo by Caren Brinkema / Fred Hutch.
Dr. Antonio Bedalov. Photo by Fred Hutchinson Cancer Center.
I spent the first summer of my Hutch Teacher Fellowship experience in the lab of Dr. Antonio Bedalov in the Clinical Research Division at Fred Hutchinson Cancer Center. The Bedalov lab focuses on two lines of research, X chromosome inactivation and DNA replication (which is what my research project was focused on). While in the lab, I was led through the intricacies of a yeast lab by my mentor, Carmina Lichauco (Research Technician 1).
The genome of yeast can be divided into two compartments: repetitive regions and unique regions. The lab is interested in how cells balance replication in these two compartments and has identified a group of genes that are dedicated to balancing replication between the compartments. One of the genes that promotes replication of repetitive sequences is FUN30. When yeast cells age, they put too many replication resources into replicating the repetitive sequence thus creating an imbalance in the two compartments. The cells eventually die because they can not complete the replication of the genome. When FUN30 is deleted, this imbalance is restored, the unique regions are replicated, and cells live longer.
Deleting FUN30
My goal was to delete FUN30 from the genome. To do that, I replaced FUN30 with the selection marker hygromycin. To create the initial knockout DNA fragment we completed a polymerase chain reaction (PCR) step. This process involved using a plasmid containing the selection marker (hygromycin) and two external primers to the selection marker to remove the selection marker from the plasmid.
Next, a confirmation PCR was run to confirm that the selection marker had been amplified. The primers used for this PCR contained one primer that amplifies the DNA that was in Primer 1 from the previous step and another primer that targeted the selection marker. Using primers that are internal to the selection marker and contain part of the plasmid ensures that the proper PCR product was made. A gel was then run to confirm the correct PCR fragment was generated. An expected band size for the PCR product is approximately 1.9KB in size. Since the sample still contained the plasmid from the original PCR, the plasmid is still visible as the larger band.
Now I needed to get this PCR amplified marker into the yeast. Transformation is one of the many ways that a DNA fragment can be introduced into another organism. In this case, a heat shock transformation was performed to move the DNA fragment with the selection marker into yeast. After the DNA fragment was introduced, the new strand of DNA is then integrated into the yeast DNA through homologous recombination (the process where DNA is exchanged between genetic material that has overlapping sequences).
Blue=Yeast DNA, Dark Red=homology to plasmid
My mentor, Carmina Lichauco, and I examine a yeast growth plate. Photo by Caren Brinkema / Fred Hutch.
Was FUN30 knocked out?
Yeast from the transformation step were plated out on plates with selection media. Cells that do not have the selection gene in them did not grow on the selection media and cells that have the selection marker (successful transformation) did grow, thus indicating that the target gene has been successfully replaced with the selection marker.
Colonies were then selected and plated on selection plates to make sure there was a single colony present. These colonies were then re-checked with another check PCR to ensure they were still the FUN30 knockout yeast. These strains were then frozen down at -80C to be used at a future time to look at therapeutics targeting DNA replication.
Transformed cells on selection media (left).
No transformation cells on selection media (right).
Students always want to know if what they are doing in class is related to what “real” scientists do. One of the ways we can do that is by providing authentic laboratory activities and connecting them to the work that research scientists are doing in the lab. Some of the real scientist practices that I was (re)introduced to during my time in the Bedalov lab this summer include designing detailed experimental instructions so someone else could follow how I set up my experiment, accurately recording data during and after the experiment so experimental results could be shared with others, and troubleshooting experiment that didn’t work to determine what went wrong. One of the biggest things I learned this summer was to ask questions. My favorite question is just the simple “why?” If you understand why you set up the experiment the way you did and if you understand why the results are the way they are, you can understand what the next step in your experiment should be or try to determine why an experiment didn’t work.
Dean Thompson teaches Introduction to Biology and Chemistry courses at Newport High School and has taught Biochemistry in the Bellevue School District. He is a former research scientist who studied autoimmune diseases and tuberculosis. He lives with his family near Seattle in Washington state.
Cover photo by Caren Brinkema / Fred Hutch. Photos of yeast colonies on plates by Photo by Carmina Lichauco, Fred Hutchinson Cancer Center, 2022.