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Time to Spill the Beads: Learning Happens Outside of your Comfort Zone
August 2023
When I graduated from college with degrees in biology and chemistry, I still had no idea what I wanted to do as a grown up. At the time I was working for a wildlife rehabilitation center, taking care of non-releasable birds of prey, bats, and reptiles. I loved being around animals, but my favorite part of that job was taking the animals into classrooms and teaching students about the importance of conservation. I realized I wanted to pursue education full time and went straight into a teacher training program and started substitute teaching. While I missed the opportunity to work in a lab, I have had many amazing opportunities in education and I have had the chance to teach all over the world. I was an ESL teacher for Mini American Summer School in Russia for two summers, a middle and high school science teacher in Shanghai, China, and a high school science teacher in Ho Chi Minh City, Vietnam. This year I am starting my 14th year of teaching, and my second year at The Northwest School, an independent school in the Capitol Hill neighborhood of Seattle.
Danielle working at Talking Talons Youth Leadership, 2009.
Danielle Thal with Dr. Denise Galloway and Dr. Jody Carter. Photo by Caren Brinkema / FHCC.
I wanted to participate in the Hutch Fellowship for Excellence in STEM Teaching because I felt like I was missing experience that would make my science teaching more relevant and authentic for my students. I was excited to be placed in the lab of Dr. Denise Galloway. This lab researches cancers caused by viruses, including the human papillomavirus (HPV) and their work contributed greatly to developing the HPV vaccine. Infections with strains of HPV that can cause cancer (including cervical, vaginal, vulvar, penile, anal, and throat) have dropped by 81-88% in teen girls and young women since the vaccine became available in the US. There is a strong negative correlation between HPV vaccine uptake and cervical cancer incidence. Despite being so effective, only 60% of American teens aged 13-17 were fully vaccinated against HPV in 2021. I want to raise awareness of the vaccine, as well as the importance of regular screening, through my classroom curriculum project that I have been working on over the course of this summer.
My mentor is Dr. Jody Carter and he works on another cancer causing virus called Merkel cell polyomavirus (MCPyV). This virus causes Merkel cell carcinoma, which is a rare but very aggressive type of skin cancer that can spread to other areas of the body.
When a virus enters the human body, our immune system identifies it as a pathogen because it has markers on it that are different from any of those found in the body. These are called antigens and are usually composed of proteins that are embedded in the protein coat of the virus, or made by the virus during its lifecycle. MCPyV makes antigens, T antigens, or tumor antigens, after infecting the Merkel cells of the skin. These antigens cause cancer because they inhibit tumor suppressor proteins, which are made by the cell to keep the cell progressing through its cycle at the correct pace. When the tumor suppressor proteins are inhibited, the cells begin dividing rapidly, and cancer occurs. There are two T antigens in MCPyV – Large T and Small T. Many of our experiments this summer dealt with Small T.
The immune system has a way of remembering which antigens it has been exposed to by creating antibodies that match the antigens. These antibodies can then help destroy or neutralize pathogens so that they don’t make us sick. About 80% of MCC tumors have antibodies against the MCPyV T antigens, which is how scientists first deduced that the virus is involved in cancer. An epitope is the region of the antigen that the antibody attaches itself to (kind of like two complementary puzzle pieces). When scientists know the location of the epitope of an antigen, they can use that information to develop treatment, vaccines, or diagnostic tools.
The antigens can be made in the lab by using bacteria to transcribe and translate a specific sequence of DNA. A genetic database contains the DNA sequence of the ‘wild type’, or prevailing sequence found in the virus. Dr. Carter then used a different website to change this sequence to create our own mutant DNA sequences. He ordered the DNA with the new sequences from a company that was able to send us our new sequence of DNA the next day! The question we are trying to answer is “what is the location of the epitope of the small T antigen?” Based on previous experiments, Dr. Carter hypothesized that the antibody attaches to amino acid position 20 on the small T antigen. We changed the DNA sequence so that the amino acid in this position was changed from an alanine to a serine and from an alanine to a phenylalanine.
In order to test this hypothesis, we have been using a process that Dr. Carter developed called the Luminex serology assay. An assay is a scientific procedure that allows a scientist to quantify the presence of something, in this case antibodies against an antigen found in blood serum. Microscopic beads are chemically bonded to the T antigens and then run through a machine called the Bioplex 200, which shines lasers at the beads. By measuring the amount of light emitted, we can see how many antibodies are bound to antigens. We can compare how many antibodies bind to the wild type Small T versus the mutant Small T with the altered amino acid position 20.
“Washing” a plate before running it through the Bioplex 200.
Photo by Caren Brinkema/Fred Hutch Cancer Center.
I have had the opportunity to practice this protocol many times this summer, and I have made about a million mistakes along the way! The beads are tiny -- one microliter (or 0.001 mL) of liquid has about a thousand beads suspended in it, and that is how many you need to couple to the antigen. Pipetting liquids in such a small amount is difficult to do. The beads are light sensitive, which means that part of the protocol must be done in the dark, which makes it even more challenging to see if you have gotten enough. At the end of the day, when the Bioplex machine is reading the beads, it will report how many were in the sample. It will also report a sampling error if there was a low bead count. It took me many tries before I was able to get a data set without any sampling errors. The protocol has many steps, and each time I would have to go back and reflect on my notes to see if I could identify where I had made an error, or identify a step that I should have followed more closely. It is easy to get discouraged when something doesn’t work the third, fourth, or fifth time, but mistakes are the best way to learn. Doing additional research or watching YouTube videos to correct my technique helped me to improve.
Dr. Carter also had a few disappointing data sets and he realized that perhaps the beads were going bad. We did some troubleshooting and ran an experiment to try storing the beads a few different ways. This was helpful to my learning because I got to see him engage in the problem solving process and try something new. He also consulted with colleagues in another lab to see how other people solve this problem.
Unfortunately, in a classroom setting we do not have the time to continue to practice something until we get it right. The curriculum dictates that we need to move on because we cannot spend the whole year specializing in one topic. I see the frustration in my students when they feel like nothing is ever working and they feel like they haven’t learned anything from a lab. I understand that feeling but I encourage students to engage in the same process of problem solving, reflecting, and learning from mistakes. While they might not be able to redo one lab over and over again, they can apply what they have learned from one activity to the next in a process of continuous improvement. The scientific method is a cycle of asking questions, trying something new, and then making a decision based on the data that you get. Even when you are finally confident in the quality of your data, anyone must be able to repeat this process many times before reaching a conclusion that would be considered scientifically valid.
When you fail a particular experiment, this failure is productive because you can learn something from it. As humans, we don’t tend to learn much when things come too easily to us, but many of us also fear the discomfort of failure because it is…uncomfortable! But this is where we grow and develop as people and scientists. I appreciated that Dr. Carter and I could laugh about my poor results. It was a reminder that failure is not personal and there is always something to be discovered from it. Unfortunately, I was not able to collect data that could answer our original research question. But I did gain so much understanding about the process of science that I cannot wait to take back to my students this year!
While the practical side of lab work has been new to me, other aspects of being a scientist are not. Collaboration, persistence, and problem solving are present in everything we do in the classroom and in the lab. I look forward to sharing these experiences with my students and hope they are inspired to get uncomfortable in their own quest for knowledge.
Two quotes from Winston Churchill that have been pasted to my lab bench:
“Success is the ability to go from one failure to another with no loss of enthusiasm.”
“Success is not final, failure is not fatal. It is the courage to continue that counts.”
The members of the Galloway lab in
Summer 2023.
I would like to thank Dr. Jody Carter for his patience and mentorship. I would like to thank Dr. Denise Galloway and the rest of the lab for welcoming me into your world this summer.
Danielle Thal teaches 10th grade biology and 11th grade chemistry at the Northwest School, an independent arts school in Seattle, WA.
She previously taught at international schools in Russia, China, and Vietnam.
References
Carter, J. J., Paulson, K. G., Wipf, G. C., Miranda, D., Madeleine, M. M., Johnson, L. G., Lemos, B. D., Lee, S., Warcola, A. H., Iyer, J. G., Nghiem, P., & Galloway, D. A. (2009). Association of Merkel Cell Polyomavirus-specific antibodies with Merkel Cell Carcinoma. JNCI: Journal of the National Cancer Institute, 101(21), 1510–1522. https://doi.org/10.1093/jnci/djp332
Galloway, Denise A. (2016). The murky life of Merkel Cell Polyomavirus. Cell Host & Microbe, 19(6), 747–748. https://doi.org/10.1016/j.chom.2016.05.021
A.D.A.M. Medical Encyclopedia [Internet]. Johns Creek (GA): Ebix, Inc., A.D.A.M.; c1997-2020. Antibodies; [reviewed 2023 Feb 11]; Available from Antibodies: MedlinePlus Medical Encyclopedia Image.
Photos by Danielle Thal and Caren Brinkema/Fred Hutch Cancer Center.
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