My Internship
At the Aoki Lab in the IUSM Department of Biochemistry and Molecular Biology we are researching the mechanisms of specific RNA-protein complexes known as P granules and their effects on regulating gene expression using the model organism C. elegans.
Project Abstract
Germ granules are membraneless organelles that regulate gene expression in the reproductive cells of multicellular animals. Despite their prevalence, we still know little about how germ granules assemble and how assembly affects their function. P granules are the germ granules of the Caenorhabditis elegans nematode worm that assemble just outside the nucleus. P granule assembly is driven by PGL-1, a structural protein that pairs up to form dimers. The mechanism of how PGL-1 dimers interact with each other to form higher ordered granule structures is unknown. I am analyzing what regions of the PGL-1 protein are required for P granule assembly and function. Using CRISPR/Cas9 gene editing, I have created mutant C. elegans worms with deleted PGL-1 protein regions. This will allow me to determine which regions of the PGL-1 protein are necessary to form P granules through various experimental methods. Performing research in simpler model organisms will give broader insight into the assembly and function of germ granules in other, more complicated, multicellular animals, like humans.
Learning and Skills
Beginning of Internship
Beginning this internship in June, I had previously had some experience from my biology and high school classes about CRISPR/Cas9 gene editing and PCR. Now that I have begun performing research using those procedures, I have had to incorporate that knowledge into what I'm doing, but also gain a better understanding of exactly how each step or component works in relation to the overall process. This understanding is something I can continue to apply in present and future work. I also have some pipetting skills from previous courses, however through practice I hope to build on those to become even more precise and efficient using a micropipette. This is especially important for loading DNA samples into the agarose gel for gel electrophoresis. Finally, I plan to use my organizational skills to keep a detailed lab notebook so I can easily track what I'm doing as well as what I need to do next.
Fall Semester:
I am currently most excited about successfully making the pgl-1 Nt deletion mutant using CRISPR and beginning to work on my next mutant: pgl-1 G12P. From here, I would like to learn how to successfully inject the worms with the CRISPR mix as part of the CRISPR process, which I hope to work towards being able to complete independently. I would also like to learn how to perform western blots on the proteins I am mutating, since it is a widely used and very transferrable skill in the research field. Most importantly, I would like to learn and develop a better understanding of how the field of research operates through publishing, conferences, grants, etc. so I am best prepared when faced with these opportunities in the future.
So far in my internship, I feel very confident in my ability to communicate with others in the lab and ask questions to better understand things. I've gained significantly more confident in my ability to perform procedures and determine the next step in an experiment independently. I have also continued to improve my organization and note taking as I have started working on multiple projects at once, meaning it has been important to be clear and specific in my lab notebook. One of the skills I would like to improve is my ability to problem solve independently. When I run into problems with my projects, such as issues with one of my PCR reactions, I am good at asking for help and collaborating on finding a potential solution with one of my mentors, however I would like to work towards being able to come up with solutions independently. I plan to do this by discussing the information I didn't know before going into a particular problem with the mentor I'm collaborating with, as well as the most effective way to approach or go about solving that kind of problem so if I face it again, I will already know what to do.
I have noticed that my work in the lab has been very beneficial in the classes that I am taking currently, especially my organic chemistry lab. Many of the organization and lab notebook keeping skills that I have been practicing through my internship have helped me keep an organized and efficient lab notebook for my chemistry lab as well. This has been especially helpful when I have had to reference back to my lab notebook to complete additional assignments related to a lab we did.
Spring Semester:
Since the beginning of the semester, I have gained a lot of new skills and confidence in my abilities. I'm much more comfortable in knowing what I need to do, how to do it, and what the next step is after I do it. I have now successfully made my second CRISPR mutant worm, which is very exciting since I was able to see this one through from the beginning to the end. Some of my favorite experiences so far is going out with the whole lab to attend research conferences. I have found them to be very enriching experiences, and I have gained a lot of insight into the field of research by attending them. My mentor and coworkers have also been very receptive and supportive of me asking questions while at the conferences, which has been really encouraging and helped me feel more comfortable.
This internship has allowed me to really expand my skill-set in the lab and has given me the tools to become even more adaptable in the face of set-backs. This is growth that I've especially noticed in the labs for some of my chemistry coursework, particularly when it comes to changes in procedure or troubleshooting problems such as broken equipment safely. I've also found that my strategy of going through the chemistry or biology behind specific experiments or procedures while learning them at my internship has subconsciously transferred over into my academic lab work. I will notice myself analyzing why reagents are added, why specific equipment is being used, what affect different reagents have on reaction, and so on. I'm really proud of the growth that I've noticed and I can't wait to continue seeing similar growth in my future classes.
The Workplace
From my time in the workplace, I have observed a great deal of collaboration between lab members. People aren't afraid to ask for help, and someone is almost always willing to step up and help when asked. I really respect this type of teamwork in the lab and hope to continue working towards emulating that same professional collaboration. I have also noticed how many of the lab maintenance tasks, such as making sure supplies are stocked or dishes are done, is well delegated so everyone pitches in and does their part, including my mentor. This strategy for making sure everything gets done and everyone contributes has been really effective. It also contributes to the overall teamwork and makes sure no one individual is burdened with tasks that may intrude on their research time. This has definitely made me a better professional and team member because it gives me a sense of purpose when it's my turn to do such tasks, and it has improved my communication with my coworkers to make sure everything that needs to get done does. I very much would like to continue contributing to this amazing workplace environment, and hopefully as I continue to gain more experience, I will be able to take on more tasks that benefit the lab as a whole.
One of the things I enjoy about working at the lab is how everyone has very different backgrounds that led them here, and how openly its discussed. Everyone frequently talks about whats going on in their lives and their ideas on different topics (though sports and music are favorites) in a really respectful way. In particular we've had discussions about how our different backgrounds and cultures have shaped our perspectives on everything from travel to family, which I consider to be very enriching and now consider to be very valuable to me in a workplace environment. I've found Scott's reflections on going through veterinary school and how he made his way into research from there to be very insightful, especially since I am planning on going through medical school and potentially incorporating research into my career. Experiencing working with all of my coworkers has made me much more confident talking with new people and networking as a whole, through watching them as example as well as personal practice.
Successes and Challenges
Fall Semester:
Over the summer, I worked with three other summer interns towards making a specific CRISPR mutant, pgl-1 (Nt deletion)::mHaloTag, C. elegans. By the end of the summer, right before the semester started, I successfully made the correct mutant and confirmed it with DNA sequencing. Since then, during my first semester of the internship, I have successfully made another C. elegans mutant: pgl-1 G12P::mHaloTag. This most recent mutation was a single base pair mutation as opposed to the large insert mutation I did over the summer, which involved a slightly different process that was interesting to learn. One of the biggest challenges I've faced so far is outcrossing the mutant worm I made over the summer, to make sure there are no additional mutations, and troubleshooting the problems that have arisen. So far, the outcrossing has taken months longer than initially expected due to various issues with primers, DNA polymerases, and more. Despite the frustrations this has caused, I believe that having to work through and try various strategies to solve it has given me a much more in depth understanding of the scientific process, as well as the mechanisms behind what I'm doing for me research. For example, issues with the primers not working for my PCR meant that I got to learn about and even design my own primers.
Spring Semester:
Despite continuing issues with the pgl-1 (Nt deletion)::mHaloTag outcross, I've continued working through them with Chris. We were able to figure out a way to test some new primers with the PCR reaction before restarting the whole outcross in order to save time. So far this has proven really effective and we hope to finish the outcross very soon. In the meantime, I was able to successfully outcross the pgl-1 G12P::mHaloTag mutant and begin performing experiments with it. One of the experiments is a fertility assay, and we were able to perform a successful test run for the method I'm going to use for the full experiment. Moving forward with this mutant has given me much more insight into the research process as well as my project as a whole.
After troubleshooting for four months, I was able to successfully finish outcrossing the pgl-1 (Nt deletion)::mHaloTag strain. I'm extremely proud of myself and I'm excited to start doing experiments and collecting data with this strain alongside the others I've generated and outcrossed. I will now get to start learning how to do immunoblots, which is a goal I've had since the beginning of my internship!