Throughout my eight weeks as a BSURFer, we had various faculty members present their research along with their inspiring stories about their journey to a faculty postition. An overall theme in each of their stories was the uncertainty about their path in life. Most of the professors had never thought about entering academia and a few had gone to medical school only to fall in love with research later. Of these faculty seminars, my favorite was Nutrition: the Human Microbiome (and me) by Dr. Lawrence David.
Dr. David’s research focuses on how dietary compounds stimulate growth and metabolism of gut microbes. Previous experiments explored the differences between high-fiber plant-based diets and low-carb animal-based diets through the analyzing of stool samples. Currently, his lab is investigating the most beneficial pre-biotic supplements to feed the microbes in a human gut. Since a large part of staying healthy and preventing disease in a majority of the population is based on diet, the experiments conducted in the David Lab are important for the overall health of the masses.
While his research was amazing to learn about, the advice portion of his presentation is what impacted me the most. He advised us to stay with our undergraduate lab for a long period of time, because good science can’t be done in just a semester. He also discussed his experience with graduate school because though he had some uncertainties at first, he realized that the experiments he was conducting made him happy and he enjoyed the process of scientific research. His final piece of advice was about knowing when to graduate. Once he finally felt like he knew what he was doing and became comfortable, his PI told him it was time to graduate. When he felt comfortable in his understanding of research, he stopped learning new things and stopped growing as a person, so it was time to take on a new adventure and learn something different. This constant sacrifice of intellectual comfort for new experiences is the difference between good and great, so I’ll be sure to carry that piece of advice with me through the rest of my journey in science.
A majority of Duchenne’s muscular dystrophy cases are caused by a frameshift mutation in the DMD gene and the deletion of exon 51 with CRISPR-Cas9 can restore the reading frame in many patients. However, there are thousands of gRNA sequences to choose from with a deletion frequency of only 2%, so which gRNAs in introns 50 and 51 are the most efficient? To test this, I designed 10 gRNAs in both introns and transfected 293T cells with these gRNAs and a CjCas9 plasmid. I harvested these cells, ran a PCR with designed surveyor primers and ran these fragments on a gel. No new bands were seen that were different from my no-gRNA control lane so the gRNAs I designed did not make an indel. I will test 2 new gRNA sequences with the same method and expect to see two bands unique to the gRNA-transfected cells. The results from my experiment will be positive controls for my mentor’s project of developing a high-throughput screening method of hDMD-targeted gRNAs. With broader and quicker gRNA screening, the CRISPR-Cas9 tool will be more efficient and used as a human medical treatment option for patients with DMD.
A normal day in the lab usually begins with checking on my 293T cell lines to see if they are confluent enough to passage to a new plate. If the cells are 80-90% confluent, then I will thaw my media and trypsin, prepare the cell culture hood and then passage 10% of my cells to a new plate so that they don’t become overconfluent and die. I usually need to split my cell lines every other day.
After I do this, sometimes I will have a mini prep or a midi prep of bacteria with my designed gRNA plasmids culturing in the incubator overnight, so I will take that out of the 37° shaker. I will begin the protocol by centrifuging the tubes with my transformed bacteria for 10 minutes, so during this time I check in with my mentor. We will go over the procedures I will need to do that day and why they are necessary for my project. This is usually a good time for me to ask questions about the various protocols for the day’s tasks and be sure I understand each step. After this check-in, I carry on with the mini prep, or whatever procedure I need to complete first for the day.
By the time I am done with the day’s first assignment, it is usually time for lunch. Some days, the lab will all go out to lunch or someone will order pizza for everyone. These are the best days because I get to hear about the cutting-edge research they do and also get to know them outside of the lab setting. After lunch, I will complete the rest of my tasks, whether that includes transfecting cells, changing media, harvesting cells, PCR, restriction digests, surveyors or designing new guides for my project.
Before I leave for the day, I will transform one of my plasmids into competent cells or start a mini prep from bacterial plates that I have already prepared. This is done at the end of the day so that the bacteria can grow overnight. Overall, the lab is a relaxed, collaborative environment where I don’t feel afraid to ask questions.
This week, I heard about the amazing research that my fellow BSURFers are conducting while listening to their riveting chalk talks. I learned about neurobiology, plant biology and all the different topics in between. Though all of the presentations were phenomenal, the one that stood out to me was Michelle’s talk. Her project this summer is on knocking out the gene Endo16 in Sea Urchins and whether or not that would affect gut development.
Endo16 is a gene in sea urchins that is fundamental in gut development. Her question is whether knocking out Endo16 will result in reduced gut development in sea urchins. To do this, she will design guide RNAs and inject them along with the Cas9 complex into sea urchin embryos with the goal of causing an indel in that particular gene. With a large deletion or insertion, a frameshift mutation may occur, causing a premature stop codon, and rendering the gene useless thus “knocking it out”. She will then monitor the embryos with a knocked out Endo16 gene to determine if gut development is able to carry on as it normally would or if the lack of the gene prevents the formation of a gut. Determining the function of this gene will aid in understanding how embryonic development works in sea urchins and could lead to information about its interactions with other genes. Her project will expand the Genomic Regulatory Network and she can continue to test other genes with unknown functions.
I enjoyed Michelle’s presentation because I saw the way it related to my own research project. The use of a CRISPR-Cas9 system to knockout a gene and test its function is a real-life application for the technique I am studying. While using CRISPR for medical treatment is what my lab focuses on, hearing about the many other ways it can be used for research makes me appreciate my project even more. I loved hearing about the different methods and aims for her project because I understood everything she discussed. I’ve had to design my own guides and I can relate to the various steps in her procedures. I am amazed by the various ways this genomic tool can be used, as determining the function of a gene is important for understanding its part in genetic diseases. By doing a gene knockout and finding its function, we grow one step closer to unraveling the mysteries of biology.
Dr. Charles Gersbach began his training as a biomedical engineer while he was an undergraduate at the Georgia Institute of Technology. As the son and nephew of many engineers, he decided to major in chemical engineering and was never exposed to biomedical research. After watching his friends enter the workforce at companies like Doritos, he realized that those jobs never interested him and he searched for other career options. During his last summer as an undergraduate, Dr. Gersbach worked in a lab and fell in love with research. Due to his lack of experience, he had trouble applying for graduate programs, but was admitted to Georgia Tech and got a PhD in Biomedical Engineering.
Now, Dr. Gersbach is a professor of Biomedical Engineering at Duke where he teaches two classes: one is a requirement for BME undergraduates and the other is a BioDesign elective course. He enjoys teaching the design course because the students are much more engaged. Since it is an elective and the course material includes innovative, cutting-edge methods, the students have a desire to learn which makes teaching it much more enjoyable. His favorite part about teaching though is watching his students go off to do amazing things in science. Watching graduate students that he mentored in his lab earn faculty positions at other universities and publish papers gives him the satisfaction of knowing that he taught them well. Nurturing young students to become contributors in his field is the best part of teaching for him.
Though Dr. Gersbach is well-established in his field, he has no lack of embarrassing stories within the lab. Due to his busy schedule, he has less time to spend in the lab doing actual experiments but is sometimes asked to help when his students go out of town. During one Christmas season, a few of his graduate students asked him to passage their cells while they went home for a few days. He forgot and the students returned to find all of their cells overconfluent and dead. They asked him to help the next year as well, but he accidentally contaminated the cells and again they died. Since then, they’ve stopped asking him to passage their cells and found other arrangements.
When asked about any advice he has for young undergraduates looking to get started in research, Dr. Gersbach told me to take advantage of the many opportunities offered at Duke. Since he was late in entering research, it took him nearly ten years to catch up, so programs such as BSURF are important to take advantage of. He also advises to try many different things and explore the various topics within science. He never thought he would like research until he worked in a lab, so sampling diverse subjects can help young students to learn about the careers they may want in the future. Overall, I enjoyed interviewing Dr. Gersbach and learning about the background of my mentor. It taught me there is always time to change directions as I discover my interests and to take advantage of the multitude of opportunities that Duke has to offer.
The Gersbach lab focuses on developing innovative methods in molecular and genetic engineering for applications in regenerative medicine, treating genetic disease, and enhancing understanding of fundamental biological processes. More specifically, many members of the lab aim to treat Duchenne’s muscular dystrophy with CRISPR-Cas9 genetic engineering tools by creating an insertion or deletion mutation that restores the reading frame of the DMD gene.
So far, their methods have been successful in skipping an exon to allow the production of dystrophin, though the deletion efficiency has been close to 2%. Though this is satisfactory for restoring muscle strength in mice, 2% is too low for humans and the methods would not be applicable to other genetic diseases. This inefficiency may be caused by the choice in guide RNAs that lead the Cas9 to the desired deletion site, as some gRNAs have been shown to have higher mutation rates than other. However, there are thousands of possible gRNAs and gRNA combinations for each exon and testing each one is tedious and time-consuming. My mentor’s project is developing a high-throughput strategy for screening pairs of gRNAs for hDMD exon deletions. For my project this summer, I will be testing 5 gRNAs on each side of exon 51 to determine which is most efficient to use as a positive control for my mentor’s screening techniques. By targeting both sides, my designed Cas9 will delete the exon and restore the reading frame shift that was caused by a mutation in an earlier exon. I can then sequence the DNA to see how many of the cells I transfected have exon 51 deleted and how well each gRNA worked.
In the future, this will hopefully lead to more frequent deletions with the use of CRISPR and Cas9 so that these genetic engineering tools can be used to treat those born with genetic diseases.
After having spent a few days in the Gersbach lab, I am looking forward to a multitude of things this summer. Primarily, I am excited to complete my project and though it may be small, be able to make a positive contribution to both my lab and science. After taking numerous science classes in high school and during my first year, I’ve learned about many common lab procedures and attempted them within our class setting. I was also a Work Study in a lab during the school year, so I was able to observe certain processes but not assist. Now I can finally apply my scientific knowledge to a project that might contribute to my field while understanding and performing the experiments myself.
Second, I hope to learn more about common techniques in a lab and gain practice with technical skills. Soon I will be doing these procedures on my own, so this is a good time to practice the protocols and learn from my mistakes. There are so many brilliant graduate students and postdocs in my lab that would love to answer any questions I have, especially my mentor Veronica, so I know I should take advantage of the collaborative environment as I learn to eventually clone guide RNAs or culture cells on my own.
Finally, I’m excited for the unexpected. Each lab I performed in class had an expected result and the teacher knew exactly what would happen. We had studied the biology and chemistry behind each step and could determine why an experiment failed based on our knowledge. In a research lab, a hypothesis can be made, but often the results are surprising or disappointing. I could make a discovery that leads to plenty of new questions or my experiment could fail and I would need to start all over. I anticipate failure at some point, but I must be prepared for any outcome because scientific research is often unpredictable. Overall, I hope this summer will teach me a lot about both scientific research and myself in a lab environment that will cultivate my passion for discovering the unknown.
There’s only more pipetting from here on out…
Posted in BSURF 2018
Tagged Week 1