Over the past year I have worked with Dr. Marcello and have learned so much about the research process, time management and teamwork. When we first started on this project there were many setbacks such as waiting for the correct bacteria strain to arrive in the lab and learning the proper way to pick the worms. When we finally had all of our reagents and necessary materials, I was able to begin RNAi trials and perfect my technique as time went on. However, when I felt the most comfortable in this process and believed I had sufficient data to switch to the next phase of the project, the lab closed due to COVID-19 and we were unable to continue the research plan. After a while, I realized this is what comes with research. There will be setbacks and as much as you can have a plan, you have to be ready and willing to make adjustments as you go. Having a schedule is important but also having room for error, flexibility, and open communication has been the biggest lesson of all. I am incredibly grateful for this opportunity.
We studied the influence of the prohibitin on fertility in C. elegans as this gene is known to play a key role in the production and maintenance of mitochondria, a necessary organelle for cellular energy. Through a series of RNAi experiments we expected for fewer progeny to be produced by mutant worms lacking this gene. The data collected supports our hypothesis by demonstrating that the phb-2 mutants produced fewer worms, differing significantly from the positive control. Surprisingly the data also showed that the reduced progeny amount is not a result of embryos arresting at the egg stage. Compared to the negative control, the phb-2 mutants had significantly fewer unhatched eggs on the plate after treatment.
We initially planned to analyze the mitochondria in the phb-2 mutants to compare to that of wild type worms to see if there were any noticeable abnormalities across the different stages in the life cycle. However, due to unforeseen circumstances we were unable to conduct these trials. The expected changes in mitochondria could potentially result in a reduced number of quality gametes in the hermaphrodite worms therefore limiting the number of progeny able to be produced. Additional tests viewing the gametes of the mutant and wild type worms would need to be conducted in order to confirm this. Fewer quality gametes would further support a relationship between humans and C. elegans in terms of diminished oocyte quality, prohibitin loss, and infertility.
At this point in my research I am getting ready to switch phases and move forward with experimentation. I have been completing more RNAi trials this semester to understand the effect the prohibitin gene has on Caenorhabditis elegans fecundity. I have had a few successful trials that show that nematodes lacking this gene produce fewer eggs than my positive control and fewer worms than my negative control. This overall goes to show that this gene likely plays a large role in the ability of these worms to reproduce normally. I will continue to do these trials in order to ensure that the results are true and there is plenty of evidence to support this conclusion. The next phase involves taking a closer look at the mitochondria in these nematodes that lack this gene. Due to the nematodes being transparent in nature, with special microscopes, GFP tagging, or splicing techniques, we can take a closer look at what is happening at the cellular level rather than just the phenotypic level.
So far I have learned that working in a lab is a group effort. There has to be constant communication to ensure that all equipment is in working order, the station is cleaned and maintained, and the supplies are continuously stocked. An example of a problem encountered due to a lack of communication earlier in the semester is when the lab was very low on RNAi agar plates. For this we had to share controls and limit the number of trials we could complete until we made more plates to replenish the supply. We have since then been more open about the tasks needing to be completed so that everyone has a job to do in order to help out. This is also important for when someone has to take time off from lab work one week and cannot complete a certain task, we all know to pitch in and maintain the work space.
Throughout the fall semester I have been working with Dr. Marcello to study prohibitin and mitochondrial significance in Caenorhabditis elegans. While waiting for the bacteria strain needed to perform the RNA interference (RNAi) experiments to arrive in the lab, I practiced my worm picking technique to preserve the wild type worms. Moving a few worms from one plate to a new one provided a diverse range of life stages for my lab mates to use for their experiments. My lab mates were an integral part of helping me perfect lab techniques and procedures. Additionally, it helped provide the necessary speed for my RNAi experiments because the special RNAi agar must be kept out of the light as much as possible. These experiments knock out the prohibitin gene so that I can observe the changes in progeny production in the mutant nematodes compared to the wild type.
When beginning the RNAi experiments, there were a couple problems we ran into. For example, condensation developing on some of the agar plates lead to contamination and chunking starved worms lead to a reduced number of viable worms in the L4 life stage. However, from the data we have collected, there does appear to be a difference in the amount of progeny produced by the mutants lacking prohibitin compared to the wild type worms. These experiments will need to be repeated in the future to ensure the validity of this finding.
After we have collected sufficient results from the RNAi experiments we hope to begin mitochondrial splicing in the spring semester. Prohibitin, the gene of interest, greatly affects mitochondria and we expect to see morphological differences in the mitochondria of the mutants lacking prohibitin compared to the wild type nematodes. Varying the treatment length of these experiments could possibly show significant variations in the reproductive capabilities of the nematode.
The title of my project is Prohibitin and Mitochondrial Significance for Viable Progeny in Caenorhabditis elegans. Dr. Marcello and I are studying the prohibitin protein complex (PHB) which is known to maintain the stability and function of mitochondria. Mitochondrial function is essential to having sufficient energy produced within cells as well as being linked to high fertility rates in humans. Accumulation of mutations in mitochondrial DNA is correlated with poor human egg quality as an adult female ages. C. elegans is an excellent model organism to study the function of this complex and its relationship to mitochondria as the nematode is transparent, small, and easily maintained. It is also excellent to study fertility and development as it has about a two week long lifespan and a single worm can produce between 300 to 1000 progeny. This research is important because the results will provide more information about the interactions and regulation of genes necessary for the maintenance of mitochondria. This in turn will can provide context for more effective treatment options created for mitochondrial dysfunction and infertility in humans.
My methods will include RNA interference where I will knocking out genes associated with the prohibitin protein complex, such as PHB-1 and PHB-2, then observe physical changes in the nematodes and progeny production. I can count the eggs and larvae produced on the agar plates and compare differences in these mutant nematodes and wild type nematodes. Using MitoTracker and splicing techniques, I can observe the physical changes in mitochondria morphology as a result of knocking out the different genes. Additionally, I can change the times of treatment length to observe for any significant variations in C elegans reproductive capabilities.
With these methods we hope to better understand the effect prohibitin and mitochondria have on developmental abilities in C elegans. I am thrilled to work on this project and present my findings at the end of the year. I hope to learn more about managing the research process and how to balance research with other academic and personal responsibilities throughout this academic year.