My Internship
I will be working with Dr. Scott Aoki and his lab team during my time with LHSI. Dr. Aoki's lab studies RNA complexes, or systems of RNA synthesis, recognition, and metabolism. We will be studying the effect of certain genes on RNA metabolism and the hereditary role of RNA as a modulating molecule in C. Elegans. Broadly, my site is investigating small RNAs and how they are metabolized by organisms. My site is also investigating the role of a system called RNAi (RNA interference) in managing the metabolism of the aforementioned RNA. As living systems, we interact with DNA and RNA everyday whether through aerosolized particulate matter or through eating food. Understanding how our bodies process our own RNA and foreign RNA may lead to a better understanding of gene regulation.
The Workplace
I have observed a lot of open communication and mentoring while at my site. This is both observation through personal experience as well as watching the grad students and other undergrads working with our site’s principal investigator. I think that both are skills that I want to work on for my professional development as my discipline is highly interpersonal and collaborative.
I want to grow my technical understanding of my field first and foremost. Biology, and particularly the research-oriented aspects of it, is a very technical field. Understanding the basic concepts of how organisms can be and are studied is at the top of my professional development goals. I would also like to grow my understanding of PhD programs and what kinds of things are necessary to be successful in them.
When I first arrived at my site, I assumed there would be a moderate amount of supervision given my lack of experience and the specificity of the procedures that I would be doing. However, the supervision at my site is retroactive. There is free reign to make mistakes and active encouragement to apply my knowledge to see if I can solve challenges I run into myself. That said, there is active guidance, but it consists of questions and answers between me and my mentor to help me figure out what may have gone wrong myself instead of directly giving me an answer. The idea I had of laboratory research was highly influenced by how I thought of industry driven work. I assumed that I would be working on something that needed to be executed perfectly in order to have validity and use. However, my site has made my learning and progression the main focus of my time with them, and I have greatly appreciated their dedication to my learning.
Describing My Work
My work at my site mainly consists of running a lot of PCR analyses on nematode samples. In addition to analytical procedures, I spend a portion of my time at my site maintaining my nematode strains to ensure that I have ample samples to test and retest. My current project is centered around a strain of nematodes that have a mutation on a gene called rde-4 that results in the gene not functioning; the strain also has a fluorescent protein tag which is important for the experimental part of the project. My goal is to use an exogenous ribozyme (foreign RNA based enzyme) to “attack” the worms and see how the transgenic strain fares in comparison to a wildtype strain. I will be using a combination of PCR, immunoblots, and confocal imaging to quantify and qualify my results. PCR is a procedure that is done to assess whether a sample contains a particular section of DNA. Immunoblots similarly assess the protein levels present in a sample. Confocal imaging is a form of microscopic imaging that allows for very detailed images to be produced using different colored lasers as well as fluorescent protein tagging.
These procedures are important because the goal of my project is to measure how the ribozyme affects the nematodes and also assess what role the gene of interest plays in ribozyme metabolism.
Schroeder, K. D. “File:Caenorhabditis Elegans Hermaphrodite Adult-En.svg.” Caenorhabditis Elegans Hermaphrodite Adult-En.svg, Wikimedia Commons, 14 July 2019
My Project
Project Summary
RNA has long been a point of fascination within the fields of molecular and developmental biology. This is because of its ability to act as both a substrate (binding site) and as an enzyme; essentially RNA can catalyze its own reactions. Adding to the fascination researchers have long held with RNA enters RNA interference (RNAi), a mechanism of gene regulation that was discovered in a species of nematodes, C. elegans, by two scientists, Andrew Fire and Craig Mello, in 1998. One of these pathways involves a gene, rde-4, which produces a protein that recognizes and binds to double-stranded RNA (dsRNA). However, rde-4’s interaction with ribozymes is poorly understood. Thus, the goal of the present study was to determine whether rde-4 is also able to recognize and metabolize ribozymes. Ribozymes are RNA molecules that act on messengerRNA (mRNA) and possess a characteristic hairpin shape which creates a double-stranded structure. Understanding the metabolism of ribozymes through this pathway is important because many of the genes in C. elegans are conserved within the human genome, including those involved in RNAi. This work then will provide insights on how ribozymes are metabolized in a model multicellular organism and broaden our understanding of developmental biology, immunology, and virology.
. . . So what does all that mean for the average person?
The work Aoki lab is doing may seem a sort of ethereal work, far from anything the average person should care about. In reality, scientific and medical advancements often begin as simple observations and the questions that follow them. The genome of humans has proteomic homologs (protein producing genes that originate from the same ancestral DNA) for around 83% of C. elegans genome. While these genes may serve different functions in humans than they do in C. elegans, this genetic overlap means that the study of gene function in C. elegans can translate to discoveries which are directly or indirectly applicable to humans. That’s pretty cool!
Hrobsky_LHSI_Showcase_Draft.pptxLearning and Skills
The biggest learning goal I have for this internship experience is to learn about the scientific process in detail. More specifically, how experiments are designed, how questions are asked, how previous research is drawn upon, and how I can progress the current work available. In addition, I want to learn how STEM majors who are seeking post-graduate education choose their PhD program, what to look for in a program, and how different institutions can offer different experiences. I am also looking forward to learning about the different experimental techniques that are used in genetics research. Many of the most basic skills include PCR, immunoblots, specimen cultivation and maintenance, and time management in relation to ensuring the highest experimental efficacy possible. These are all transferable skills that will aid me in my future career. I expect to contribute to my research team's current work. I am working on a project that parallels one being completed by another undergrad and, if the experiments produce the results that they are hypothesized to, then our work will overall contribute to a paper to be published by our PI.
Given my interest in drug development and gene therapy, working with Dr. Aoki's lab will give me the unique experience of examining how biological systems respond to and utilize endogenous and foreign RNA in a model organism (C. Elegans). In addition, I will learn basic and high-skill lab procedures that are common in the fields outlined above. This will give me a running start in the field and allow me to develop a sense of properly executed and documented research.
I am most excited about learning how to create experimental models. At my site, I have begun to learn how to do this by crossing two strains of nematodes with different gene edits to produce a strain that has both a fluorescent reporter (a protein that fluoresces when exposed to a certain kind of light or auto-fluoresces when in the presence of a specific molecule) and a nonsense mutation in a specific gene region associated with RNAi (nonsense mutations are a genetic mutation that results in a premature termination of a genetic sequence translation).
Using my previous knowledge as well as what I learn from mistakes has been something I have used a lot since starting at my site. I am much more confident in my ability to complete procedures myself using guides and instructions. I am also learning to be more independent and troubleshoot issues myself with confidence.
I would like to learn to communicate more effectively. Some of my shortfalls in communication have to do with the complexity of the subject matter that is discussed at my internship site. It can feel daunting discussing such concepts because my understanding of them is still very rudimentary. That being said, my mentor on site has helped me begin to work through this by providing me for some resources after I asked for them. There is a lot of ground to cover but learning and understanding starts with small steps which I have already begun taking.
In all honesty, the concepts that I am learning in class are very rough approximations of the actual concepts I have been exposed to so far in my internship. Having said that, learning is a multistep process that involves building a strong foundation of the basics so that as concepts become more complicated one is able to move forward without getting lost in the new information. I have a feeling that as I progress into more specified courses such as cell biology, the concepts I am being exposed to at my site will be closer in-line with what I am learning in class. Even so, taking introductory biology has been absolutely essential for me as I have explored the complexities of genetics further at my lab. I still remember in Biology 101 there was a section where we went over RNAi in very basic terms. I did not honestly think a lot about it during that period in time but as I have moved forward in my learning through my internship, I have come to realize RNAi is one of the most interesting aspects of genetics research.
As I said above, my time with Dr. Aoki’s lab has given me new passions to explore and a deeper understanding of topics I had previously learned about. I am very grateful to be able to explore the area of interest I want to pursue in my future career. As I have learned more about basic procedures, I have also learned more about cellular biology and consequently I have learned about topics that directly align with certain chapters of my classes. Things I have learned have been relevant to chemistry coursework, biology coursework, the publishing of research studies, and even broadly applicable to how my future career could be of benefit to the general public.
Most of my time learning at my site has been spent learning very technical information that sometimes gives me pause. There have been so many moments where I actually got goosebumps after learning something new. For example, in relation to RNAi; it is possible to remodel chromatin (genetic material present in the nucleus) using DSRNA (double-stranded RNA) by taking advantage of RNA interference pathways. This means that hypothetically speaking, it may be possible to one day use RNAi to treat everything from cancer to autoimmune disorders by silencing genes that cause such conditions. Learning this fascinated me greatly. Beyond technical information, I have also learned a lot about procedures that are used in labs to produce data. I have learned both how to do the procedures and how they work biochemically.
One of my favorite experiences as an intern so far has been when I was running a PCR reaction and accidentally used the wrong stain for my gel. When I went to image the PCR gel, it produced a blank image except for the reference DNA ladder. I was completely perplexed as I had never gotten a result like this before, so I went to discuss it with my mentor. In the middle of our conversation, I stopped talking, sighed heavily, and told him I used the wrong dye. Biology is an uncertain science and as such there were so many places where my experiment could have gone wrong and produced this result. However, because I was able to go back and assess the steps I took that led to it, I only had to repeat the PCR reaction instead of starting with new samples, etc. Failure has truly been an area that gives me the space to prove that I have learned something through my time at my site and I am glad to say that even though I have made mistakes several times, I have been able to identify where I went wrong and troubleshoot a solution.
Learning New Things
New Skills and Strengths:
Since beginning at the lab, I have learned many new things. Much of this new knowledge has been centered around the technical skills and fundamental understanding one would need as a burgeoning scientist; this includes how and why procedures work and what procedures would be must effective in any given instance. Hard skills like perfecting protocol execution and manipulating values depending on sample size are essential baseline skills for my field and I am proud to say that I have gained them with the help of the lab. I also had decent exposure to SnapGene, a software used for, but not limited to: plasmid design, primer design, and identification of mutations within a gene.
Protocols I have become comfortable with:
Polymerase Chain Reaction (PCR), Sample lysis, Western Blots, Nematode strain maintenance, Agarose gels, SDS-Page gels,
NGM + HB101 Agar plate pouring (+ RNAi plates), HB101 Transformation using plasmids and Culture
Beyond this growth in fundamental of my field, I have also learned how to effectively collaborate within a lab space and how to track my progress professionally using an online lab journal. Effective and timely communication is an important part of any workspace but it is essential to the schedule and efficiency of projects within a lab, and as such mastering these skills has been a large part of my learning journey.
Contributing to the Lab
My work at the lab was done parallel to the work of another undergraduate student. I had a few questions that needed to be answered by my project. Did the ribozymes designed by the lab work as they were intended? If they do work as intended, does rde-4 function to disable them through the same RNAi pathway as dsRNA? With these questions in mind, my task was to answer them experimentally. If the lab hypothesis was proven correct, the mechanism established would directly contribute to the work being done by our other undergraduate intern and our graduate student mentors. Regardless of success, all of the work completed would help our PI by contributing to publishable work. During my time at the lab I also created a transgenic strain of C. elegans that the lab may continue to use far into the future as it was frozen down for later use.
Successes and Challenges
Over the course of my internship, one procedure has given me the most success while another has been the most challenging. I will discuss this in greater depth during my presentation, but to summarize the PCR protocols I ran through the course of my internship have yielded the highest quality images and have also provided the most conclusive data. The procedure I struggled with the most was the experimental procedure at the end of my project. It required very precise timing and altogether it took about two weeks to set up and complete. The procedure being used to produce experimental data was a Western Blot and this particular procedure requires something called biological replicates. My project was intended to produce at least three biological replicates as this is the minimum sample size that produces statistical validity. Beyond timing, another issue that I experienced was sample contamination due to fungal growth in assorted media and buffers. A particularly bad case of fungal growth cut my available plates in half which resulted in a limited amount of procedures being able to be run. I will include an image I took of the contamination below so you can see what fun-guys scientists collaborate with, even unintentionally!
