As summer research winds down, I have taken some time to really appreciate the marvel that is Barnard Chemistry as well as the Summer Research Institute. Likewise, I have been able to reflect on my progress as a researcher and ceaseless learner. The SRI poster session really substantiated all the positive feelings I had regarding the program. It was quite remarkable to see over one hundred posters displaying the research of about one hundred and sixty undergraduate women! It was not until the provost mentioned that the poster session of undergraduate women presenting their work in STEM was probably one of the largest events in the country, that the significance of the implication really set in. While discussing other students' research with them at the poster session I realized just how much science I have learned in the past year both in the classroom and in the lab. Not only is it kind of impressive to recall and apply the chemistry, biology, and other sciences I have learned, but it really rewarding to be able to have a conversation with professors and researchers regarding a range of topics. Even when I or my peers do not understand certain aspects of someone's research, we are able to ask intelligent questions which can help clarify our confusion or unfamiliarity with the subject. I often find myself recalling various course lessons from different classes to put things in perspective. I really enjoyed moments this summer when a friend and I bounced ideas off one another, drawing from past lectures or labs. Moments like these were helpful for brainstorming research ideas, as well as confirming and testing your own scientific knowledge.
In anticipation of the poster session, I had to spend some time planning my presentation. I had to consider the story I wanted to tell based on the research question I had originally set out to answer and the data I had collected. Once I compiled all my data and really sat down to look at it, I saw the story in front of me and where it may lead to. The background information consisting of literature and work done by previous researchers helped dictate my experiments this summer. The bulk of my story stems from my results from this current summer, and the future directions of the project are influenced by the questions I have and conclusions I can make about my results.
For instance, I have been getting varying results concerning one of the proteins of interest in different cell lines. I am unsure if the results I have been getting are differing based on the cell line or based on my experimental procedure. In the past, I have experienced many problems with immunoblotting, but need to rule out any inconsistencies I have seen by repeating the experiment multiple times in all the three cell lines to get a more definitive answer. Additionally, I have seen some results that do not exactly line up with or support the results from a previous researcher and will need to continue to either establish the pattern I am seeing or get similar results as the former student. Future work will hopefully clarify the results and thus the conclusions we can draw from it. When I first started conducting research, I would have asked these questions to my mentor, but now I can plan my own experiments aimed at answering these research questions I have. I have definitely grown as a researcher by learning which questions to ask, how to set up an experiment to properly address my research question, and how to analyze the results.
The next step in preparing for my presentation was to design my poster to best represent the research story I wanted to tell. I had a template from the previous year's poster session but needed to update a lot of my results. Originally, I tabulate data based on an individual experiment, but in order to tell my story more concisely, I needed to compile results from experiments under the same conditions. This took more time than I anticipated, but it really helped when presenting my work to more definitively show a pattern. While compiling my data, I learned that I am very much a visual learner. I retain information better when I can read over the experiment and see the specific conditions set. I always keep a pen and small notebook next to me whenever I step into meetings even for something very quick because on many occasions my research advisor and I would bounce ideas off each other and I would leave unsure of our final decisions.
I also practiced my presentation tailoring it to various audiences based on timing, content, results, and future directions. For some people, they are more interested in the conclusions and outcomes of the research while others are interested in the specific details of the procedure and results. Throughout this process of preparing and presenting at the poster session, I found that I became more dynamic and flexible in diverse environments. During this summer and especially at the science symposium, I found that I was improving as a presenter and possibly and educator. With group meetings aiding as additional practice, I learned how to present my results in a comprehensible way. Being able to teach or talk about a very specific topic in a coherent way confirms my understanding of the material let alone my ability to present to others.
At the end of the summer, it is extremely important that I make sure all my files of raw and analyzed data are comprehensive, organized, and match the work documented in my lab notebook. Similar to the time when I needed to compile results from various trials of the same experiment, I needed to thoroughly and carefully go through all the files I had generated this summer and make sure that others such as my research advisor and new student researchers could easily refer back to my work. Even in a month, when I return to the lab, I will appreciate the work I did to clarify my procedure and experiment conditions. I couldn't stress enough the significance of an up-to-date lab notebook.
I look forward to continuing doing research in the Fall. I really enjoyed running my own experiments and planning the course of my inquiry. Conducting research over the summer allowed me the opportunity to work full time, pursue questions, and run experiments which I would not be able to during the school semester. This summer, as well as my past semesters conducting research, has been a great learning experience where I overcame challenges, developed new skills, and grew as a student and researcher.
There have been a lot of ups and downs during this past month conducting research. I have experienced the same imaging problem with Western Blots which I thought I had overcome. Unfortunately, I am still having trouble visualizing the protein of interest and am unsure where in the process something has been going wrong. As a check between steps in the long process of western blotting, we are able to stain the protein membrane after the protein has been transferred from the gel to the membrane. Normally we use a red solution called Ponceau S, but we have started to use a new protein membrane staining kit which should be more accurate and easier to wash off. The new kit has been very helpful in indicating the presence of protein and whether it is worth the time and reagents to continue with the rest of the procedure. This new kit has quite a few more steps than the original solution we used, but its all a matter of reading the protocol ahead of time. Much of lab work is following a procedure or replicating a technique. My mentor often recommends looking up the suggested instructions for various reagents and printing out the company’s protocol. We have also referenced many journals, reading for more insight into groups’ successes with a certain procedure. While reading journals, we noticed that two groups noted using a higher concentration of one reagent that was used in a solution for the western blot process. The researchers mentioned that increasing the concentration from something as small as 3% to 5% caused a decrease in the background color of the protein membrane and would allow for a clearer reading of the protein bands. Having increased the concentration, we did see better contrast between the protein membrane and the protein of interest.
This past month I tried to transfect cells for the first time. Transfection is a process of inserting genetic material into cells. In my case, I was injecting microRNA (miRNA) into neuronal cells. I had to continually reread the protocol and previous lab members’ lab notebooks for this experiment. I was trying to replicate the transfection done by a previous lab member, but in a different cell line and with different reagents. Since we were unsure of the degree of success for each reagent we also tested the dosage of reagent used. Acting as a no treatment control, I used the transfection reagents on the cells but did not include the miRNA. Then I had prepared a low dose and a high dose solution for the two different reagents and included the miRNA of interest. The cells were given some time to sit in the transfection solution before I lysed them and extracted the RNA. I then set up a miRNA quantitative polymerase chain reaction (qPCR) in order to quantify the amount of miRNA of interest in the different conditions. If there was an increase in the miRNA that we added to the cells compared to the cells that did not receive any miRNA then we know the transfection worked. We could also compare the two different reagents and the doses to see if one showed a relative increase in the amount of miRNA. With this preliminary experiment we were able to determine which reagent and method would be best for our cell line. Moving forward, we plan to transfect cells with the miRNA of interest and then also treat the cells with excess metal ions to better figure out the molecular pathway leading to the overexpression and hyperphosphorylation of Alzheimer’s disease related proteins.
So far I am still working on visualizing protein bands on the western blot. One of the earlier struggles I faced was that I had lower protein concentrations than we preferred for running a gel. Coupled with the fact that I was sometimes having trouble counting the number of cells for plating, my mentor checked the density of the cells on the plates and realized that I was plating at a lower density than what was recommended. I think overtime I was interpreting the density of the plate differently than what I should have been. For instance, when I thought the plate was ready to be split, my mentor showed me how it was still not confluent enough for splitting.
When splitting at a lower density than what is best for the cells, it takes longer for the cells to grow on the plate. This idea goes back to cell signaling lessons in Cell and Molecular Biology. If the cells aren’t in communication with each other then they will not continue to grow. Thankfully, we caught this mistake and can hopefully eliminate this error when considering why certain procedures have not been working out. In addition, since I needed to wait a week for the cells I was working with to grow up a little more, I was able to work with my research advisor more closely. We have a lot of various metal ions and small molecules we could treat the cells with and sometimes need to prioritize some over others, but I was able to help her extract protein and run a gel for quantifying protein because we were able to work as a team. We were testing these conditions in two different cell lines so the number of samples was doubled.
It has been a great experience working so closely with a professor in the lab. I have been able to ask questions small questions about a treatment condition or a step in the procedure which turn into greater conversations about the molecular mechanism in question and the many factors affecting it. So many enlightening conversations are in the air. I love stepping into other labs as well and hearing the different discussions about chemistry. It is also a neat way to learn more about people’s research as well as the different ways that their work may overlap with things we learned in classes or what we are working on in our lab. For instance, a friend of mine who is working in a biochemistry lab needed to extract protein with techniques we learned in our organic chemistry lab. I love the diversity of the research and the niche focus of the projects. I can’t wait to learn about everyone’s research projects at the poster session!
I am very happy to be back on the 6th floor of Altschul to continue conducting research this summer. I have been working in Professor Sever’s lab for the last year - starting as a student in SRI in 2016 and continuing through the academic year for credit. I am working on a project with the goals quantifying RNA and protein levels of molecules related to Alzheimer’s disease.
This project has been passed down through many students and I am very excited to continue and progress the work. Specifically, I am investigating the mechanism connecting miRNA expression and excess metal ion treatment with hyperphosphorylation of tau through the MAPK pathway and the cleavage of APP in AD. In the Sever lab, we carry out cell culture and treat out cells with small molecules or metals, as with my project. To see the effects of the treatments, we extract the RNA or protein of interest and quantify it. On the first day, I immediately took cells that we had previously stored in a freezer and “brought them up from frozen”. To do so, I needed to thaw the frozen cells, plate them in media, and then store in an incubator until I am able to work with them. After the cells start to grow to a preferable density, or confluency, I split and plate them in new petri dishes. Since a few passages are necessary to begin working with the cells, I am waiting for the cells to grow and reading journals in the meantime.
The project I am working on requires me to recall lessons about gene expression from my classes: the Spring semester of Intro Biology as well as Mendelian and Modern Genetics. Gene expression is the process of using information from a gene to synthesize a functional gene product. There are many steps involved in gene expression. The three main ones are DNA replication, transcription, and translation. Transcription results in RNA, especially messenger RNA (mRNA), and translation results in protein. When planning experiments, specifically the timing of experiments, I need to take into consideration these processes. For instance, translation is towards the end of the long process of gene expression so I need to wait 24 hours after treating cells with metals until I can extract protein from the cells. Likewise, I needed to find the optimal time for extracting RNA from the cell. I conducted a time trial to find the time which resulted in the most mRNA available in the cell since mRNA can degrade overtime if not used.
In addition to learning the more tangible side of research, I’ve also learned the more emotional side of research. For example, I’ve learned new techniques and procedures in lab and from journals, but I’ve also learned that a lot of time experiments don’t work or don’t produce a result you were expecting. There is a lot of problem solving involved in lab work once you get poor or unexpected results. In July of last summer and during the last academic year I was troubleshooting a procedure. I was trying to quantify protein using the Western Blot protocol. After the very long procedure which typically takes a few days, I was not seeing any protein bands when imaging. We went back to the drawing board and tested all the steps in the method which could possibly cause the result we were getting. Finally, we were able to better optimize the procedure and see protein. What I hope to do this summer is finish quantifying the proteins of interest.
Along with quantifying protein, I hope to work a step prior in the gene expression process and quantify RNA. I hope to see a similar response with specific mRNA to that of the protein they encode. For instance, if there is an increase in MAPK3 protein after treatment compared to that of non-treated cells then we would hope to see the same increase for mRNA for MAPK3. I hope I will be able to quantify the molecules of interests and be able to replicate the results at least three times. I will also be working with three different neuronal cell lines and I hope that I can replicate the results in all three.
I also plan to quantify the amount of microRNA in the cells after treatment with excess metals. MicroRNA are involved in the gene expression process and specifically can cause translational repression. The miRNA complementarily binds to mRNA and inhibits translation so no protein is produced. There are many miRNA that are related to Alzheimer’s disease. I have been reading a lot of journals to find more information about miRNA and have compared all my sources. There is a lot of information available to us and it is important to check for any new findings or inconsistencies.
Each week, my group presents our research updates in a group meeting. I have learned that presenting is just like telling a story. It is easy to jumble up the specific details you want to communicate, but it is necessary to take some time to plan and present the information logically giving plenty of background information and details. Having presented in group meeting for about a year I have gained confidence in my ability to present and communicate the work I have done. Looking back, I have noticed my presentation skills improve from just my experience over last summer. I was floored by the eloquence of the presenters when I listened to many thesis presentations in the Chemistry Department. I am glad that I can seem calm as well as experienced when presenting.
I have also learned that it is okay to ask questions. I try to reason concepts out by myself but often need to check for confirmation with Professor Sever. Often, Professor Sever answers my question by stepping me through the process in question and drawing out information from me that I can add to solve my problem. I have found this method of answering questions may take a bit of time, but it is very helpful to have the person asking the question learn the answer by working it out with some guidance. This was one of the techniques that Barnard College suggests peer tutors use. As an Organic Chemistry peer tutor, this technique, although it required more time, helped students solve problems with better understanding and retention of the information.
This past week a few of those who are eligible took a safety course in order to become C14 certified. Each lab is required to have someone on site who has this certificate of fitness and I am very excited to finally take the test and get my certification. With this certification, my research advisor said that I would be able to receive a copy of the keys to the lab. I am very excited to take on more responsibility. All in all, I am thrilled to continue working on the project I started last summer and further the research with substantial and reproducible results.