Wendy Zhan is a rising junior majoring in Biology. She was awarded a Spring 2018 Independent Grant which she used to conduct research on single-celled amoebas under Dr. Adam Marcus.
When I first got an email back from Adam (my PI) saying that I could start going into lab, I was extremely excited. I had been assigned my own project where I was going to use Dictyostelium discoideum, a single-celled amoeba, to model cancer metastasis and tumor heterogeneity. As the only lab experience I had at the time was high school biology and chemistry lab, I expected that working in an actual lab would be a similar experience - that I would follow some procedures and get my results. However, as I quickly learned, I couldn’t have been more wrong.
There were several challenges that I faced when I began my project, first of which, is the learning curve. What they don’t tell you about science is that more often than not, it doesn’t work out the way you intended. At the start of the project when I was trying to successfully culture the cells, I was faced with contamination issues that wouldn’t seem to go away even though I had followed the protocol specified for culturing Dictyostelium discoideum. It took me a couple weeks to a month of learning how to culture and developing my technique to minimize contamination; however, the bacteria was still present no matter what I did. Thus, I began to experiment outside of the protocol and read up on more papers in the field to determine how using different media and antibiotics would affect the cultures. Ultimately, I was able to discover the best mix that allowed my cells to grow without contamination and increased the dilution of the antibiotic drug (Penicillin-Streptomycin) until it was at the recommended 1:500 dilution.
Fast forward 6 months where I began to live image the amoeba in their developmental stage and create the cell lines necessary for the execution of the project. Luckily for the live cell imaging, I was able to nail it down and gain a quality video of the cells going through development after 5 tries. However, the time that the 5 tries took was over the span of 3 months. Thus, science has taught me that patience and attention to detail is key as every time it didn’t work out, I had to figure out how to improve the design to make it more successful for the next time. The process of transforming Dictyostelium with plasmids has proven to be a much more challenging feat. This entailed searching through a large database of plasmids specifically designed for Dictyostelium on “Dictybase”, a website dedicated to all information and current research involving the species. The purpose of using a plasmid is to insert it into the cells so that the cells can exhibit fluorescence and allow us to “label” the different cell types and differentiate between them when cultured together. As our lab had previously created a technique called “SaGA” which allowed us to mark cancer cells of interest with a laser and photoconvert them from fluorescent green to red, we already knew that the plasmid that needed to be inserted into the cells which allowed for photo conversion was Dendra. Upon searching through papers and the database, we found that there was only one Dendra plasmid available for Dictyostelium and that there were no published articles on it, meaning that nobody has done much work with the particular plasmid in this field. Thus, there has been a lot of trial and error with trying to insert it into the cells. As of now, we have had multiple trials using lipofectamine and electroporation in an attempt to transform the cells with no luck. For electroporation, we were faced with yet another challenge in which the Amaxa Nucleofector II machine does not allow you to set specific voltage settings, a number which is described in many protocols. Rather, the machine provides a vague list of options “A-Z” and “1-34” in which you have to choose one from each category to create an electroporation program to zap your cells. The issue with this is that there is no one in the lab who knows what the letters or numbers mean. In addition, attempts to find a manual or key that might tell us the meaning to the letters and numbers have proven futile as the company has not and does not seem to want to provide any forward. Moving forward, we are hoping to try calcium phosphate.
Thus, what I have learned is that science is not nearly as straightforward as science class labs may seem. Although you may have a protocol to follow which details exactly how to perform the experiment, things may not turn out the way you may expect and it usually takes many tries to get it right. That being said, I have also learned the importance of communication and collaboration. Without the help of the professionals in the lab as well as the Dictyostelium community, I would not have been able to learn as much as I have during the past year working at the Marcus lab. I am appreciative that the scientific community is very supportive, especially of beginner researchers like me, and are willing to answer most questions that you have and are happy to see people curious about research. In addition, I have found that the challenges that come up offer opportunities to find a new or better techniques that may improve the overall research experience with whatever you end up working with in lab. Thus, I encourage people to step out of their comfort zone and engage in research if they want to truly see how working at a lab bench feels like – it definitely is nothing like I had expected going in.
Visit the Undergraduate Research Programs website to learn more about applying for Independent Research Grants.
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