Saket Ram, SEAS ’27, Jersey City, NJ
This summer, I had the wonderful opportunity to work as an undergraduate researcher at the Bugaj Lab at Penn Engineering. This was my first time doing research, and I was a little nervous—but, Dennis, a graduate student and my mentor at the lab, and Dr. Bugaj, my PI, were both extremely patient and showed me firsthand what a future career in research could be like.
The lab has a very broad focus on synthetic biology and cell signaling, and two ways we understand how the cell works is through optogenetic (proteins that are sensitive to light, like Cry2) or thermogenetic (proteins that are temperature sensitive) tools. The projects I focused on were primarily related to this: on the optogenetic side, we were interested in creating tools that would use light-induced clustering to regulate cytoplasmic-nuclear transport. On the thermogenetic side, we explored the creation of thermosensitive proteins by inserting a temperature-sensitive fragment into predetermined locations, including a thermosensitive fluorescent protein reporter (a protein that would indicate in vivo temperatures based on brightness).
In order to synthesize these constructs, we needed to make plasmids, which are small, circular pieces of DNA that contain the base pair sequence encoding for whatever proteins we want to create. Usually, we also fuse the DNA encoding the protein we want with a fluorescent protein, so that we can visualize it with confocal microscopy. To do this, we needed to harness the power of cloning.
HEKs cotransfected with Cry2-mCherry (a red fluorescent protein) at t = 0s (left). Within seconds of blue light stimulation, clusters begin to form (middle, at t = 20s). Light stimulation is shown until t = 180s (right).
One of the first things I learned was the cloning process—even though I’ve done it dozens of times now, it still fascinates me how we can manipulate DNA that we can’t even see. In cloning, you start out with a template, where you identify a small part of interest called the insert. With PCR, you amplify the insert by designing primers, and isolate it from the template with gel electrophoresis. At the same time, you take your vector plasmid (the piece of DNA you want to add your insert into) and digest the parts that you don’t want with restriction enzymes. Then, with ligation, you connect your insert with your vector plasmids, creating a new plasmid, grow it in bacteria, and then purify it. And within a few days (if everything goes well and you didn’t miss any steps), you have a newly created plasmid!
After I mastered this, I learned how to put this DNA into mammalian cells and image them with the confocal microscope. The first time I saw the cells, it was incredible. Watching how these tiny biological machines responded in real time to light and temperature stimuli was like witnessing science fiction come to life. The ability to control cellular functions so precisely felt almost magical, and it gave me a glimpse of the vast potential of these technologies in understanding and influencing biological processes. And with the data we collected from the confocal microscope, we could quantify our results using ImageJ/FIJI, CellProfiler, and R, and see the effects of our constructs not only qualitatively, but also in terms of line graphs and box plots.
Slowly, over the weeks, I got better and more efficient at this iterative workflow—cloning, transfection, imaging, quantifying, and reevaluating—and our experiments started to become increasingly successful. I’m really excited to present our findings when I get back and see where we can go from here!
I want to thank Career Services for giving me the opportunity to spend my summer doing research, as well as Dr. Bugaj, Dennis, and the rest of the lab for making my experience so memorable. I can’t wait to continue working on my projects and exploring thermogenetics and optogenetics in the fall!
This is part of a series of posts by recipients of the 2024 Career Services Summer Funding Grant. We’ve asked funding recipients to reflect on their summer experiences and talk about the industries in which they spent their summer. You can read the entire series here
