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Elucidating the Maturation Mechanism of the
Lasso-Structured Antimicrobial Peptide Microcin J25

Adviser: A. James Link

Wai Ling Cheung

Chemical and Biological Engineering

“I was able to delve deeply into research, where I was able to be the one to discover knowledge in a way that would never be found in the classroom setting.”

cheung-wai-ling

I have never been much of a writer. From the moment I decided to attend Princeton, I dreaded the thesis. However, as a freshman, senior year seemed far off. But as most Princetonians would agree, the semesters fly by pretty quickly. Soon, the thesis was no longer three years away, but less than one. Therefore, toward the end of sophomore year, I decided to pursue independent work. I was interested in research, but I also wanted to get a feel for what a thesis would entail. I approached a couple of professors whose work I was interested in, and fortunately Professor James Link offered to advise me. He would later become my thesis adviser. Over my junior year, I learned everything from keeping a lab notebook to cloning genes into bacteria and making them produce protein. By senior fall, I had a year’s worth of lab experience. The work that I did junior year could not be included in my thesis because of department rules, but it served as background research and prepared me well in technical aspects for my thesis research; I had acquired most of the skills and learned most of the techniques I needed to use for my thesis the year before. Whatever I still needed to learn, Professor Link was always there to teach and advise me.

I would recommend doing some research before senior year if possible, primarily to get used to working in a laboratory setting. Not only did I get to learn earlier the skills that I later needed for my thesis work, but the research itself was more interesting because I was able to develop the project myself. In my case, I spent a couple of months working in the lab before I truly appreciated the excitement of what I was doing. By the time senior year began, I spent hours every day in the lab because I was eager to see the results of my experiments. Excitement over my research prevented me from procrastinating, which definitely paid off in the spring. By the time I needed to start writing, I had plenty to write about. Ultimately, starting my research early allowed me to become more immersed into my research than I would have been if I started senior year.

However, that is not to say everything went smoothly. My thesis topic was on how microcin J25 (MccJ25), an antimicrobial peptide with a special lasso structure, is produced by bacteria. I began the fall semester with a plan to isolate the precursor to MccJ25 and the two enzymes involved in creating the lasso structure to do in vitro tests. We wanted to know what each of the enzymes did and hoped to figure that out by isolating possible intermediate forms between the precursor form and the final MccJ25 structure. By November, though, I was unable to detect MccJ25 production in vitro using my positive control test.

I was disappointed, but my adviser and other lab members kept me from becoming discouraged. Professor Link always welcomed me when I stopped by his office to discuss the issues I had and what we should try next. Everyone in the lab also was friendly and willing to listen. Even though I eventually had to abandon my original approach to my thesis, I was, to my surprise, not terribly stressed. My adviser and I had discussed other possible approaches to my topic since the fall term began, and I was doing some of these other experiments on the side while I tried to troubleshoot my in vitro experiment. In November, we decided to put the in vitro approach on hold and focus on the other experiments I was doing instead.

By the end of February, I had a few more tests to do, but had the bulk of my data. It was time to write, the part that I simply did not want to do. I probably put it off for longer than I should have, and in short, it was very difficult and almost painful. Outlining, writing, making figures, and formatting all took longer than I expected. I spent all of March writing my first draft, just in time for the departmental deadline for a full draft. This deadline before the actual thesis deadline kept me sleep deprived for a month, but it was a blessing in disguise. It gave me time to edit and include comments from my adviser, who read over my first draft, before the actual thesis deadline. In the end, I had a thesis that I was proud of.

I would describe my overall thesis experience as extremely enriching. I was able to delve deeply into research, where I was able to be the one to discover knowledge in a way that would never be found in the classroom setting. I was able to make a contribution to the scientific community, publishing part of my thesis findings in the Journal of the American Chemical Society. Although I did not enjoy the tortuously slow writing process while I was doing the writing, at the end I had a tangible reflection of the work that I did enjoy. The most important piece of advice that I can give to seniors is to choose a topic that actually interests you, as it made my experience that much more enjoyable and gave me the motivation to keep myself on top of my research.

Elucidating the Maturation Mechanism of the
Lasso-Structured Antimicrobial Peptide Microcin J25

Wai Ling Cheung

A. James Link

Assistant Professor of Chemical and
Biological Engineering

“Whereas my classroom teaching is focused on disseminating knowledge, the interaction between a senior and an adviser in a thesis project results in the creation of new knowledge.”

The senior thesis is a major defining aspect of a Princeton undergraduate education. Because of this central role it plays, I make sure to always host several students in my laboratory each year for senior thesis work. My career path was steered toward research in large part because of my senior thesis experience at Princeton 11 years ago. I enjoy advising undergraduates in research because these students can work on high-risk/high-reward projects. Because these projects are offshoots of the main research programs in my lab, the contributions made by senior thesis students can push these programs in new and different directions. Whereas my classroom teaching is focused on disseminating knowledge, the interaction between a senior and an adviser in a thesis project results in the creation of new knowledge, and I find these interactions particularly rewarding.

In the case of Wai Ling Cheung’s senior thesis, she was able to solve a challenging puzzle relating to the biosynthesis of a class of short, knotted proteins called lasso peptides. As their name implies, lasso peptides are tied into a slipknot by enzymes in certain bacteria. Lasso peptides start out as a precursor protein, a linear chain of amino acids that can be conceptualized as a rope. This rope is then chemically “tied” into its lasso shape by enzymes. Bacteria make a precursor protein that is much longer than the final lasso peptide product; in other words, the precursor has a lot of extra rope that is not needed to tie the peptide knot. What Wai Ling figured out was that the majority of this “extra rope” could be trimmed off of the precursor without affecting the ability of the enzymes to tie the peptide knot. This work resulted in a publication in the Journal of the American Chemical Society, a prestigious chemistry journal.

My interactions with Wai Ling began well before her senior year. She started working in my lab on an independent work project at the beginning of her junior year. The summer between junior and senior years she served as a Stoll fellow, continuing her junior independent work and laying the foundations for her thesis. In this time leading up to her thesis, Wai Ling learned all of the laboratory techniques she would need for her thesis work, so she hit the ground running in September. If I can offer one piece of advice to rising seniors reading this, it would be to get started as early as you can on your thesis. The demonstration of a strong interest in your project is always appreciated by those of us who serve as thesis advisers, and the sooner you can get through the “learning curve” portion of your project, the sooner you can start to make real progress.

During the thesis, Wai Ling functioned as a full member of my group, showing up to the lab essentially every day (including a lot of nights and weekends) and attending our weekly group meetings on Wednesday nights. Wai Ling worked so diligently on her thesis that sometimes it was easy to forget that she was still enrolled in a full set of courses! In addition to these formal weekly meetings, Wai Ling and I would often informally meet a couple of times per week to discuss the progress on her project. These informal meetings increased in frequency to essentially daily toward the end of the fall semester as Wai Ling’s project really started to take off and we prepared a manuscript for publication. Wai Ling was a particularly strong writer, and the sections of the manuscript I asked her to write were done quickly and impeccably, requiring minimal editing by me. Perhaps the only difficult part of Wai Ling’s thesis experience was that her main project was completed (and submitted for publication!) in January, so we needed to decide on something for her to work on for the spring semester. The work she did in the spring was also of very high quality, and is being continued by students in my lab.

Wai Ling was one of the strongest senior thesis students I’ve seen because of a unique combination of traits. As I’ve mentioned above, she had an indefatigable work ethic but was successful at balancing that work ethic with a constantly positive attitude. I think I can honestly say that Wai Ling had fun in the lab. When an experiment didn’t work out, she never let a negative result get her down; she simply went back into the lab to troubleshoot and move on. Wai Ling’s thesis also was particularly strong because she was able to convey her results well to people who are not specialists. In our department, the seniors present a poster, and these posters are judged by several professors. Since chemical and biological engineering professors have many different research expertises, Wai Ling’s ability to explain her research to a diverse crowd was essential to her success.

In summary, Wai Ling’s excellent thesis experience can be used as a blueprint for success in future senior theses. Get started as soon as you can and work hard. Practice your writing and make sure you can communicate your results to a broad audience. Keeping these suggestions in mind will not only help you with your thesis, but also in whatever career you end up pursuing.