Author: Grant Robinson     Majors: Biochemistry, Biophysics

Working with gel and creating SPE solutions

Throughout the 2020 fall research grant semester, I worked under the guidance of my research mentor, Dr. Julie Stenken, to finalize my previous research projects and begin to work toward publishing a paper with my senior research graduate and toward working on my thesis. The Stenken lab has pursued a greater understanding of the interactions between the antimicrobial peptide LL-37 and biofilm-forming diseases such as those from S. aureus and P. aeruginosa. Within the broad aim of the lab, I have sought to develop a novel method to isolate and quantitate LL-37 within a complex media resembling that of the human body. After many unique challenges resulting from the charged nature of the peptide, I was finally able to solve the problems I faced and determine a novel method to quantitate the peptide. Now, having optimized my designs, I am looking toward completing my honors thesis and finalizing my undergraduate research.

This semester has presented itself with a host of unusual challenges. All of my classes were online, and going into the laboratory was far more challenging than it had ever been before. This forced me to become more deliberate and efficient in my few lab visits. Following previous semesters of research, I had already determined my method of isolating and quantifying LL-37. My lab time this semester focused on optimizing the method and determining the data analysis of the trials I had run. Using the guidance and data from my graduate lab assistant, Alda Diaz Perez, we managed to slowly accomplish these goals.

Previous studies had led our work toward utilizing a weak-cation exchange solid phase extraction device. Due to the positively charged nature of LL-37, methods of isolation such as size exclusion and typical solid phase extraction were shown to not be viable. However, the weak-cation exchange column for our solid phase extraction was not enough to fully isolate the peptide of interest. This led to considerable challenges with no prior work serving as a guide for our project. Eventually, we utilized gel-electrophoresis following the solid phase extraction method. This breakthrough allowed method validation using UHPLC/MSxMS to quantify the isolated LL-37. This semester, I worked to perfect the method we had determined. It was found that when acidic conditions were used to condition the samples, the largest percent recoveries were found. Additionally, several trials were run of the gel-electrophoresis to most precisely and accurately remove only the LL-37 from the gels for trials.

Following this optimization work, most of my work this semester was focused on writing. Working closely with Alda, I spent considerable time editing a paper that we hope will be published soon. This required considerable knowledge of all the work that I had completed over the past three semesters working in the lab. Being able to compact all the work that we had done down into its most important experiments while convincingly exploring the importance of the research challenged me in ways that traditional lab papers never could. Furthermore, this paper helped to prepare me for another challenge I have begun to face, my thesis. Now that my research has largely become wrapped up, I have started to write my thesis. This unique style of writing has pushed me even further to learn how to understand my own work and how to portray that work in a manner others will gain from.

This research grant has allowed me to explore a topic of study that I have been highly passionate for in ways I never imagined prior to being an undergraduate. The grant has funded experiments and trips that showcased my research. This has allowed me to spend considerable time working toward furthering my research project and to fully appreciate the difficulties of the project. Thanks to this help, I have now fulfilled my goals I initially set out to accomplish, and I intend to move onward into the final semester of my undergraduate career where I will use all the research I have completed to write and defend my thesis. I intend to move on to medical school following this semester where I hope I will continue to learn about topics of study similar to what I have researched and will hopefully be given more opportunity to conduct further research.