I had the opportunity to spend the summer in the Ju Research Group as a student research assistant for my STEP Signature Project. Specifically, my research focused on elucidating the biosynthetic pathway of the production of two nitro-containing natural products, 2-nitroimidazole (commonly referred to as azomycin) and N-nitroglycine. These two natural products exhibit antimicrobial activity and have a unique chemical moiety of the nitro group.
Prior to this experience, I have had some experience doing research but I have never invested that much time into it nor did I have the skills to independently complete my own experiment. With some knowledge regarding the topic of biosynthetic pathways, I entered my STEP project believing that I could accomplish my goal with relative ease. I had read papers about determining the biosynthetic pathways of natural products before and was confident that I knew how to do this. However, this project opened me to reality. During my project, I realized that while being optimistic is good, you cannot be overconfident. I thought that I had the skills and mental fortitude to complete the experiment, but I was faced with many obstacles. Though I know that not all experiments, or even events in life, work out the way you want them to at first, I was not prepared to meet an onset of failures. It was very disheartening to repeat experiments to no avail. At some point, I even began to lose hope and wanted to give up. However, I tried to find a solution to my problem and realized that with will, anything is possible. Doing research is not easy—in fact it involves many failures that you cannot anticipate. I now know that I have to be mentally prepared for experiments not working the way you want them to, and that eventually it is possible to overcome these issues by finding a solution.
I started the project by first creating a cosmid library for 2-nitroimidazole. This was very difficult for me as I have never used the molecular genetic techniques involved in this before. Thankfully, with the guidance of my principal investigator (PI), I was able to successfully create a cosmid library on my first try though it was smaller than it should have been. I then had to design primers for the first time to screen the library for the putative gene cluster. Although the actual designing of the primers went smoothly, I faced my major failure in trying to screen the cosmid library via Polymerase Chain Reaction (PCR). I screened 960 cosmids from the library (though I managed to condense them to 96 actual reactions) using PCR, but there were no DNA bands when I analyzed the DNA gel. I knew that statistically speaking, I should have had at least two positive hits out of the 96 I had done, yet somehow there was not anything. At first, I thought that perhaps I had not correctly assembled the PCR reactions, so the next day I repeated them again, only to find that I had failed again. Again, I did the same PCR except changing the annealing temperature in hopes of getting some kind of result. Somehow, I had managed to waste an entire day and many materials on an experiment that had completely failed. I went home that night thinking of all the possible ways that I could possibly fix this issue.
At this point, I wanted to give up. I was overwhelmed with feelings of inadequacy and uselessness, and thought that there was no way that I could achieve my goal. I did not want to talk to my fellow lab mates nor my PI because I felt that I had failed them. However, with the advice of my friend who is also in a research lab, I decided to approach my PI about how we could solve this issue. The next day my PI and I discussed ways that we could optimize the PCR reaction. Our line of reasoning was that there was nothing fundamentally wrong with the cosmid library nor the primers, but perhaps the PCR reaction was not the ideal conditions for my template and primers. Thus, I did several different PCR tests where I changed the reaction composition and even the PCR cycle itself until finally, one set worked. With this success, I was renewed with hope and screened the 960 cosmids once again, with 7 different positive results.
Although I had experienced that major failure, I had been able to overcome it by thinking about the reason behind its failures and coming up with a solution to overcome them. Suddenly I felt like I was able to do whatever I put my mind to. I managed to successfully create the cosmid library for the strain that produces N-nitroglycine on my own, and it was even larger than the first one. Because of these successes, I was able to do experiments without the need to rely on someone else, but if I ever got confused I had many people who I could go to for advice or guidance like my lab mates or PI. Though I had not completed my actual research project of elucidating the biosynthetic pathways of 2-nitroimidazole and N-nitroglycine during the STEP Signature Project, I am confident that I will be able to in the coming year.
Overall, I think that my STEP Signature Project has transformed me for the better as a scientist and person, and these will all help me in my future career as a pharmacist. Due to this experience, I am now equipped with more skills and knowledge that are necessary for performing research. I know more techniques and skills, but I also know that failures are not the end of a project. Even in the face of failures, I can troubleshoot to come up with possible solutions. In addition, I can independently perform my own experiments. These skills will better help me in my future research as I complete my research project. However, the most important thing that has changed in me is my confidence. I have a tendency to feel incompetent in the face of failure but because of this experience I feel more confident in my abilities. In addition, I am no longer scared to ask for help from those close to me. As I plan on becoming a pharmacist, I think that these qualities will be fundamental in my career. I will have to work in a hospital where I will undoubtedly be left in situations where I will be unsure of what to do. Now, I am better prepared to anticipate problems, find solutions, and ask for advice.