Meet Swamy Singaravelu
Major: Biology
Graduation Year: May 2016 Field of Study: Microbiology/Molecular Biology Abstract: Multidrug-resistant bacteria and the lack of drugs to combat them are serious issues threatening public health. Beginning in the 1980s, the emergence of vancomycin resistance in Enterococcus faecalis, a gram-positive bacterium and opportunistic pathogen, initiated the alarm for a closer look at antibiotic resistance in microbes colonizing the human body. At the end of that decade, a system analogous to an adaptive immune system was discovered in bacteria: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) with CRISPR associated genes (Cas). Approximately 45% of bacteria encode CRISPR-Cas systems, which defend cells from mobile genetic elements (MGEs) introduced through horizontal gene transfer. CRISPR-Cas systems utilize a three-stage mechanism to provide adaptive immunity to microbes: adaptation, expression, and interference. It has been demonstrated in Staphylococcus aureus that Cas-encoded RNA-guided nucleases (RGNs), specifically Cas9, can be utilized in conjunction with an engineered CRISPR to target the bacterial chromosome, thereby acting as antimicrobials. Here, we investigate the potential for Cas9-guided antimicrobials in E. faecalis. E. faecalis T11 possesses a Type II CRISPR-Cas system, which contains the CRISPR2 and CRISPR3-cas loci. The CRISPR3-cas locus has the cas9 gene that encodes for Cas9, a RGN that aids in the cleavage of MGEs. We hypothesize that the introduction of pTCV-lac containing a chromosome-targeting CRISPR into E. faecalis T11 will activate the endogenous CRISPR3-Cas system and induce bacterial cell death by cleavage of the bacterial chromosome at the target gene by Cas9. In this study, our aims were to: 1) generate engineered “auto-immunity” CRISPR constructs under the control of the cas9 and CRISPR promoter regions, and 2) determine whether these constructs confer an antimicrobial effect when introduced to E. faecalis. We find that when ddl, mtlD, and murAA genes are targeted using engineered CRISPR constructs under the control of the cas9 promoter, there is a growth delay in wild type T11 but no growth delay in T11 Δcas9, a strain that lacks Cas9. Our results suggest that engineered CRISPR-Cas constructs can be used to kill E. faecalis. |
My Experience:
Research was something that I have always wanted to do, even as a high school student. However, I did not come across any opportunities to engage in research until my college years. I first heard about the Green Fellowship when I was in high school. The experience offered by the program combined with my interest in scientific research motivated me to plan my years at UT Dallas so that I would be able to devote a semester to research as a Green Fellow. I thought the toughest part would be getting accepted into the program. To my surprise, though, it was actually the actual research experience that was demanding but, at the same time, rewarding.
Even though I had a few months of experience working in a lab prior to becoming a Green Fellow, I still had my fair share of up’s and down’s. The dedication and responsibility that were required for working full-time on an actual scientific research project were the most surprising aspects of my experience. Since I was working with bacteria, my project was divided into multiple stages. Each of these stages had to yield good results, or I had to repeat each stage until I got satisfying results. Sometimes I had to start over from the very beginning due to factors beyond my control. But the excitement that came with moving from one stage to another (when they worked that is!) pushed me to advance further in my project. Since I was able to work with Ph.D. student, Valerie Price, and others in the Palmer lab, I also learned a great deal about teamwork and the extent to which teamwork is ingrained within research.
Although my Green Fellowship experience started off a bit unsteady, I slowly came to enjoy my role as a student researcher and came to understand the true meaning of research. I would like to thank Dr. Kelli Palmer and her lab for teaching me a lot about microbiology and helping me improve my research skills. I look forward to incorporating research into some aspect of my future medical career.
Research was something that I have always wanted to do, even as a high school student. However, I did not come across any opportunities to engage in research until my college years. I first heard about the Green Fellowship when I was in high school. The experience offered by the program combined with my interest in scientific research motivated me to plan my years at UT Dallas so that I would be able to devote a semester to research as a Green Fellow. I thought the toughest part would be getting accepted into the program. To my surprise, though, it was actually the actual research experience that was demanding but, at the same time, rewarding.
Even though I had a few months of experience working in a lab prior to becoming a Green Fellow, I still had my fair share of up’s and down’s. The dedication and responsibility that were required for working full-time on an actual scientific research project were the most surprising aspects of my experience. Since I was working with bacteria, my project was divided into multiple stages. Each of these stages had to yield good results, or I had to repeat each stage until I got satisfying results. Sometimes I had to start over from the very beginning due to factors beyond my control. But the excitement that came with moving from one stage to another (when they worked that is!) pushed me to advance further in my project. Since I was able to work with Ph.D. student, Valerie Price, and others in the Palmer lab, I also learned a great deal about teamwork and the extent to which teamwork is ingrained within research.
Although my Green Fellowship experience started off a bit unsteady, I slowly came to enjoy my role as a student researcher and came to understand the true meaning of research. I would like to thank Dr. Kelli Palmer and her lab for teaching me a lot about microbiology and helping me improve my research skills. I look forward to incorporating research into some aspect of my future medical career.
My Advice:
I highly recommend future Green Fellows to have some idea of what field of research they are interested in before applying, keep in touch with their UTD mentors, and complete a senior honors thesis. I think one of the toughest decisions to make as a Green Fellow is choosing a field of research. Every field is important and is changing everyday, in terms of techniques and discoveries. However, it would be a good idea for anyone interested in the program to choose some fields that interest them the most and read a few papers from each one. This will help, when you’ve to narrow down your interest to one field of research. It is always a good idea to keep in touch with your UTD mentor, because often they will be the one who will guide you and help you compile your project. Many view the senior honors thesis as a lot of work and do not understand the importance of it. While writing a thesis, you are exposed to the fundamentals of scientific writing, which may help you when you publish a paper in the future. Even though your project may not yield amazing results, I highly advise future Green Fellows to complete a thesis. The though process involved in writing one will prepare you for professions that require constant exposure to scientific and medical literature. Finally, and most importantly, make sure that you like what you do and are comfortable working on your project. |