About Me: I am a high school science teacher. I have been teaching for the past 7 years. Currently, I teach college-prep biology, anatomy & physiology, zoology, and biomedical science at Douglas High School.
I signed up for the RET program because I want to incorporate authentic research into my curriculum and share it with my students and colleagues. Over the past few years, the science department and I have been working to build our science program, introducing five new elective courses in the past two years. By taking part in this program, I will have an opportunity to present real-world problems to students and give them the opportunity to research sustainable and equitable solutions. Students who choose to take these courses are looking to pursue future careers in the health sciences. Providing them with research experience will prepare them for research they will complete as part of their college education and beyond. I also want to share my experience with my colleagues so that we as a department can work together to create research experiences for our students.
About the Lab: We are a microbial molecular biology lab seeking to address basic research questions that hold the keys to advancements in human health and understanding the natural world. Our central goal is to understand the regulatory mechanisms that underlie mycobacterial stress tolerance. We combine genetics, genomics, transcriptomics, and biochemistry to understand how mycobacteria respond to, and ultimately survive, stressful conditions. Our guiding principles are curiosity, respect, and scientific rigor. Together we strive to push the boundaries of knowledge and advance our field for the ultimate benefit of humanity. We are located in a broad multidisciplinary department in a collaborative science and engineering institution in the heart of the vibrant Massachusetts life sciences community.
Project: Mycobacterium tuberculosis (Mtb) is the most lethal single infectious bacteria (Kreutzfeldt et al., 2022) affecting our planet. It is so successful as an infection due to its successful ability to resist and tolerate antibiotics and stress conditions. This makes it crucial to determine whether an antibiotic is successful at treating Mtb infections. Current methods for determining whether Mtb cells have undergone apoptosis are cumbersome due to the bacteria’s colony-forming nature, although some studies have shown that Mtb cells don’t always form colonies on growth media (Barr et al., 2021). Therefore a new method is needed to determine whether Mtb cells are alive or dead after antibiotic exposure.
The goal of our research is to develop a live-dead fluorescent reporter system using the mScarlet fluorescent protein. In doing so, a new sequence containing specific promoter regions, UTR sequences, tet operon sequences, GFP, and mScarlet has to be annealed using a previously made plasmid. This sequence then has to be input to Mycobacterium smegmatis, a model organism for Mtb that behaves similarly. After doing so, flow cytometry will be used to detect which cells underwent apoptosis and which are still in an active and alive state. The cells that developed a resistance will be fluorescent, and the ones that were killed will not. We will then be able to use this data to determine the success of the developed system.
Weekly Updates:
- Week 1: This week I learned how to use Benchling in order to determine primer sequences to amplify the mScarlet gene and the pTB12 promoter. I also read journal articles to get some background information on Mtb and using fluorescent proteins as a live-dead reporters.
- Week 2: We finally ordered the primers and they came in. Yay! Liz and I did Q5 PCR to amplify the different DNA fragments we would need for our new plasmid.
- Week 3: We ran a gel electrophoresis on our PCR products to confirm that our PCR products are the correct size. Then we cut the bands from the gel to purify the PCR products.
- Week 4: We were unable to get enough PCR products from the purification. We learned that some of our primers were not working the way we had hoped, so we met with Scarlet to redesign them.
- Week 5: We did PCR of the PSS470 plasmid, mScarlet gene, and pTB21 promoter using the new primers. We did gel electrophoresis on them and purified the DNA fragments from the gel. We finally got enough PCR products to do the HiFi assembly. We also worked on a draft of our final poster and draft of the lesson plan
- We did the HiFi assembly to make the new plasmid, then we transformed the new plasmid into E. coli. Then we cloned the plasmid, and extracted it from the cells to be sent out for sequencing. We worked on our final poster and lesson plan.
Poster and Lesson Plan: