About me: Hello everyone! My name is Abigail Prisby, and I currently teach Biology and Environmental Science studies at Groton-Dunstable Regional High School in Groton, Massachusetts. This is my first year participating in the RET program at WPI, and it has been an amazing opportunity to learn about much of the current research studies going on in the life sciences and engineering fields. It has also been a great way to develop new lab skills that will be taught to incoming students at the high school level. It is my hope to use the knowledge, lab skills and techniques learned to create engaging and meaningful lessons for students that will inspire them to become more involved in the sciences.

About the Lab: I will be working in the Shell lab this summer working on the research to understand innovative ways to combat the pathogenic strains of bacteria that cause infection during Tuberculosis. Tuberculosis affects the human population significantly in countries around the world. Without proper treatment, it can be fatal. Before the Covid pandemic, tuberculosis was the leading cause of mortality by a single infectious agent worldwide! TB is a contagious bacterial respiratory infection that spreads through the air. Treatment typically involves multiple antibiotics taken for six months, but the type of drugs used depends on the form of TB. Treating this infection can be an extremely cumbersome process, as some forms of TB are resistant to certain antibiotics, so a combination of drugs may be necessary. It can be difficult for specific antibiotics to work effectively against these cells because typically antibiotics target actively growing cells and proteins that regulate cellular processes required for the cell to stay alive. Normally, by targeting ways to shut down these essential cellular processes, the cells causing infection can be eliminated within the body, mitigating the symptoms caused during infection. However, for these nonactive, slow growing cells involved in TB, antibiotics cannot inhibit these nonactive, slow growing cellular processes efficiently, making antibiotics less effective during treatment.

This lab focuses its research towards understanding how the bacteria that cause tuberculosis (TB), Mycobacterium tuberculosis (Mtb), are able to survive a variety of microenvironments within the human body during infection, and the role of evolution of antibiotic resistance. Specifically, this lab is isolating and analyzing the proteins coded within a non-pathogenic model species, Mycolicibacterium smegmatis, that may allow the pathogenic mycobacterium species to withstand many modern treatments for tuberculosis (TB). The overall goal is to understand new innovative ways to combat drug-resistant strains of M. tuberculosis in order to reduce the negative health effects caused during TB.

Project Title: Investigating the Minimum Inhibitory Concentrations of Translation-Related Antibiotics Against Mycolicibacterium smegmatis

Project Overview: The portion of the research that I will be working on is performing growth assays for testing the minimum inhibitory concentration (MIC) of antibiotics required to prevent cell growth. We will be working with a nonpathogenic strain of mycobacteria, M. smegmatis, that is evolutionarily similar to Mtb to test the phenotype of a novel protein called 5691 whose function is not well understood. The Shell Lab has recently discovered that this protein plays a role in ribosomal assembly and potentially RNA degradation, which are importance cellular processes needed by the cell to function. It is suspected that in the absence of 5691, the RNAs that comprise ribosomes (rRNA) is synthesized and processed differently from wildtype cells. Therefore, the ribosomes may function less efficiently during cellular translation. We hypothesize that any antibiotics that target translation will have a greater affect on these genetically altered TB cells since the rRNA is different.

Weekly Updates:

• Week 1: This week has been a busy time getting assimilated into the RET program! We began our first days with orientation and team building to meet the other RET participants, then were introduced to some of the graduate students working within our lab spaces. I was able to spend some time the first two days touring Dr. Shell’s lab space, and was able to shadow a couple of the graduate students working on their research! During this time, I was able to start refreshing some of my prior knowledge of RNA transcription and translation processes, while pushing myself to advance my knowledge to understand each of the graduate students’ projects and the overall goals of the lab’s research. We also had the opportunity to tour the chemical engineering lab, and see some of the current projects and engineering equipment to date. I was able to shadow one researcher working on isolating and testing the effect of different proteins involved in RNA degradation, which is a main focus point in the lab. I was also able to sit in on Dr. Shell’s weekly lab meeting, where one graduate student presented their paper to the group for feedback. It was a challenge to keep up with the level of topics being discussed, but very informative to see how research is presented and critiqued!
• On my third and fourth day, I was able to start working with my graduate mentor on the current project that they have been working on with testing the dosages of antibiotics on modified strains of bacteria. I was able to start learning sterilization techniques, and we started to prep our first trial run experiment.

 

• Week 2: This week I have spent more time in the lab learning how we will set up our experiment using three strains of our bacteria. We started to set up our first MIC growth assay by collecting and preparing our three strains of bacteria. The first cell strain will be our wildtype strain, in which the bacteria contain the 5691 gene and should grow normally under controlled conditions. This wildtype strain will serve as our control and be used for growth comparison. The other two strains consist of the 5691 “knockout” cells, meaning they have had the gene coding for 5691 removed from their genome. The purpose of these knockout cells is to observe differences in physiology in the absence of the 5691 gene. The final strain, called the complemented strain, will contain the 5691 gene added back into the genome to confirm whether differences in the knockout were solely due to that gene, and not other factors (such as random mutations or off-target effects of the knockout process). Our first growth assay turned into a trial run once we analyzed the results and determined there were errors made along the way. We then redid the experiment setup to determine what went wrong, and made adjustments to our protocol. It was a great experiment to learn not only how to set up MIC growth assays, but also to understand the importance of failure. It was informative to learn the steps that go into setting up a new type of experiment, and how to learn from the mistakes we make in the lab!
• I was also able to shadow other students within the Shell lab working on protein isolation. It was particularly interesting to shadow a student’s experiment that was referred to in our group’s lab meeting this week. I was able to observe the actual experiment, and start brainstorming ways to connect these experiences to my own curriculum. I was also able to tour some of the other RET teachers labs.

 

• Week 3: This week I was able to run two MIC tests using antibiotics Kanamycin and Chloramphenicol. Setting up the two plates and running the tests took the full week, and I was able to analyze results at the end of the week. The test using Kanamycin yielded the best results thus far, showing that the knockout strain grew differently from the wildtype and complemented strains. There were still visible areas of error, including a few wells within the plate that showed pipetting/technical issues. One of the replicate knockout strains seemed to grow less than the other, so we will remake this strain the following week, in case this is a source of error. Our Chloramphenicol test also showed unusual overgrowth patterns, indicating that there was a technical error made in creating the plate, or incorrect dosages of the antibiotic on the plate. We will test Chloramphenicol again next week and alter the concentration of antibiotic to see our new results.

 

• Week 4: This week I ran two more tests with antibiotics Chloramphenicol and Tetracycline. In our final weeks, our goal is to test as wide variety of antibiotics as we can that alter translational processes. I started with the second trial of Chloramphenicol, where we tried a different concentration of the drug. By the end of the week, there were less visible technical errors on the plate compared to the previous test. Setting up MIC plates requires much technical ability with pipetting, so it is often easy to make errors. The test for Tetracycline showed similar results. On both plates, we noticed that all three strains grew about the same. The knockout strains surprisingly showed no major differences in growth compared to the wildtype and complements, indicating these antibiotics did not have a major impact in the absence of protein 5691. We are curious to try two new antibiotics next week, or retest the kanamycin.

 

• Week 5: This week we tested two more types of antibiotics, Erythromycin and Streptomycin. Both MICs showed slight errors, but both showed much cell growth occurring. We also ran one final test for Kanamycin (as tested in previous weeks). Thus far, Kanamycin has been the only test showing different cell growth patterns in the knockout strain. This potentially indicates that the class of antibiotics, Aminoglycosides, might have an effect on inhibiting cell growth when protein 5691 has been removed from the genome. If more of these types of antibiotics are tested, this could indicate that this specific protein might play a key role during translation in helping the cells to survive different types of conditions during infection. I was also able to attend my gradate mentor’s lab meeting presentation, which was exciting because she is currently performing deeper studies into 5691. Our lab has been focused this summer in studying proteins such as 5691 that interact with a RNase complex during transcription and translation. This RNase complex is theorized to degrade RNA at different rates, which could play a role in how TB cells can survive and also adapt to harsh conditions. I was able to learn at this meeting that they have found some new exciting advancements on this protein complex, such as how 5691 has a sulfur-iron containing structure. This could be an evolutionary adaptation to sense sensitive changing conditions of oxygen, iron, and sulfur in the environment, and regulating homeostasis. I was also able to start finalizing my research poster and lesson plan, where I plan to have students construct models of mutations that can occur during cellular transcription and translation that lead to types of genetic disorders. The students will also be able to practice using some of the equipment used in the lab such as gel electrophoresis, to understand how scientists discover changes in genetic code. We will also use these hands on activities to investigate the process and advantages to gene editing tools such as CRISPR to help cure some of these genetic diseases.

Our Final Poster: