About Me: Hi everyone! This summer I am excited to be a returning RET participant working with the Shell lab again on cellular studies related to Tuberculosis infection. I am currently entering my fifth year of teaching at Groton-Dunstable Regional High School, focusing mainly on teaching Biology and Environmental Science at various levels. I am excited to once again join the RET team to conduct authentic research this summer to better my lab skills, network with other educators and researchers, and bring more interesting examples and concepts back to my district. This program has made a huge influence on the way I approach my lesson planning in STEM, to focus more on problem-solving, inquiry based lessons that are inspired by the Engineering Design Process. Since my previous year, I have enjoyed making many adjustments to our current labs and other activities to promote more skill building and collaboration between my students.

About the Lab: The Shell lab is geared 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. 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.

To understand how to combat and eliminate bacterial cells that cause TB, we need to understand what makes these cells so tolerant of antibiotics. When a person has been infected with tuberculosis, one type of immune response in the body is to trap foreign Mtb cells within granuloma complexes to kill the cells- exposing them to unideal conditions such as low pH, hypoxia (where there is very little oxygen available) or low nutrients available. However with Mtb, these cells are able to remain in a slow, nongrowing state that does not need to expend as much energy. This allows them to stay alive within the human body until conditions become more favorable, where they can start the infection again. A central goal of the Shell Lab is understanding how these Mtb cells are able to adapt to these stressful conditions by altering their gene expression as a survival mechanism. How these gene regulations and survival mechanisms work in Mtb is still being researched, but may allow future researchers to design better and faster treatment regimes for patients.

Within each main focus, lab members are conducting experiments to isolate and analyze the proteins coded within a non-pathogenic model species, Mycobacterium smegmatis, that may allow the pathogenic mycobacterium species to withstand many modern treatments for tuberculosis (TB). Our protein of focus this summer called Sre, is believed to play a role in stimulating an important cellular complex called RNase E, which may play a role in detecting stress responses and allowing Mtb cells to be more tolerant of drugs (meaning they are not efficiently killed by antibiotics). Sre is a protein that is conserved throughout mycobacteria and close relatives, but its function is not well understood. This summer, a fellow in-service teacher, Em Beeler, and I will focus our project on trying to understand Sre’s functions during cell growth when exposed to various antibiotics.

Project Title: Investigating the Impact of a Novel Protein (Sre) on Antibiotic Tolerance in Mycobacterium smegmatis

Weekly Updates:

Week 1:

• This week was our first week starting the RET program, and being able to return back to the WPI campus. The day started with introductions to the program, overviews, and touring of the labs. By the second day, we were granted access into the Shell lab, and started to draft our experiments that Em and I will be working on this summer, called timed-kill assays. These assays will allow us to study how our nonpathogenic, but related species of bacteria called Mycobacteria smegmatis grow with or without our protein of interest (Sre) when exposed to different antibiotic treatments. This lab is similar to my experiment last year, where I performed MICs, or Minimum Inhibitory Concentration tests to initally understand Sre’s role.
• Many modifications were made to this protein of interest over the year, so I am excited to jump back into a follow up experiment where more has been understood about this protein. I am also excited to renew some of my lab skills gained from last year ans rejoin my lab members. This week we have started to write our experiment and culture our cells of interest, however since our experiment is timed, we will start real trials next week. This week we have focused on deciding which strains of bacteria we want to use, culturing them, and getting them prepped for next week. We have also worked on deciding which types of antibiotics would be the best to test for our assays.

Week 2:

• For our second week at RET, Em, myself, and our graduate mentor spent the first day determining the setup for our first time-kill assay that we would be running for our first antibiotic, Erythromycin. This particular run of the experiment was a bit tricky, as few people in our lab have run them for the strains of bacteria that we would be working with, so a challenge was determining which time stamps we would be using to plate our cells and check for growth. We decided for our first practice run this week, we would test out 3 time points (0, 4 hrs, 24 hrs), and use the results of this assay as a baseline for our future assays, which would have more accuracy.
• On the previous Friday, we had began growing our cultures of our 3 wildtype (or normal cells that contain the Sre gene of interest) and our 3 “Knockout” strains (or cells that had been genetically modified to remove the Sre gene from the genome, to see if this would have a greater impact on the growth and survival of our mycobacterium cells). We obtained our original starter cells from the freezer, and heated and stimulated them over the weekend for use this week.
• On Monday, we “normalized” our cells, meaning growing the cells to have the same approximate density between the different test tubes. This can be a tricky step, as cells can be unpredictable! Once they were normalized to the approximate density, we placed them back into the incubator for the next day for use. In the meantime, we learned how to create the petri dishes which we would be using to grow and count the bacteria for our experiment.
• The next few days we began our first time-kill assay once our cells had been normalized. The idea behind our assays is at different time points, we take “snapshots” of the cultures by diluting each strain down and plating it. We would plate cells with the wildtype strains 1, 2, and 3 with drug and no drug, and then plate our Sre Knockout strains #706, #709, and #712 with drug vs no drug. By Thursday we were able to check our first plates for growth by counting “CFUs” (colony forming units) on the plates, or tiny dots on the petri dish that each contain millions of invidual bacteria cells. We ended up having several plates, where certain time points worked better than others. For example, our first initial plates for our wildtype strains without the drug ended up overgrowing (colonies are so large and clumped together that it is difficult to count how many dots are within that cluster), so by the time we checked for analysis, and were not usable due to low accuracey. Other plates such as our second timepoint, which actually need more time over the weekend to grow in order for us to count the cultures. However these errors in our test will provide valuable information for planning our assays moving forward! Not only do they allow us to lock in better time stamps for analysis, but this test also allows us to see how much we should be diluting our cultures. We will retest next week and see how our results change!

Week 3:

• This week we continued to create another time-kill assay for Erythromycin. We experienced difficulties this week as two of our cell cultures were not growing in a way we predicted! Normally, in order to begin the experiment and plate the bacteria, they have to be at a specific starting density range, which our cells did not want to do this week! This required us having to re-normalize (meaning, calculation dilutions to get our cells to reach a certain density) almost 3 separate times to get it to a usable range. This put a delay on starting the experiment until Thursday, but we will try our assay again this week to see if our dilutions were correct. Next week, we will start our cultures from scratch and try again.
• On the RET side of things, this week we begun PD sessions where we discussed what the engineering design process would look like in the classroom, with some creative examples! The focus will be on skill building important critical thinking and collaborative skills with the students using the EDP as a model for inquiry activities. We will continue to discuss our goals for the lessons as well as types of assessment methods in the coming weeks.

Week 4:

• This week Em and I continued to figure out the experiment parameters for setting up our time-kill assays. Last week we had experienced some difficulties as our cell’s densities and growths were not aligning as we expected. This delayed the start of the experiment to Thursday, where we made the decision to continue to gather data using these strains regardless. This continued into this week, where we continued to plate

Week 5: 

Week 6:

Final Poster:

Lesson Plan: