In the Fall of 2016, I took the course Microbiology. I wasn’t particularly interested in the subject at first, the class was fast paced, and the laboratory component was twice a week. After the first couple weeks though, I loved the lab. Every day we did a new technique to look for presence of microorganisms. It amazed me that millions of these little creatures surround everything we touch, see, and are inside our bodies for better or worse. I loved learning about the subject so much, I decided to reach out to my teacher and ask for a spot on a research team. I was paired with Dr. Secott for research who had several projects he was experimenting with at the time. He gave brief explanations of the topics and asked which one I was most interested in. I chose to work with Mycobacterium avium subspecies paratuberculosis or MAP. I wanted to work on this project as it causes a disease called Johne’s in cattle and I come from a dairy farm in Central Minnesota.
To begin the semester, Dr. Secott and I met twice a week in his office and he would give me articles on MAP. We discussed research he had already done on the mycobacterium and how he wanted to move forward. One of the articles I was given was about quorum sensing, a way for bacteria to control gene expression to produce beneficial cell functions based on cell density. It was Dr. Secott’s thought that MAP cells could potentially be driven out of dormancy if we stimulated them with a quorum sensing molecule and cause them to start replicating.
To test this thought, I researched acyl homoserine lactones or AHL, a classical quorum sensing molecule, and how they worked in other organisms. I used the genome sequence for MAP and compared it against different gene homologues for AHL receptors called Lux sequences from other organisms using a program/database called BLAST, Basic Local Alignment Search Tool. The purpose of this was to see if I could find regions of similarity between biological sequences in MAP and AHL gene homologues. Upon finding a couple good sequence matches for several different Lux sequences, Dr. Secott had an acyl homoserine lactone made by another faculty member that was specific for LuxS. This sequence was picked because there was a very similar sequence of LuxS in the MAP genome, meaning it might use that type of AHL for quorum sensing. I do not know how to protein was made, but I can take an educated guess that it was either purchased from a database of proteins online or made from bacteria cells with transplanted plasmids in their DNA for AHL. I hypothesized that the MAP cell line being tested would remain dormant despite being grown in culture with a autoinducer. I chose this based upon the difficulty to get MAP to replicate in culture in other experiments and I don’t think one protein would make a difference, however, it was a possibility that Dr. Secott wanted to rule out. A study like this as far as we know hadn’t been conducted and published so it would extend what we knew about the organism even if the results were negative.
I learned various techniques to complete this experiment, many I hadn’t learned before, but now have done in multiple other classes. Some techniques I learned were how to make dilute solutions, sterilization processes, pipetting, and how to record data through the plate reader and computer. After sterilizing everything I was using, I mixed MAP cells and LuxS protein in medium using dilution techniques to disperse an equal number of cells into a 96 well plate. I then read the plate several times a week over the course of the semester using a machine that measured absorbance inside the cell plates. I made one plate to read for a couple weeks and then repeated the procedure for another plate. I then acid-stained the cell culture for the presence of contamination after I was done with the second plate. The results did not show an increased presence of cells at the end of the semester, but I would like to repeat the experiment with fresh solutions to make sure of the results.
Although my hypothesis appeared correct, it did not mean I proved anything. Science is the process of studying what is known and unknown to add to existing theories, not to prove what is true in the world. I added some new information to the scientific community about how MAP contains some AHL synthase gene homologues, something that hadn’t been studied before and it doesn’t appear to affect the MAP dormant cells tested. The biggest problem with the mycobacterium is that is extremely hard to grow in culture, making it hard to test what antibiotics/vaccines might be able to rid animals of the Johne’s disease. While it’s important to help cure cattle, as infected animals who show outward signs of the disease eventually die, it also essential to human health as it has been linked to Crohn’s disease. If by some reason these cells were able to be cultured more easily, more extensive research could be done to eradicate these diseases.