Right now I am in the middle of my NAD+/NADH assay. Each assay I need to conduct three times and take measurements during specific days during the growth curve. The hardest aspect of this experiment is keeping the bacteria alive. Mycobacterium bovis-BCG has always been a difficult bacterium to grow. Cell death can arise from tainted media, bad culture, all the way to poor incubation. Everything needs to be as precise as possible to have the best chance to get accurate data. Coming into the lab every day is tiring which is prone to mistakes. But with the support and guidance of my primary investigator, it’s not too difficult. One positive is that we finally have preliminary data that we can work with to obtain newer and better data.
Another difficult task I am faced with this semester is finding the balance between class, research, and studying for the MCAT. Scheduling and planning ahead have never been so difficult. If I focus too much on one responsibility the other two lack. One thing I am trying to do is to keep up with a strict schedule.
One extra responsibility I’ve been tasked by my primary investigator to teach the upcoming new researchers in my team. Performing a lab experiment is a challenge but stepping back and watching someone perform and trusting they can do the job right is ten times harder. This was because one year ago I was the new recruit proving myself to my peers and showing I was dedicated to the project. That mindset was what pushed me to come to Pace in the summer and work on my project. I had to learn to trust the ability of my peers and have patience. But I’m working on it.
Throughout the semester I have been testing the growth rate as well a viability of Mycobacterium bovis BCG in several different media. Each growth curve requires a daily optical density measurement for two weeks. For weeks I kept on making new media varying up the recipe and calculations and finally about three weeks ago we might have stumbled upon a media that is suitable for M. bovis BCG growth in the presence of glutathione. I have conducted three growth curves using this media and plan on conducting a viability test by plating the cultures onto agar plates every day for 14 days.
After the viability test, I will perform an NADH assay, which will determine the relative NADH concentrations of my cultures. Running through each failed trial was frustrating but it taught me a lot about being meticulous and patient. Reading more literature was key to modifying the media. My next step would be to use transcriptomics to figure out which genes are up-regulated when M. bovis BCG is under non-replicative persistence. For this process, I would need to extract the RNA and then create a complementary DNA. Then I input the genetic information into a machine that scans and processes the genes. This would then tell me which genes are up-regulated.
One other aspect of my research also was to teach and help my other lab-mates with their own project. This gave me a better grasp of techniques needed to perform RNA extractions, feeding assays as well as inoculations.
According to the US Centers for Disease Control (CDC) approximately one-third of the world’s population is infected with the bacterium, Mycobacterium tuberculosis, which causes tuberculosis (TB). Once a person has Tuberculosis the bacterium still remains in the body. This is because when the active mycobacteria enter our body, our immune response activates and tries to combat the foreign invaders with cells called macrophages, but the macrophages are not able to kill all of the bacteria. For the bacteria, the macrophage cannot kill the macrophages surrounds it and trap it in an airtight sac, forming a granuloma. The macrophages release harmful chemical agents, Glutathione, in the granuloma. In the granuloma, the M. tuberculosis is completely harmless, but it entered into the non-replicative persistence stage (NRP). In this stage, the bacteria basically dormant and drastically decreases its metabolism drastically. The bacteria can be dormant for years and years and if our immune system is weakened or compromised nutrients can enter the granuloma and the bacteria can “reawaken” and behaves as if it was never trapped. The title of my project is “The influence of Cholesterol on NAD+ production in Mycobacterium bovis-BCG”. Mycobacterium bovis-BCG shares 99% of its genome with M.tuberculosis, but it is safe to work in an undergraduate lab. We believe that the when the bacteria enters NRP it starts to create NAD+ in order to offset the reductive stress caused by glutathione. The purpose of my project is to better understand the mechanism, non-replicative persistence, that allows for Mycobacterium bovis-BCG to survive in a nutrient deprived environment. If we figure out how non-replicative persistence works we can end Tuberculosis.
I am working with Dr. Kelly with growth curves, testing what media would expose Mycobacterium bovis-BCG to glutathione without killing it. Then we would conduct an NAD+ Assay to test the concentration of NAD+ using Promega. After the assay, we would run a Polymerase Chain Reaction in order to find out what genes are up-regulated and down-regulated.