Jared Balaich and Dr. Richard Robison, MMBIO
Spore forming bacteria are of great medical importance because of their ability to infect, and sustain infection in the human host. A spore consists of the bacterium’s DNA surrounded by thick membranes and is resistant to desiccation, disinfectants, radiation. The spore can then form a living bacterium after remaining dormant for thousands or even millions of years. Although much is now known about the structure and pathways involved in spore formation, much mystery remains around exactly what triggers spore formation to occur. The focus of this research project is to culture spore forming bacteria on medias of differing composition and then to identify which ingredients, or the lack thereof, cause an increase or decrease in the levels of sporulation.
The first and what proved to be the most difficult step of the project was finding a way to quickly quantify the percentage of bacterial cells that are sporulating. Methods employed in most labs involve taking two samples, killing all the living cells in one culture, leaving only the spores. The two samples are then grown, counted by hand and then the difference is calculated. This methodology is very labor intensive and time consuming and requires a few days to get any results.
Using flow cytometry would allow for more accurate data to be collected in a fraction of the time. The main issue that we came across was being able to differentiate between spores and living cells with no spores. We tried sorting the sample based on size, but they seemed to be too closely related in size. Next we attempted to use live dead stains and size separation together but the results were still lacking. After this we used a strain of Bacillus anthracis that had been previously engineered to incorporate green fluorescent protein. The problem was that all the cells were stained not only the spores.
The project then focused on creating a strain of Bacillus subtilis, a harmless well known bacterium, that would incorporate this green fluorescent protein only into the spores. All cells with spores would fluoresce allowing easy differentiation and counting of sporulating cells versus those cells without spores. There was some literature on how the Cot family of proteins is a large spore specific protein on the outer coat of the spore and when tagged with GFP only the spore appeared to glow. Specefic proteins in this family were now our target and work began on engineering this strain. Once engineered it would allow for fast, easy quantification of spores. Data could then be gathered quickly and efficiently in order to answer the original question of what substances affect the levels of sporulation.
Sadly I was not able to finish the project, but have given all my data and work over to other members of the lab so that they can finish the work that has been started. I was accepted into Johns Hopkins University and after some complications with my wife’s job due to the fact that we were leaving for Baltimore, we were left with no source of income and so were forced to move in with family out of state right after graduation. Even with this occurrence, I am confident in those who are still working on the project that they will collect some interesting and relevant data and be able to publish data collected in this project. I have heard from them recently assuring me that the project is moving forward. I wish to thank all those donors who support the ORCA grants and all those who helped in the project.