David Schlesinger and Dr. Alan R. Harker, Microbiology
Being that the scope of this project was much larger than anticipated I was unable to complete my research in its entirety. However I did make substantial progress and was able to answer many of my original questions.
Originally the intent of my research was to recreate a previous experiment of Dr. Alan Harker’s (1) and then to take it one step further. I was successful in completing the first part of my research, with a few differences. I was able to induce the mutation in the plasmid pJP4 by forcing it to grow on phenoxyacetic acid (PAA) as a sole carbon source. I did this by growing the bacterium on Noble Agar plates that contained .05% PAA. After several days of incubation, bacterial growth appeared on the plates. I isolated several colonies and grew each of them in bulk liquid culture in order to successfully isolate the plasmid. Isolating the plasmid proved more difficult than originally thought. It took many different protocols and modifications of these procedures in order to successfully isolate the plasmid. This is the point at which my experiment strayed from the original.
The plasmid was then cut by several different restriction enzymes and examined by gel electrophoresis. The results were extremely different from the original. The difference was so substantial that originally I had thought there had been contamination and that the plasmid I had isolated was not a mutated pJP4, but something entirely different. Where as the original experiment produced a single deletion mutation my experiment produced what appeared to be many deletions and possible duplications. If this plasmid is a derivative of pJP4 then some interesting questions are raised. What exactly is going on? What do such drastic changes to the plasmid mean? How and why does the bacterium do this? My results so far have opened a dozen new questions for every answer I have yet to receive.
To make sure that my results were correct the first part of the experiment was repeated two more times. Each time the results were the same. So, in order to be absolutely sure that the plasmid I had isolated was in fact a mutated pJP4 I decided to perform a Southern hybridization using pieces of pJP4 as a probe. This is where my research ended. I was in fact able to bind my plasmid fragments to a nylon membrane but was not able to probe the membrane. This technique required that I take a radio safety test to be certified to perform the experiment and I was not able to do so. Although I am confident that the plasmid isolated is a mutated form of pJP4 I need to be absolutely sure before I can proceed with my research.
References
- Harker, A. R., R. H. Olsen, and R. J. Seidler. 1989. Phenoxyacetic Acid Degradation by the 2,4-Dichlorophenoxyacetic Acid (TFD) Pathway of Plasmid pJP4: Mapping and Characterization of the TFD Regulatory Gene, tfdR. J. Bacteriol. 171:314-320