During this summer I have worked hard to obtain data that will be used to quantify the mutation rate of the hyper mutable region of interest in the genome of H. pylori. I have done procedures that have put me closer to inserting the hyper mutable region into the bacterium E. coli so that the mutation rates can be compared between these two bacterial species. The data gathered from the AFLP protocols done this summer have consistently shown the same number of cytosines within the hyper mutable region of interest in both the bacterial colonies and bacterial populations analyzed. It will be interesting to see if this result holds up when the bacterial environment is experimentally stressed. This will be the topic of experiments to come in the near future.
At the same time that I was doing the procedures talked about in the previous blog post, I was also continuing the procedures that lead up to the AFLP protocol. After some initial troubles with the sequencing machine that carries out the protocol, I obtained results that were consistent with results from previous AFLP protocols done. So far, each AFLP has revealed the same number of dominant poly-C lengths within each bacterial colony and population. In the future, we will try to stress the environment of the bacteria to see if the number of dominant poly-C lengths will increase or decrease due to environmental stress.
During the previous week, I picked transformed E. coli colonies that were mentioned in the previous blog post. I put these colonies into media and incubated them at a temperature that would allow them to grow at optimal reproductive capacity. After approximately 24 hours of growth, I created a pellet of the cells in a centrifuge and performed a plasmid preparation procedure on the pellet. This procedure resulted in purified plasmids, on which I then performed a restriction digestion. After this I performed gel electrophoresis on the restriction digestion in order to see if the reaction worked and found out that it did. In the following day, I extracted this restriction product from the gel, and executed a procedure on it in order to purify the DNA in the product. In the future (most likely the beginning of fall semester) I will do procedures to incorporate this purified DNA product into a viral genome that will be used to infect E. coli cells in a process called transduction. During transduction, a viral vector will incorporate it’s genome, that includes the hyper mutable region of interest, into E. coli cells. Once this is done, I will begin performing the same mutation rate procedures done on H. pylori on these E. coli cells.
This previous week of lab work has been exciting because I have moved on to a new portion of my project. I ran PCR on a bacterial population in order to make several copies of my gene of interest (arsS). The PCR product was then used to perform a cloning reaction, which essentially placed the amplified arsS genes into plasmids, which are independent circular strands of DNA. These plasmids were then introduced to E. coli cells in a transformation procedure that caused the E. coli cells to incorporate the plasmids into their cytoplasm. As of now, I am growing these E. coli cells on Agar plates and will pluck the colonies once they have grown large enough. These procedures are exciting because I am one step closer to being able to perform tests for the mutation rate of the poly-C region in E. coli so that I can compare it to the mutation rate of H. pylori.
For the last few weeks I have been growing isolated colonies and using these colonies to run Polymerase Chain reactions (PCR), which were then used to run Amplified Fragment Length polymorphisms (AFLP). These procedures were also done on a bacterial population swabbed from the identical bacterial plate used to create the isolated colonies. The AFLP results from the isolated colonies and the bacterial population were consistent in that each showed the same number of dominant poly-C lengths of our gene of interest, arsS. Necessary procedures have also been done on the isolated colonies and bacterial population in order to calculate a titer for each sample that PCR and AFLP have been done on. The titer will ultimately be used to calculate a mutation rate for H. pylori. The next few weeks will involve continuing trials of the procedures just described and also preparing reagents that will be needed to clone the poly-C region of H. pylori’s genome and insert it into the bacterium E. coli.