Great presentation! Did you encounter any limitations while conducting your research? Did you have to make any adjustments to your methods because of these?
Yes, during our initial round of DNA isolation we discovered that our DNA was of high quality and had a lot of smearing so we had to redo DNA isolation. In order to prevent the degradation of our DNA from the nuclease (which was one possible reason our quality control proved our DNA wasn’t sufficient) we added 15microliters of EDTA to extra inactivate the nuclease instead of the listed 10 microliters from the lab manual.
Hi Sophia. Yes, during DNA isolation after running a quality control we determined our DNA to be insufficient since the was a lot of smearing on the bands showing it was of low quality. We hypothesized that one reason might be degradation from the nuclease. To counteract this we adjusted our methods by adding 15 microliters of EDTA to sufficiently deactivate the nuclease instead of the instructed 10 microliters.
You mentioned that your phage would need to be genetically engineered before it could be used in phage therapy; How much effort is that? Does it add a significant amount of time or effort to the process, or how much simpler would it be to use a phage that is already lytic? (also, what was the inspiration for the name RozMI?)
Hi Logan, using a lytic phage would eliminate a portion of the work that it would take to lyse a bacteria cell since we wouldn’t need to genetically edit any integrase or repressor proteins, but it’s still important to know how to modify temperate phage in case there isn’t a lytic phage that characterizes the same host range as the temperate one needed to infect a pathogenic bacteria cell.
The A, B and K cluster infect similar related bacteria such as M. smeg, siphoviridae morphology and temperate plaque morphology. The main differences would have to do within genetic sequencing.
Hi Nate, we determined the cluster from our PCR results by analyzing the band which shows amplified DNA in depicted in the A4 enzyme lane. Since A4 is a subcluster of the A cluster we determined RozMI found it’s home in the A cluster.
Great presentation! Did you encounter any limitations while conducting your research? Did you have to make any adjustments to your methods because of these?
Yes, during our initial round of DNA isolation we discovered that our DNA was of high quality and had a lot of smearing so we had to redo DNA isolation. In order to prevent the degradation of our DNA from the nuclease (which was one possible reason our quality control proved our DNA wasn’t sufficient) we added 15microliters of EDTA to extra inactivate the nuclease instead of the listed 10 microliters from the lab manual.
Hi Sophia. Yes, during DNA isolation after running a quality control we determined our DNA to be insufficient since the was a lot of smearing on the bands showing it was of low quality. We hypothesized that one reason might be degradation from the nuclease. To counteract this we adjusted our methods by adding 15 microliters of EDTA to sufficiently deactivate the nuclease instead of the instructed 10 microliters.
You mentioned that your phage would need to be genetically engineered before it could be used in phage therapy; How much effort is that? Does it add a significant amount of time or effort to the process, or how much simpler would it be to use a phage that is already lytic? (also, what was the inspiration for the name RozMI?)
Hi Logan, using a lytic phage would eliminate a portion of the work that it would take to lyse a bacteria cell since we wouldn’t need to genetically edit any integrase or repressor proteins, but it’s still important to know how to modify temperate phage in case there isn’t a lytic phage that characterizes the same host range as the temperate one needed to infect a pathogenic bacteria cell.
This presentation was really good and interesting! How do the A, B, & K clusters differ?
The A, B and K cluster infect similar related bacteria such as M. smeg, siphoviridae morphology and temperate plaque morphology. The main differences would have to do within genetic sequencing.
How did you determine the cluster from the PCR digest result?
Hi Nate, we determined the cluster from our PCR results by analyzing the band which shows amplified DNA in depicted in the A4 enzyme lane. Since A4 is a subcluster of the A cluster we determined RozMI found it’s home in the A cluster.