Mar 18, 2021
Abby Mueller and Marley Fuhrman
Introduction:
We are working in the Bacteria and Disease Stream lead by Dr. Zhu at Minnesota State University Mankato. We are focusing on the bacteria (Flavobacterium psychrophilum) in charge of causing Bacterial Coldwater Disease and Rainbow Trout Fry Syndrome. We are working with a motility adhesin encoding gene. Our goal is to mutate the adhesin gene to help prevent the spread of the disease.
Specific Content:
Our first task was to amplify the upstream region of our gene by PCR. There were some problems with getting the PCR product, however. We thought it could be due to pipetting errors, but after some trouble shooting, we found that changing the annealing temperature yielded a faint band. When we ran a gel for the PCR product of the downstream region, and a bright band showed up. Since the band was so bright, we decided to switch our focus to the downstream region and come back to the upstream region later. When we focus back on the upstream region, we will most likely have to modify the PCR conditions to improve the concentration of DNA.
After we had a successful gel image of the downstream region of the adhesin gene, we purified that DNA to remove any contaminants we didn’t want. We also performed a plasmid extraction with the E. coli and also purified it to get just the plasmid DNA. We then used the nanodrop machine to determine the concentration of the DNA for both products. Since we saw good concentrations, we decided to move forward.
We then did a restriction enzyme digestion for both the plasmid and the downstream region of the adhesin gene. This process cuts at two spots on the DNA strand and produces sticky ends. After this we used the nanodrop machine again and we also ran a gel to make sure we didn’t lose the DNA and we had an okay concentration. Unfortunately, the concentrations for both the plasmid and the downstream PCR product were very low and the gel we ran was inconclusive. Probably somewhere a mistake was made and most of the DNA was lost. However, we decided to perform the ligation procedure anyway. In theory this process will fuse the plasmid with the adhesin gene. However, since the concentrations of our products are so low, positive results are less likely.
Conclusion/Reflection:
So far in our research, we have made mistakes and had setbacks, but we have learned from them, and found a bright side to it all. For example, we had to redo the PCR and agarose electrophoresis a lot due to a band not showing up, but the practice improved our lab skills, and we eventually got a bright band on the agarose gel once we experimented and switched up our process. We are excited to continue researching and perhaps achieve our goal of mutating the adhesin gene.
