By: Reika Hallett & Zach Leaf
Introduction
We are part of the “Bacteria and Disease” research stream led by Dr. Zhu in Minnesota State University Mankato. Our primary purpose is to investigate the pathogenic mechanisms of Flavobacterium psychrophilum, which causes “Rainbow Trout Fry Syndrome and bacterial cold-water disease in wild and aquaculture-reared fish. While not much is known about this disease, it requires a type IX secretion system for its virulence or its ability to cause disease. Our group specifically is observing an enzyme called Peptidase which is responsible for breaking down peptides into amino acids.
Research
First, we had to amplify the peptidase encoding gene’s downstream region. We did this by running a PCR and a DNA agarose gel electrophoresis. After getting the results of the gel we ran we saw a bright white band under UV light. We then purified our sample to remove most enzymes, nucleotides, primers, and buffer components. We were ahead of the other groups at this point, so we decided to do a PCR gel electrophoresis on our gene’s upstream region; the results were a bright white band. Afterwards, we ran a gel electrophoresis with our purified downstream sample, and this is when things went a little downhill.
A pipetting error might have occurred during the purification process of our downstream PCR sample. Due to this error, we lost the DNA and had to create a whole new PCR and then purify it again. So, we were back where we used to be and ran another gel electrophoresis with our purified sample and this time it worked. While looking at the results we saw a bright white band which means there was a high amount of PCR product within our sample. We then performed a plasmid extraction using E. coli and purified the plasmid sample after. Then we ran a nano drop with both our PCR and plasmid samples to measure their concentrations. After seeing a good concentration for both from the nano drop, we decided to move on to the next steps.
Then, we did a restriction enzyme digestion for both the plasmid and the upstream sample. This digestion cuts two spots in the strand of DNA allowing us to combine them together. After the digestion we measured the DNA concentrations again using nano drop and performed another gel electrophoresis to see if the digestion was successful. After getting the results we saw that the DNA was digested as expected. Some DNA was lost but that is expected while handling it and pipetting to so many different times. We then performed a ligation procedure to combine these two samples into one.
Conclusion
While we have experienced issues during the purification procedure with our upstream sample and had to redo the PCR for it, we have made up for it with the use of extra time and through being more precautious with our handling and pipetting of our samples. Nevertheless, we have successfully performed all our procedures for our peptidase gene and will continue with further research regarding it.
