Over the summer of 2017 the research I have been conducting involved the Cryspovirus, which is found within the parasite Cryptosporidium parvum. The overall outcome of my project was to insert a green fluorescent protein, GFP, into the virus to make it glow and be easier to see the virus and how much of it there is.
The first thing I had to do before beginning anything else was to extract the RNA from the Cryspovirus. Breaking the oocysts, or shells, of the Cryptosporidium parvum, which is where the Cryspovirus is contained, did this. This was a success for me as I was able to extract the RNA using this method. With this RNA I was able to do a polymerase chain reaction with it, PCR, using different reaction conditions. The PCR was done to copy a specific portion of the virus RNA. After the PCR was complete I then made an agarose gel and injected the PCR samples into the gel and let it run for a half hour. This was done with intentions to see a specific band on the gel that would mean that the PCR had worked and the RNA was successfully copied. Unfortunately this came out with negative results.
The reason why I think there was no band showing up for the RNA is because the hb, ns, and cpv primers I used were not cutting the RNA correctly and therefore the PCR was copying the wrong sections of the RNA. I think the primers are the problem because I have used them to do PCR with many different times using different reaction conditions and all of them came out with negative results when running a gel. So, to fix this problem I designed new primers that would hopefully bind to the correct sequence of the RNA and therefore the correct sequence will be copied when running the PCR. Just last week these primers arrived. They consisted of two hb primers and two ns primers whereas before I was only using one ns primer and one hb primer. These primers were used to do a PCR using a new set of reaction conditions and thankfully the results came out positive for the ns primers. As for the hb primers the results still came out negative. This means that I am going to have to repeat PCR using the hb primers with different reaction conditions to see if I can get positive results. If that does not work after doing multiple different PCR’s then I will most likely need to design and order new primers.
To be able to extract GFP from the plasmid I had to swab E. coli onto an agar plate containing ampicillin. These plates were left to grow overnight and once they had grown, one colony was picked off of the plate and put into a tube containing agar broth and left to grow more overnight by being incubated with shaking. When the tube turned cloudy is when I knew it had grown enough to extract. The GFP DNA was extracted using the QIAprep Spin Miniprep Kit and then the extracted DNA was cleaned using the QIAquick Gel Extraction Kit. After the DNA was cleaned, I ran a gel with it to make sure that there was DNA present. Unfortunately the gel came out negative indicating that there was no DNA present. So to see if there was any GFP present at all I used vecotrs and restriction enzymes to see if the DNA was the correct size. These results came out negative as well meaning that there was no GFP present in my sample at all. I am still not sure why there is no GFP present in the sample.
Along with the GFP I also ran a gel using cas9, CRISPR-associated protein-9 nuclease, which is an enzyme that will help insert the GFP into the virus RNA so that it can fluoresce. I used vectors and restriction enzymes to make sure that there was cas9 present and these results came out positive. Since the cas9 is used to help the GFP be inserted into the virus we had to be able to extract the GFP successfully before being able to use the cas9 so I just froze down the sample until it is ready to be used.
Moving forward, I will hopefully be able to get the PCR to work for the hb primers. From there, then I will try to extract the RNA from the gel again and hopefully it will work since I have the new primers that work. If that works then the GFP will be inserted into the Cryspovirus with the use of the cas9 to help it be inserted properly. If this step works then the virus will fluoresce green and my research project will be complete.
Overall, I am very thankful to have had this opportunity as I learned a lot. First of all, I have learned to be very meticulous while working in the lab because it is very easy to make small mistakes that can cause your research to be affected. I have also learned that there are not always straight forward answers as to why something went wrong and that it takes a lot of time and thinking to solve an issue. However, even if you think you have found the answer as to why something went wrong you may not always be correct. There is a lot of patience, critical thinking, and trial and error that goes into research.