Final Blog Post

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.

Blog Post 2

 

Since the last blog post, my research has run into many roadblocks unfortunately. These challenges were to be expected though as scientific research comes with much trial and error. The one success that has come out of my research so far this summer was extracting the RNA from the Cryspovirus itself. This was done by first breaking the oocysts, or shells, of Cryptosporidium parvum, which is where the Cryspovirus is housed. However, when attempting to extract the GFP, green fluorescent protein, from the gel it did not work. This raised many questions as to if there was any GFP present at all. To be sure that I did in fact extract GFP present we used vectors and restriction enzymes to be sure that there was GFP present and that the correct size I was looking for. These results came out negative causing me to think of other problems as to why I could not successfully extract GFP.

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 with cas9 also to see if it was present and the results came out positive. So, cas9 was present but GFP was not. Without the GFP being present there would be no use for the cas9 at this point so I just froze down the rest of the cas9 to keep as a stock solution for when the GFP eventually gets extracted from the gel successfully.

I think the reason why there is no band showing up on the gel for RNA is because the hb, ns, and cpv primers are not cutting the RNA correctly meaning the PCR is not copying the correct sequence of RNA. The next step in my research is to design and order new primers that will be used with the RNA extracted from the Cryspovirus. These primers have been designed specifically to cut the RNA at the exact positions, but of course there is no guarantee they will work until I use them to do a PCR. If the new primers do not work then new ones will have to be made until I get primers that bind to the right positions on the RNA to cut the correct sequence.

Throughout my time conducting this summer research I have come to learn many things. 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 critical thinking and trial and error that goes into research.

Finding Ways to Manipulate Genetic Information in the Cryspovirus

The purpose of my research project being carried out during Summer 2017 is to find ways to manipulate genetic information in the Cryspovirus. The Cryspovirus is found in Cryptosporidium parvum, which is a parasite. This parasite is known to infect many mammals, birds, and reptiles, and also causes diarrheal disease in humans.

There are many steps that must be taken to be able to manipulate the genetic information of the Cryspovirus. First, RNA must be extracted from the Cryspovirus and then be used to do a polymerase chain reaction, or PCR. Before being able to extract RNA the oocysts, or shells, of Cryptosporidium parvum must be broken so that the Cryspovirus will be accessible and then the RNA can be extracted. With this RNA many PCR’s will be done with different reaction conditions. After the PCR is complete, a gel will be run using primers and the RNA extracted from the Cryspovirus. Polymerase chain reactions of the RNA will continue to be done until a specific band is shown on the gel, which indicates that the PCR worked and that the fragment of RNA extracted from the Cryspovirus was successfully copied.

To be able to see the Cryspovirus in the Cryptosporidium parvum, green fluorescent protein (GFP) gene will be extracted from the plasmid and then cloned and inserted into Cryptosporidium parvum to make the Cryspovirus glow green. To extract the GFP gene, E. coli will be swabbed onto agar plates containing ampicillin and left to grow over night. Once the E. coli has grown, one colony will be picked off of the plate and put into a tube containing agar broth and then left to incubate with shaking overnight until it becomes cloudy indicating growth of the bacteria. After growth has occurred in the tubes, the GFP gene will be extracted using the QIAprep Spin Miniprep Kit and then the extracted DNA will be cleaned using the QIAquick Gel Extraction Kit. The clean DNA will then be used to run a gel with to be sure DNA was extracted. If everything goes as planned then the GFP gene will be cut out of the gel and inserted into Cryptosporidium parvum where it will glow and be easier to see the virus and how it reacts to manipulating its genetic information.