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.