The effect of Amyloid Binding Dyes on the Cryspovirus in Cryptosporidium Parvum

Since my last blog post, I have encountered some challenges and successes. My main success was extracting the RNA from the cryspovirus found in Cryptosporidium Parvum. We achieved this by breaking the oocytes and removing the RNA from the sporotizes. My other success was growing GFP and CAS proteins in E.coli. With these cultures, I was then able to extract the DNA from the CAS cultures. I first grew the samples on agar plates containing ampicillin and then I grew them on agar plates that contained kanamycin.  Ampicillin and kanamycin are both used on the agar plates because the degradation can lead to the formation of the colonies. I had grown enough cells to be able to freeze them so they can be used again in the future. For the CAS protein, the DNA was extracted and will be used to help insert the GFP protein into the right part of the sequence in the RNA.

My main challenge is finding the right band in our 1% agarose gel for our RNA extraction from the Cryspovirus in Cryptosporidium Parvum. I believe this is because of the primers I am using. This is because I have three different primers with all different PCR settings to see if the RNA shows up, so far I have not had any luck with the NS, HB, or CPV primers. Instead of the RNA being shown on the gel just bands of the primers are being shown.  The primers are used to cut a specific sequence of RNA that will be shown on the gel and compared to an RNA ladder. So as seen on the picture below you can see how my gels have been showing.

(The two ladders are shown correctly and all the other bands are just the primers. The purple bands are just the loading dye put in our samples when they were injected. I am looking for very distinct bands containing the RNA.)

I have tested someone else RNA sample from the Cryspovirus in Cryptosporidium Parvum with all three primers and PCR conditions and their gel showed the same results. Right now I have new primer sets in the route of shipping to test other options and see if the problem truly relies on my primers or if something else is affecting our gel results. The other primers will be NS and HB, but they will be cutting the RNA in a different spot to see if that will effect the band. Another issue is with my GFP DNA. I have not been able to successfully extract the DNA from the GFP cultures. I ran a gel and saw a smear in my band so I tried extracting DNA from it, but this also didn’t work. I was only able to extract the DNA from the CAS cultures. The DNA from the gel is needed to be inserted into the RNA of the Cryspovirus in Cryptosporidium Parvum to fluorescent it. My main question now was is if the GFP was even present in the gel. To test to see if the GFP was present and the size we were looking for in the gel we cut our DNA with restriction enzymes (vectors). This showed me that there indeed was no DNA present in the GFP sample, but there was in the CAS.  Overall, the two main things that need to be figured out is if the RNA is present and if it is what are the right primers needed to cut it at the right spot. Also to figure out how to get the DNA from the GFP cultures and eventually extracting it from the gel. Once both of those steps are figured out I will be able to inject the fluorescent GFP protein into the RNA sample with the aid from CAS.

This project has shown me and taught me a lot of different things. The first thing is that not every problem is easily detected. For example, I have been struggling with this primer issue from the beginning and still dealing with how to figure it out. Another thing is that details matter. At first, I didn’t realize the small details and explanations are as important as the main details. I didn’t take as detailed or elaborate notes and I feel like this made me a little lost in the beginning, but now I feel like I have a firm grip on what I should keep an eye out for and to take notes of. I am so grateful for everything I learned especially on how it is going to impact my future. I plan on attending graduate school to earn a Ph.D. To do so, research will become a very valuable and important part of the next couple of years. The project I am working on I feel like is a great way to start such a career. I feel like my professor is going to prepare me and help me a lot to get me ready for future research projects. I want to study Forensic Anthropology in graduate school and eventually get a job through different government opportunities. With this job comes to a lot of opportunities to help people and my project I am working on right now is doing the same. This project will show me how much work it takes to put into helping people’s lives.

Blog Post #1: The effects of Amyloid Binding dyes on the Cryspovirus in Cryptosporidium Parvum

The purpose of my project is to detect the cryspovirus in Cryptosporidium Parvum. This will be done by cloning GFP, a green fluorescent protein into the virus dsRNA. The GFP gene is inserted into the cryspovirus will make it easier to enumerate the number of virus in Cryptosporidium Parvum.
First I clone out the GFP gene from the pCMV-GFP vector using PCR. I will then extract the PCR product out by the gel extraction technique. From that gel, we will be removing the GFP band containing the restriction sites needed and inserting it into the cryspovirus. To obtain GFP we first need to extract the DNA as well. We will be extracting the DNA from E. coli. The first thing that needs to be done is E.coli with GFP needs to be grown on an agar plate. So the Agar broth and agar were made and Ampicillin was added to both solutions. The agar was poured into an agar plate and left for solidification. Once solidified GFP was streaked onto the plate and left to incubate. The GFP grew very well. Test tubes were made with the agar broth made and tips containing cultures from the E.coli plate. The test tubes have to be incubated while being shaken.  When they grow the liquid will be cloudy. After they grow, QIAprep spin miniprep kit is used to extract the DNA. Once the DNA is extracted it needs to be cleaned by using the DNA clean and concentrator kit. Once our DNA is ready we will PCR it and use it on our gel. We are looking for a specific band and if that band shows up we will remove it from the gel and remove the DNA from the gel. A gel extraction kit is used to remove the DNA. Once the DNA is removed it will be inserted into the cryspovirus dsRNA. I have to extract dsRNA by excystation of Cryptosporidium Parvum oocysts. This breaks the oocysts (shells) that hold the sporozoites. The RNA in the sporozoites is then extracted. The RNA is extracted so we can use PCR to clone in restriction sites. We are currently using different PCR conditions to clone out the different regions of the viral dsRNA.