Blog Post #2: Update

Following up from the prior blog post, this research project requires a lot of trial and error, which has proven to be very tedious and requires a lot of patience. Since the last blog post, we have yet to successfully extract the RNA required to perform the gene insertion into. This could be due to the fact that the primers that have been designed are not binding to the specified binding sites and cutting the RNA enough for it to be visible in the gel.

As previously mentioned, there have been a lot of PCR conditions that were previously tested and manipulated but we have yet to find the right one for the experiment that will give us the results we need. Other changes we have made to the experiment were creating dilutions of the RNA because it actually found in a capsid – or shell, that may be hard to break if using too much concentration of RNA. When using the dilution of the RNA, there is less to work with, therefore may be easier to break the capsid. Once the dilutions were made – 5x,10x, 20x; a gel was run but the results we hoped to obtain did not work.

The two main research goals for the rest of the summer and the rest of the research project is to extract RNA by creating the right primers and conditions, and successfully inserting the GFP gene into the RNA of the parasite.

From this research, I have found that the patience is definitely key because we aren’t just tweaking one part of the research for the results we require, but are actually tweaking multiple aspects of it at the same time and seeing what works and where. Also, this research helped me to be a lot more focused on minor details because it could be the smallest thing that needs to be adjusted or changed to create a very major difference.

Thankfully, I believe that my professor and this research project has prepared me for the steps I will be taking once I graduate in the spring. This research has helped to perfect my skills in various techniques such as PCR and proper pipetting that will be useful when I work in a lab as a forensic scientist. Hopefully, the results in this research will positively impact the science community and further studies and research will be conducted to expand on this topic.

Quantifying the Number of GFP Expressing Trichomonas Vaginalis Adhering to the Vaginal Epithelial Cells Treated with Amyloid Dyes

Trichomonas vaginalis is a eukaryotic parasite that causes the most common non-viral sexually transmitted infection worldwide. Although it is the most common, it is also the most understudied and poorly comprehended parasite. The research that will be conducted on this organism will help to better understand host-parasite relationship as the trichomonads adhere to vaginal epithelial cells and begin to colonize. The purpose of this research is to successfully quantify the number of GFP expressing Trichomonas vaginalis adhering to the vaginal epithelial cells treated with amyloid dyes. Because there are a lot of trichomonas cells that will be seen under a microscope, the use of GFP or green fluorescent protein will help to magnify and quantify how many cells are present. The goal is to see an increase in fluorescent activity each time the cells are displayed under a microscope.

In order to carryout this research, we first must colonize GFP. To do this, we use GFP that was found in E. coli and smear it onto an agar plate with LB (Luria broth) medium and ampicillin.

(Source: http://microbeonline.com/wp-content/uploads/2016/10/Pour-Plate-and-Spread-Plate-Method.jpg)

Ampicillin is sometimes used on plates because when ampicillin breaks down, it can often lead to more colonies being formed. The plates are then incubated over night to allow for colonization to occur. Once colonization occurs, the plates are moved under a sterile hood and using a sterile pipet and tip, a single colony is extracted and placed into liquid culture made up of the same LB medium with ampicillin and relocated to incubate on a shaker plate over night. The following day, the tubes are removed from incubation and transferred to a microcentrifuge tube and centrifuged out to collect a concise pellet at the bottom of each collection tube. This pellet collected contains bacterial cells that contain GFP, which will eventually be cut out and cloned multiple times. Once the pellet is collected, the DNA is extracted from those cells using a QIA Spin Miniprep kit. The DNA collected after the end of this kit is the unclean DNA, which could possibly contain buffers and residual waste from the kit that was not completely centrifuged out. However, the unclean DNA is tested to make sure that there is indeed DNA present, and to do this, we have to set up a gel electrophoresis.

Gel electrophoresis is a technique used to separate mixtures of DNA, RNA or proteins based on molecular size. Molecules are pushed by an electric field that moves negatively charged particles through the small pores of agarose gel to the positive charge at the other end.

(Source: https://schoolworkhelper.net/gel-electrophoresis-basics-steps/)

Through this technique, we can compare the sizes of bands to a ladder, or just to simply confirm the presence or absence of a specific molecule. For this purpose, we used the gel to determine the presence of bacterial DNA from the E.coli culture. Below is a picture of the resulting gel after running this technique:

As shown by the picture above, DNA was indeed present in the unclean tubes. From those tubes, a certain amount was then collected and another kit, DNA Clean & Concentrator, was used to further clean the DNA to use for a PCR or polymerase chain reaction.

Polymerase chain reaction is used to increase a single copy or a few copies of a section of DNA. Below is a table that shows the main ingredients of a PCR and what their role is:

The main goal to achieve from the PCR is to use the primers that have been specialized to highlight the region used to code for GFP in the DNA sequence, amplify it and cut it out. Once the PCR is complete in the set conditions, another gel is run to determine if the primers worked and if the gene was amplified. However, PCR is a very sensitive technique that requires the right conditions, which have yet to be discovered. Below are the results of the gels following a PCR treatment and the conditions that were set:

1.)

 

2.)

Using these previous conditions, it will allow for us to easily manipulate the numbers to try and find the most ideal conditions. In the future, these ideal conditions will allow for us to isolate the GFP band with the correct restriction sites and insert it into the parasite.

Moving forward, the research aims to successfully obtain the GFP gene in the PCR using the specialized primers. Once this is obtained, we can perform a gel extraction that will isolate the GFP that contains the specific restriction sites that will eventually be cloned and inserted into the parasite in question. As an individual, I hope to perfect my skills in performing a PCR as well as extracting DNA in the form of a pellet. These skills will eventually aid me in my future career as a forensic scientist where the use of PCR is necessary to amplify the DNA in question to perform further tests on it.