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.

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