Intro:
Neisseria Gonorrhea is a sexually transmitted pathogen that infects the mucous membranes of the reproductive tract leading to pelvic inflammatory disease, ectopic pregnancies, and infertility. The survival of Neisseria in a human host requires an uptake of iron. This is facilitated by the TonB-dependent receptors. By using antibodies that target these receptors, Neisseria’s iron supply is depleted which greatly affects its survival in host epithelial cells.
The Cornelissen lab currently has a suite of monoclonal antibodies against the TonB-dependent transporters of Neisseria Gonorrhea. Creating these antibodies was a significant achievement and advancement to the field, so it is important that these antibodies are protected under intellectual property. However, in order to do this the antibodies need to be sequenced. The aim of this study is to sequence these monoclonal antibodies against the TonB-dependent transporters of Neisseria Gonorrhea.
The process of sequencing is multiphase and involves many different techniques that aim to remove RNA from cells, convert it into cDNA, make more copies of the cDNA to then be cloned. As this is the first-time antibody sequencing has occurred in the Cornelissen Lab, it is expected that this project will involve a lot of troubleshooting until a satisfactory procedure has been developed that can be easily understood and repeated by others. Once this project has been completed, these antibodies will be sequenced and patented to the Cornelissen Lab.
Body:
Neisseria gonorrhea is a sexually transmitted gram-negative diplococci bacterium. It is the second most frequently reported sexually transmitted infection in the world. It is known to affect the mucous membranes causing pelvic inflammatory disease, ectopic pregnancies, urethritis, and infertility. Neisseria requires certain trace metals such as zinc and iron for certain cellular processes, however the human body tries to hide these metals so that Neisseria cannot grow and spread.. Unfortunately, Neisseria gonorrhea has found a way to counter this. “During infection, pathogenic Neisseria express a repertoire of metal-responsive genes that allow the bacteria to overcome metal restriction efforts by the human host” (Maurakis & Cornelissen 2020,).This metal acquisition is facilitated by the TonB dependent transporters (TdTs). “Unlike many bacteria, gonococci do not secrete siderophores to scavenge iron, but instead express high-affinity TdTs that directly interact with host iron-binding proteins and subsequently internalize iron sequestered from these proteins” (Hagan & Cornelissen 2006)
Antibodies are large proteins of the immune system that circulate in the blood. When they encounter a foreign substance such as bacteria or a virus, the antibody attaches to it and neutralizes it.
An artist’s illustration of antibodies (blue-green structures) attempting
to latch onto the antigens on the outer surface of a coronavirus (red).
Antibodies are one of the major players in the immune system’s attack
against viruses.
The Cornelissen lab has curated monoclonal antibodies against the TonB dependent transporters for Neisseria gonorrhea. They were created using hybridoma cell lines. By using antibodies to target these TdTs, Neisseria’s ability to uptake iron from its human host is interfered with, thereby affecting its ability to grow and spread.
Sequencing of antibodies determines the order of amino acids in a given antibody. It gives important information on protein characterization such as antigen binding quality and cross reactivity. The sequencing of these antibodies is important for intellectual property reasons so that no one else can claim these antibodies as their own. In order to sequence these antibodies many different experiments have to be performed. Meyer at el., (2019) describe a simple workflow on how the amplification of antibodies from hybridoma RNA for sequencing. During this project experiments such as RNA isolation from the hybridoma cells, DNase treatment, PCR, cDNA synthesis, RT PCR, TOPO cloning, plasmid extraction and finally sequence analysis were performed.
Methodology:
In my research I started by removing the RNA from specific cells and completing a treatment to get rid of any contaminating DNA. This was then followed by a polymerase chain reaction (PCR) experiment which amplified the target RNA. I created an experimental control, positive control and no template control for this experiment. To see if the experiment was successful, all 3 controls were run on an electrophoresis agarose gel. The purpose of the gel is to separate DNA, RNA or protein molecules based on size and electrical charge.
Agarose gel of DNase treated samples. An experimental control,
negative control and no template control respectively, were ran.
Picture was captured using a ChemiDoc imaging system.
Next the RNA underwent cDNA synthesis using a Superscript First Strand kit. This process turns RNA into cDNA by using a reverse transcriptase polymerase chain reaction (RT PCR). I created 4 different samples using 4 different sets of primers for this cDNA synthesis. In addition, 4 no template controls were made using the same sets of primers. These primers were designed and ordered from scientists at Amherst College (Wang et al., 2000). Once the cDNA was amplified by the RT PCR experiment, the samples were run on another agarose gel to be visualized.
Agarose gel of RT PCR products for specific primers. Picture was
captured using a ChemiDoc imaging system.
This cDNA was then used for cloning on agar plates to insert the cDNA into a plasmid. Before the cloning process took place, the cDNA was purified in case there was any contamination after the RT PCR. The cloning experiment streaking plates with the cDNA and the plasmid. This allowed the cDNA to be inserted into the plasmid which then grew and multiplied. This step was essential to make more of the cDNA. Again, a PCR reaction took place followed by an agarose gel to ensure that the cDNA was successfully inserted. The DNA was then isolated from the plasmid and the DNA was sent to be sequenced.
"Antibody Sequencing" by Antibody Design Labs
Conclusion:
Antibodies targeting the Ton-B dependent transporter for Neisseria Gonorrhea disrupt the disease’s ability to uptake iron, in turn preventing the growth and spread. To sequence these antibodies methods such as RNA isolation, DNase treatment, PCR, cDNA synthesis, RT PCR, TOPO cloning and plasmid extraction were used. These methods were completed using a variety of different kits. As this was the first-time sequencing has been done in the Cornelissen lab, the process took a lot of troubleshooting. In the future other people can use the notes in my lab notebook to follow the procedures that produced a successful experiment. With these procedures, others should be able to sequence these antibodies much quicker and easier. Though the process was long, the antibodies are now sequenced and are protected by intellectual property rights to the Cornelissen Lab.
Reflection
My semester in this signature experience course can be split into two parts, lab experience and course work. I believe both have greatly enhanced my scientific skills and insight into the science field as well as helped me to prepare for a career in biomedical science.
Being a part of the Cornelissen lab was the first time I have gotten any practical lab experience and seen the inner workings of a lab and what goes into being a research scientist. It’s not just about copying experiments, but coming up with your own, troubleshooting, having lab meetings and presenting findings. I was able to carry out scientific methods that I have only learned about in previous courses. I have acquired both hard and soft transferrable skills. The hard skills include the lab techniques I have used. The soft skills include finding support groups, communicating with an audience and sharing my project and findings with others which was a part our career readiness competencies. These skills look really good on my graduate school applications and will help in both my future academic and career goals.
The coursework has helped me by introducing me to and giving tips for things that I will need as I continue my journey. Assignments such as writing scientific papers, writing a CV and even this E portfolio will be essential parts of my future academic and career goals. I plan to go to graduate school where I know I will need to write many scientific papers and learning how to create an e portfolio will help for when I must present my projects. The CV assignment was important for when I look to find a job after I graduate. I used the critiques I got on my CV to change and make it better so that I can use it for my graduate school and job applications. The translating skills to the job market assignment helped me to see what education and skills are required for my intended career choice as well as possibly paths to take to get there.
Overall, I think this choosing independent research course as my signature experience was the best thing for me as it has introduced me to experiences and assignments that will be a big help for my future.
Citations:
Antibody sequencing. Antibody Design Labs. (n.d.). http://www.abdesignlabs.com/services/antibody-sequencing/
Hagen, T.A. and Cornelissen, C.N. (2006), Neisseria gonorrhoeae requires expression of TonB and the putative transporter TdfF to replicate within cervical epithelial cells. Molecular Microbiology, 62: 1144-1157. https://doi.org/10.1111/j.1365-2958.2006.05429.x
Hurt, A. E. (2021, May 25). Explainer: What are antibodies?. Science News Explores. https://www.snexplores.org/article/what-are-antibodies-explainer
Maurakis, S., & Cornelissen, C. N. (2020). Metal-Limited Growth of Neisseria gonorrhoeae for Characterization of Metal-Responsive Genes and Metal Acquisition from Host Ligands. Journal of visualized experiments : JoVE, (157), 10.3791/60903. https://doi.org/10.3791/60903
Meyer, L., López, T., Espinosa, R., Arias, C. F., Vollmers, C., & DuBois, R. M. (2019). A simplified workflow for monoclonal antibody sequencing. PloS one, 14(6), e0218717. https://doi.org/10.1371/journal.pone.0218717
Wang, Z., Raifu, M., Howard, M., Smith, L., Hansen, D., Goldsby, R., & Ratner, D. (2000). Universal PCR amplification of mouse immunoglobulin gene variable regions: the design of degenerate primers and an assessment of the effect of DNA polymerase 3' to 5' exonuclease activity. Journal of immunological methods, 233(1-2), 167–177. https://doi.org/10.1016/s0022-1759(99)00184-2
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