Internship
Project Summary
Decoding Dynamin Protein Interactions Using CRISPR-Cas9 in Malaria Parasites
Parasites of the genus Plasmodium are responsible for causing malaria, the deadliest mosquito-borne parasitic disease in the world. Due to increasing concerns of drug resistance in these parasites, new targets for anti-malarial compounds are in high demand. I am helping to investigate an uncharacterized protein in P. falciparum called PfAnchor that is essential for parasite replication. When PfAnchor is absent, parasites cannot emerge from red blood cells after cytokinesis, leading to non-invasive clusters of parasites. We found that PfAnchor interacts with PfDyn2, a protein crucial for organelle fission in related organisms. I wanted to characterize this interaction, so I used CRISPR-Cas9 gene-editing technology to introduce protein tags for PfDyn2 into PfAnchor tagged parasites. This process involved designing and building CRISPR-Cas9 plasmids with the PfDyn2 gene and associated tags, purifying plasmid DNA from bacterial cultures, and transfecting P. falciparum parasites with this plasmid. The transgenic parasites can then be collected for advanced ultrastructure expansion microscopy to visualize and compare PfDyn2 localization in the presence and absence of PfAnchor. This work will enhance our understanding of organelle fission and will lead to new targets for anti-malarial drugs.
Final Project
About the Internship
Malaria was ranked as the world's 4th deadliest infectious disease in 2022, only following COVID-19, tuberculosis, and HIV/AIDS. Anti-malarial drug resistance is an emerging global health concern that has greatly impacted the effectiveness of existing treatments. New chemotherapeutic agents are needed to continue battling against malaria. At my internship site, we study Plasmodium falciparum biology to uncover potential anti-malarial targets. More specifically, our current focus is on PfDYN2, a dynamin-related protein that is essential for organelle growth and fission, thus affecting erythrocytic schizogony.
Project Explanation
DNA Cloning
Forming a recombinant plasmid containing the PfDYN2 gene, antibiotic resistance gene, selection marker, and aptamer.
DNA Isolation
PfDYN2, gRNA1, and gRNA2 transformed bacterial cultures were grown. The DNA or RNA of each was extracted and purified for parasite transfections.
CRISPR-Cas9 Transfection
CRISPR-Cas9 is being used as a gene-editing tool to perform transfections. The goal is to introduce the recombinant plasmid into the parasite's genome, forming genetically modified parasite cell lines. The PfDyn2 plasmid will be introduced into PfAnchor tagged parasites to characterize interactions between both proteins.
Ultrastructure Expansion Microscopy & Immunofluorescence Staining
Ultrastructure expansion microscopy is a method of expanding samples on gels to attain clear images with enhanced resolution. This tool will be used with immunofluorescence staining to characterize PfDYN2 interactions with PfAnchor and the apicoplast.
Workplace
Strengths as a Team Member
As a team member, some of my strengths include collaboration, communication, and organizational skills. I have held different positions that require working independently and with others. When working alone, I remain self-disciplined and organized to execute a task. My communication skills allow me to work effectively in collaborative environments. I enjoy providing input and asking questions when working in a team - it does not matter if it is in class or the workplace. I highly value structure and organization, which enable me to manage my time wisely, pay great attention to detail, and meet deadlines.
My team role has evolved to give me more flexibility and independence. As I work with my mentor, I am being exposed to different tasks and lab experiments. I find it very fascinating because I am getting a much better idea of how all of our work connects.
Growth as a Professional and Teammate
This year I would like to grow on my skills as a scientist. I want to work on thinking analytically like a scientist and improving my lab note taking and record keeping skills. I am interested in learning more about current research being conducted in parasitology and the infectious diseases field. Lastly, I would like to build some independence in the lab environment. Each of these skills will aid to my success as a future graduate student and biomedical scientist.
Over the past few months, I have grown as a professional by taking on individual tasks, keeping a lab journal, and reading about ongoing research in the field. My skill set has expanded as I take on new tasks such as maintaining tissue cultures of Plasmodium parasites, performing ultrastructure expansion microscopy, and isolating DNA and RNA for CRISPR-Cas9 based transfections. Each of these tasks are necessary for culturing genetically modified parasite cell lines and performing gene knockdowns.
Successes and Challenges
Success
Some of my successes include maintaining tissue cultures and transfection samples, improving my blood smearing technique, keeping a lab journal, gaining a better understanding of malaria parasite biology by reading papers, and expanding my skillset by being exposed to an extensive range of tasks including DNA cloning, DNA isolation, tissue culturing, PCR, gel electrophoresis, DNA quantification, CRISPR-Cas9 gene-editing tools, and ultrastructure expansion microscopy. All of these skills will be beneficial to my future as a graduate student and scientist since I plan to work in infectious disease research.
Challenge
My biggest challenge thus far has been understanding the extensive details of the work we do in the lab. Parasitology is a very complex field and it is something that I have not been exposed to in any of my courses. Malaria parasites in particular undergo multiple life cycles in mosquitos and humans. Each life cycle consists of various phases, and the terminology was all very new to me at the start of my internship. I have spent some time reading papers over some of the life cycles. The paper I am reading next covers all of the lifecycles and it brings everything together. In the spring semester, I plan to continue reading articles in order to become more familiar with all of the work.