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Investigating Zinc Metalloprotease Protein Mutations in Clostridioides difficile through Multiple Sequence Alignment

Project by Polygence alum Matthew

Investigating Zinc Metalloprotease Protein Mutations in Clostridioides difficile through Multiple Sequence Alignment

Project's result

Matthew created a poster and slides presentation of the results of his project. Matthew will also be using these to compete in his local NCSEF Science Fair.

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Purpose: Antibiotic resistance is a growing health crisis projected to cause more deaths annually than cancer by 2050. Per CDC’s 2019 report, Clostridioides difficile qualifies as an urgent antimicrobial resistance threat. To aid in understanding antibiotic resistance, we studied C. diff protein mutations using all the available sequences on the NCBI website. This approach enabled us to find certain regions of the zinc metalloprotease (Zmp1) protein displaying high rates of mutation, and contributes to our growing understanding of how microorganisms evolve in the context of modern antibiotic use.

Procedure: We inputted the Zmp1 reference sequence into the NCBI BLAST database to search for other similar Zmp1 protein sequences (N=93). Using those sequences, we conducted a multiple sequence alignment via Clustal Omega’s website, then viewed the alignment results in Jalview. Here, we were able to highlight important mutations and count the number of appearances of each amino acid by position using a Python program my mentor, Jacob, had written. Finally, we used ChimeraX, a protein visualization software, to pinpoint the locations of these mutations on a molecular scale.

Data collected & Conclusions: We collected data on the protein’s conservation scores and notable mutations near its active site. The results mostly supported my hypothesis, as the active site in the Zmp1 protein was mostly well conserved. However, a few mutations in the zinc binding site demonstrated significant differences in amino acid structure. For example, the glutamic acid to lysine mutation is very interesting because their side chains have completely different charges.



Polygence mentor

MD/PhD Doctor of Medicine and of Philosophy candidate


Medicine, Biology


Structural Biology, Cryo-EM, Cryo-ET, Ligand Docking, Chemical Biology, Bioinformatics, Computational Biology, In Silico Screening




High school student interested in researching biochemistry protein structure to study antibiotic resistance and cancer immunology.


Durham Academy

Graduation Year


About my mentor

“My mentor was extremely knowledgeable in his subject fields (biology, chemistry, compsci, protein structure, coding, etc.) and helped me tremendously by guiding me throughout the whole research process and helping my project ideas come into fruition. Our scheduling was pretty smooth, and my mentor always was on time to meetings and sending me resources to work on. My mentor is also a very nice person overall, who, as a former student, definitely understands the stress that students face during their time in the program. Aside from research, my mentor is also a very fun person to be around; he always asks me how my week/my classes have been and we talk about some other common interests as well. The only thing I would mention is that my mentor was a lot more into computer science than I was, so we incorporated a lot more code into our project than I would've liked, but other than that, our match was near-perfect in terms of subjects of interest.”