Biomedical Projects
February 2020 — May 2020
pSK41/pGO1-family conjugative plasmids of Staphylococcus aureus encode a cryptic repressor of replication
The majority of large multiresistance plasmids of Staphylococcus aureus utilise a RepA_N-type replication initiation protein, the expression of which is regulated by a small antisense RNA (RNAI) that overlaps the rep mRNA leader. The pSK41/pGO1-family of conjugative plasmids additionally possess a small (86 codon) divergently transcribed ORF (orf86) located upstream of the rep locus. The product of pSK41 orf86 was predicted to have a helix-turn-helix motif suggestive of a likely function in transcriptional repression. In this study, we investigated the effect of Orf86 on transcription of thirteen pSK41 backbone promoters.
We found that Orf86 only repressed transcription from the rep promoter, and hence now redesignate the product as Cop. Over-expression of Cop in trans reduced the copy number of pSK41 mini-replicons, both in the presence and absence of rnaI. in vitro protein-DNA binding experiments with purified 6 × His-Cop demonstrated specific DNA binding, adjacent to, and partially overlapping the −35 hexamer of the rep promoter. The crystal structure of Cop revealed a dimeric structure similar to other known transcriptional regulators. Cop mRNA was found to result from “read-through” transcription from the strong RNAI promoter that escapes the rnaI terminator. Thus, PrnaI is responsible for transcription of two distinct negative regulators of plasmid copy number; the antisense RNAI that primarily represses Rep translation, and Cop protein that can repress rep transcription. Deletion of cop in a native plasmid did not appear to impact copy number, indicating a cryptic auxiliary role. For more, see the paper, here.
June 2020 — August 2020
Database Analysis of Antimicrobial Peptides for Use in a Nanoparticle Antibiotic
Antimicrobial Peptides (AMPs) are a diverse group of naturally-occurring molecules that act as first responders in multicellular organisms. Through years of research and discoveries, scientists now believe that AMPs may have widespread use as antibacterial agents. To date, nearly 3,000 AMPs have been discovered, created, and chemically modified, yet only seven have been approved by the U.S. Food and Drug Administration for medicinal use. Previous research that demonstrates topical application of AMPs as effective against bacterial infections have set the groundwork for the possibility of pairing AMPs with delivery systems targeted to various tissues in the body.
Dr. Charles Roth’s laboratory is looking to embody this idea by using AMPs to eradicate the presence of biofilms in the lung tissues of Cystic Fibrosis patients. Our group has found success in our approach in encapsulating AMPs in synthetic polymers that self-assemble into bioactive nanoparticles. The goal of the current work is to determine which among the thousands of candidate AMPs would be the most effective in this nanoparticle delivery system.
This work utilizes a database-guided review to combat this issue. We used several databases to complete a general search for natural, synthetic, and modified peptides with anti-bacterial activities, honing in on ones that were annotated as anti-biofilm. Next, the AMPs were clustered by various criteria such as their physicochemical properties (qualities like hydrophobicity, charge, length, structure, etc.) as well as their source and notated their effectiveness against numerous species of bacteria. Finally, in comparing AMPs within each cluster, the best were selected to move on for review. The goal is to begin utilizing the ability of these AMPs to penetrate and eradicate bacterial biofilms at micromolar concentrations. We hope to extend this work by formally testing the AMPs and analyzing how the physicochemical properties affect their efficacy in vitro. Listen to the audio presentation here.
September 2019 — Mar 2020
Protein Analysis of T. thermophilia
Under the guidance of Professor J. Bill Straus, I analyzed the membrane and associated protein compositions of Tetrahymena thermophila, researching how synthesizing novel fluorescent reagents can trace how and when different proteases are delivered to maturing phagosomes.
Here, I began the analysis of soluble membrane proteins by extractions leading to an investigation by polyacrylamide gel electrophoresis and mass spectrometry.
January 2019 — May 2019
Investigating the Role of the PhoP/PhoQ Regulatory System on Headgroup-acylated Glycerophospholipid levels of Escherichia coli
Under the guidance of Professor T. Garrett, I examined the role of the PhoP/PhoQ regulatory system on headgroup-acylated glycerophospholipids in Escherichia coli and these mutations on virulence, cell division, and shape. I collected data through various biochemical and microbial techniques and have demonstrated aptitude in biosafety, laboratory management, and documentation/reporting within academic research settings.
Here, I learned basic experimentation techniques focused on analyzing an organism for specific enzymatic activity and membrane composition (i.e TLC, HPLC, etc.), became adept at finding, reading, and analyzing science literature and replicating experiments.