Vibrio cholerae, a pathogenic bacteria, form biofilms as a way to protect themselves from environmental threats and enable adhesion to host surfaces. The biofilm is a composite of organized clusters of cells, vibrio polysaccharides (VPS), biofilm matrix proteins, and other matrix components. My project focuses on RbmC, a biofilm matrix protein that may have an adhesive role in biofilms. Previous studies identified another biofilm matrix protein, Bap 1 with high sequence homology to RbmC, that has domains that are involved in biofilm and environmental surface adhesion and other domains that are involved in protein to VPS adhesion. Both proteins play dominant but differing adhesion roles in the Vibrio biofilm. We want to explore whether RmbC are able to bind to the VPS that make up a majority of the biofilm structure, as well as other sugars that may be found on surfaces of hosts. I will be cloning, expressing, and purifying different combinations of domain segments of RbmC. Larger soluble fragments of RbmC will be useful in structural and functional explorations into the roles of RmbC in the biofilm. Understanding the roles of matrix proteins in cell-cell interactions and cell-surface adhesions in biofilm formation is crucial to the study of how pathogenic bacterium such as Vibrio cholerae endure environmental threats and transmit diseases to human hosts.

Proteins are crucial to the development of multicellular organisms. These microscopic machines serve a wide range of functionality—from transporting small molecules to catalyzing biochemical reactions. In recent years, advances in technology and knowledge of proteins have significantly progressed to the point where we can now create artificial proteins. The current project aims to optimize the functionality of a de novo protein known as mini-Fluorescence Activating Protein (mFAP), which stabilizes a small chromophore known as DFHBI, allowing it to fluoresce. We utilize the Rosetta molecular modeling software to develop a protocol that predicts which mutations increase mFAP-DFHBI brightness. From there, we can then run these predictions in molecular dynamic simulations and ultimately, experimentally test promising candidates that increase mFAP-DFHBI brightness. Our research provides many useful applications, for instance, mFAP-DFHBI complex can serve as a sensor or fluorescence-tagging tool in other disciplines.

HU, a histone-like protein, is one of the most abundant proteins in Escherichia coli and has significant roles in DNA packaging, recombination, replication, and repair. Previous studies have shown that HU binds with high affinity, in a non-sequence specific manner, to an important intermediate in recombination and repair, known as the Holliday or 4-Way Junction. HU binds to the Holliday junction with nanomolar affinity, suggesting that recognition of specific structural elements may help to facilitate the binding between the protein and DNA. Our research aims to elucidate the structure of the HU-J20 complex to clarify the structural elements that lead to HU recognition of the Holliday Junction. In our crystallography studies, an immobile Holliday junction with 20 base pairs per strand, referred to as J20, is used to reduce the flexibility of the construct. If diffraction quality crystals are obtained, X-ray crystallography can be used to visualize structural features of the protein-DNA complex and how HU binds to J20. To develop diffraction-quality crystals, we will screen conditions that promote crystal formation. Conditions will be optimized to produce crystals that lead to high resolution diffraction patterns. The crystallographic information furthers our understanding of how HU interacts with the Holliday junction as well as its other binding partners. Additionally, by understanding how HU binds to J20, we may be able to comprehend how other proteins bind to the 4-Way Junction. Overall, the goal of this project is to solve a piece of the puzzle in HU-junction interactions in the context of recombination.

One of the earliest identified human coronaviruses is the human OC43 coronavirus (HCoV-OC43). This endemic virus is associated with the common cold. Protein synthesis is a critical step in the virus life cycle. To make a subset of viral proteins, the ribosome must slip at a specific point in translation. When the ribosome slips backward by a single nucleotide, it continues translation in an alternate -1 reading frame to produce this subset of proteins. Although this event is rare in cellular mRNAs, the proteins produced from the frameshift are necessary for viral replication. While the coronavirus research field has made substantial progress in understanding the basic principles of coronavirus replication and disease development, the frameshift efficiency of the HCoV-OC43 has not been measured. The frameshift efficiency describes how often the ribosome slips at the frameshift site. We hypothesize that the HCoV-OC43 frameshift efficiency will be similar in magnitude to SARS-COV-2 (~36%) because their frameshift sites are comparable. To fill this gap in knowledge, we used a well-established dual-luciferase assay to measure the HCoV-OC43, SARS-CoV-2, and HIV-1 frameshift efficiencies. The SARS-CoV-2 and HIV-1 frameshift sites served as positive controls. A negative control lacking a frameshift site structure was also included. Preliminary results from a single biological replicate suggest that the HCoV-OC43 frameshift efficiency is roughly 35-43%. Future work will include the additional replicates needed to conclusively quantify the frameshift efficiency. Our investigation will ultimately provide insight on a critical step in the HCoV-OC43 life cycle.

Standard diagnostic tests for common bacterial and viral infections are invasive and uncomfortable. Difficulty performing these tests may delay or prevent diagnosis in children which leads to more severe consequences. Delays may occur due to children’s noncompliance or difficulties of access to a clinic. There is a need for more accessible diagnostics to prevent these complications. This research investigates strep throat as a disease caused by an infection of the bacterium Streptococcus pyogenes. Our goal is to create a novel saliva collection platform that is designed and engineered to be child-friendly, effective at pathogen collection, and is suitable for home use to address this problem. The device is uniquely designed to be attractive to children as well as optimized for collection of the pathogen. The prototype devices are developed using 3D printing, computer numerical control (CNC milling), and molding. Human subjects studies were performed to examine usability and inform further design. Preliminary results show the device’s ability to capture the bacteria has been optimized. Participants in the studies were satisfied with device and consistency in usage. Once the device and system are established, it has potential to be adapted for additional diseases which increases the versatility.