Some of our 2022 seniors and their research
Aseman Bagheri Sheshdeh – Advisor: Dr. Samantha Glazier
Analysis of Binding Affinity of Nogalamycin to various DNA Motifs based on Gel Electrophoresis Assay
Anthracycline antibiotics are DNA intercalators with effective anticancer properties with one of these mechanisms being inhibition of replication by binding to chromosomal DNA. Studies have suggested low specificity of this intercalation, which leads to side effects such as dose-dependent cardiotoxicity yielding in lower commercial use of anthracyclines in cancer therapy. Yet, more recent effort on investigations some types of anthracyclines are proposing ways to prevent the cardiotoxic side effect of these drugs. From anthracyclines, nogalamycin is a naturally occurring anthracycline antibiotic from streptomyces nogalator with significantly improved replication inhibition, which bind to DNA by threading between its base pairs with unique properties. This improved mechanism is suggested to be due to the electrostatic interaction of positive bicyclo amino sugar with DNA backbone and dynamic sliding between the base pairs.
We plan to use a circular dichroism-based assay to better understand the sequence dependent binding of nogalamycin. Circular plasmid DNA is a small double stranded deoxyribonucleic acid that assumes variable degrees of supercoiling in the presence of topoisomerase I and intercalating anthracyclines such as nogalamycin. The degree of supercoiling will be assessed using a gel electrophoresis. For example, an increase in intercalation introduces more negative supercoiling of circular plasmid DNA once DNA is exposed to topoisomerase I. Therefore, the levels of negative supercoiling indicate the degree of relaxation of circular plasmid, which is influenced by nogalamycin concentration. Once DNA is relaxed, it moves a shorter distance across the agarose gel electrophoresis and can therefore be used to assess the levels of relaxation due to intercalation with nogalamycin as a function of concentration. The aim is to refine and utilize this method to assess how the binding affinity and even the binding mechanism of nogalamycin to various DNA motifs compare. Even though, nogalamycin is not commercially used due to inducing cardiotoxicity, the study of its binding characteristics given its unique low dissociation rate provides valuable information of property of similar potential anti-tumor drugs.
Hunter Dean – Advisors: Dr. Lorraine Olendzenski and Dr. Nadia Marano
The Formation of Biofilm and Amyloid Proteins in Different Strains and Species of Microbacterium
Bacteria produce extracellular biofilms to help aid in the function, resilience, and proliferation of the bacterial population. Biofilms are composed of eDNA, lipids, polysaccharides, and proteins. One of the protein components of biofilms can be amyloid fibrils that are integral to the structure and resilience of the biofilms. Amyloid proteins are characterized by repeating beta sheet monomers which aggregate to form amyloid fibrils. We aim to compare the development of biofilm and amyloid proteins in four strains of Microbacterium (M. oryzae strains 2, 29, and 23396 and M. paraoxydans) isolated from soil. To study the growth and development of these bacteria, their biofilms and accompanying amyloid fibrils, I am growing cultures in liquid R2A media in round bottom plates for 5 days. At 24-hour timepoints, I measure the absorbance at 600nm to quantify growth and a Thioflavin-T assay to measure amyloid content in the liquid portion of the culture. I perform a crystal violet biofilm assay to measure the quantity of biofilm adhered to the round bottom well. Understanding the timing of amyloid production and its relation to biofilm abundance will help in understanding the role of amyloid proteins in biofilms of these strains of Microbacterium. Results so far indicate that strains 2 and 29, isolated from agricultural soils on pig farms in Northern NY produce the most biofilm, while strain 29 and M. paraoxydans produce the most amyloid.
Erin Kumler - Advisor: Dr. Emily Dixon
Investigation of Effects of Oxidative Stress on SEF1 Yeast Gene and its Relationship with Other Genes
SEF1 stands for Suppressor of Essential Function, and is a gene found in Saccharomyces cerevisiae yeast that little is known about. Based on previous research, it is predicted to code for a nuclear transcription factor and seems to be upregulated by cells when under stress. This research aims to push that hypothesis further by investigating how other stressors such as oxidative stress can affect cells with and without the SEF1 gene, as well as utilize mRNA sequencing and analysis to further study the gene. Oxidative stress applied to wild type yeast cells with a functioning SEF1 gene and mutant cells is done with 4mM of hydrogen peroxide. The results are then analyzed via growth assays to see if the functionality of SEF1 led to a significant increase in survivability of Saccharomyces cerevisiae cells. This treatment will be replicated a few times to determine if results are consistent. Lastly, the mRNA that was sent out for sequencing by Dr. Dixon’s previous research students, following treatment of mutant and wild type SEF1 yeast cells with heat shock, is sequenced and must be analyzed. This analysis of the mRNA should hopefully reveal crucial information regarding the specific functionality of SEF1 by providing information on its relationship to other genes by looking at what genes have transcription/ translation levels similar to SEF1 and may therefore be connected.
Steph Sauve - Advisor: Dr. Matthew Skeels
Investigation of the chemical and thermal stability of the coral cyan fluorescent protein from Anemonia majano
Photodamage of symbiotic dinoflagellates exposed to thermal stress is involved in coral bleaching, a major cause of reef decline. When coral reefs die, the ecosystem the reef supports and the biodiverse species they protect die along with it. In addition to concerns surrounding biodiversity, economic goods and services provided by corals, valued at over US $20 trillion annually, would be lost if the reefs were to disappear. Photoprotection is therefore a vital aspect of coral stress physiology. Corals produce a variety of fluorescent proteins (FPs), some of which screen the symbiotic algae from excess sun light, and others enhance light availability for photosynthesis of the algal symbionts by converting longer wavelengths of light into a more usable form of light for the dinoflagellates. Cyan FPs have characteristically broad absorption/excitation spectra with peaks at 440–460 nm that overlap with the absorption spectrum of photosynthetic pigments of the symbiotic dinoflagellates, making these FPs photoprotective. The question arises about whether the thermodynamic stability of these cyan FPs are related to reef decline. To determine the thermodynamic stability of a cyan FPs, the CFP from Anemonia majano was cloned into an E. coli expression vector with an 6x His affinity tag. The 6xHis-CFP was overexpressed and purified from E. coli. Thermal and chemical denaturation curves of this protein were monitored using intrinsic tryptophan fluorescence. Additionally, mutagenesis studies will be performed to understand which interactions within the protein have the greatest impact on the protein’s stability.
Carter Tracy – Advisor Dr. Samantha Glazier
Complete characterization of the methyl green-DNA interaction to determine possible major groove binding for competition studies
Methyl green is a cationic dye that is believed to bind to the major groove when interacting with DNA. While binding to the minor groove is common, there are virtually no reports of small molecules that bind to the major groove of DNA. However, there is not a thorough understanding of the interactions of methyl green and DNA, including the possibility of other binding modes like intercalation. While methyl green has historically been used as a DNA staining agent, where the exact binding mode is unimportant, methyl green has the potential to be used in competitive binding studies to learn more about the role of the major groove in mechanisms of intercalation. Intercalation has long been assumed to involve the minor groove, but there is little experimental evidence to support the exclusion of major groove involvement. Therefore, if methyl green does bind in the major groove, more could be discovered about the binding mechanisms of medicinally important drugs like doxorubicin, a chemotherapeutic. The characterization methods will include melting point and viscosity studies, as well as using circular dichroism spectroscopy to understand the binding mode. Additionally, determining water exchange values will aid in the interpretation of the thermodynamic quantities calculated using the Van ’t Hoff method. Lastly, a home-built temperature jump method will be used to characterize the kinetic pathway of methyl green-DNA binding. Together, these techniques will allow for the determination of the binding mode(s) and characterization of the associated thermodynamic and kinetics properties of binding.