Festival of Science, Scholarship & Creativity 2022
At St. Lawrence’s Festival of Science, Scholarship, and Creativity on April 29, 2022, Biochemistry majors and first-year students doing research in the Chemistry Department presented their results.
Carter Tracy, Faculty Samantha Glazier, Biochemistry
“Complete Characterization of the Methyl Green-DNA Interaction to Determine Possible Major Groove Binding for Competition Studies”
Abstract: 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.
Hunter Dean, Faculty Nadia Marano and Lorraine Olendzenski, Biochemistry
“The Formation of Biofilm and Amyloid Proteins in Different Strains and Species of Microbacterium”
Abstract: Bacterial biofilms aid in the function, resilience, and proliferation of the bacterial population. Biofilms are composed of eDNA, lipids, polysaccharides, and proteins. Amyloid fibrils are a common protein component of biofilms. We compared the development of biofilm and amyloid proteins in four isolates of Microbacterium (M. oryzae strains 2 and 29 isolated from agricultural soils on pig farms in Northern NY), M.oryzae strain 23396, and M. paraoxydans. Cultures were grown in liquid R2A media for 5 days. At 24-hour timepoints, I measured the absorbance at 600nm to quantify growth and used a Thioflavin-T assay to measure amyloid content in the liquid portion of the culture. I used a crystal violet biofilm assay to quantify the biofilm adhered to the wells. Strains 2 and 29 produce the most biofilm, while strain 29 and M. paraoxydans produce the most amyloid protein. I attempted to isolate the amyloid protein from M. oryzae strain 29 using sonication, DNase and Triton-X100 treatment, ultracentrifugation, and SDS-PAGE tube gel. I obtained ThT-positive material from the top of the tube gel for further analysis.
Aseman Bagheri Sheshdeh, Faculty Emily Dixon and Samantha Glazier, Biochemistry
“Analysis of Binding Affinity of Nogalamycin to various DNA Motifs based on Gel Electrophoresis Assay”
Abstract: 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 the lower commercial use of anthracyclines in cancer therapy. Yet, more recent efforts to investigate 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 binds 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 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 on properties of similar potential anti-tumor drugs.
Erin Kumler, Faculty Emily Dixon, Biochemistry
“Effects of Oxidative Stress on the SEF1 Gene in Yeast”
Abstract: 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 functioning SEF1 gene, as well as research possible connections to other well-known genes to provide a starting point for future students to do mRNA analysis. Oxidative stress applied to wild type yeast cells with a functioning SEF1 gene and mutant cells was done with hydrogen peroxide of a 4mM concentration. The results were 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. The growth levels of the two cell lines were also analyzed by tracking their growth overtime in medium compared to control and the other, and a spot test was done with higher concentrations of hydrogen peroxide to investigate whether sef1 is connected to survivability. Since not much is known about the function of the SEF1 gene, this research is important for developing a better understanding of the gene and producing new questions and possible areas of research related to the gene.
