DNA damage occurs regularly, and although the cell has numerous repair mechanisms to counteract it, some DNA lesions may persist. Many of these lesions can be bypassed by DNA and RNA polymerase, with replicational mutagenesis leading to permanent mutations in the genomic sequence, and transcriptional mutagenesis leading to mutant transcripts that may direct the production of mutant proteins. We hypothesized that transcriptional mutagenesis is a mechanism for initiating adaptive mutagenesis, which occurs in nondividing cells in direct response to a selective pressure, allowing cells to overcome the selection and resume cell division. Our lab has previously demonstrated that DNA damage leading to adaptive mutagenesis in yeast is biased to the template strand relative to transcription, supporting this hypothesis. Unexpectedly, DNA damage leading to replicational mutations in our system is similarly skewed to this strand, although the sequences surrounding these two classes of mutations seems to differ. We hypothesize that sequence context is important for determining whether certain mutation sites are more likely to contribute to replicational or adaptive mutagenesis. To address this hypothesis, our aim was to create Trp5 mutant yeast strains using CRISPR/Cas9 in which the mutation occurred in a sequence context-specific manner, and the reversion of which could be scored for both replicational and adaptive mutagenesis. The CRISPR/Cas9 system required generation of a plasmid containing the target sgRNA (via molecular cloning techniques) as well as a linear repair template with the mutation of interest (via a two-step PCR protocol). Creating both of these components proved challenging, and optimization of the protocols will be required. Once we have all the components for CRISPR/Cas9-mediated mutagenesis, future research involves creating the mutant strains and assessing whether the sequence context affects the mutation frequency with respect to either replicational or adaptive mutagenesis.

In the US over 100 million people live with diabetes or pre-diabetes. The economic burden of this is approximately $327 billion every year. This study seeks to establish an alternative mode of insulin production using a polyethylene glycol (PEG) transformation of Pleurotus ostreatus. P. ostreatus is a valuable target for genetic transformation due to its lack of endotoxins, rapid growth, and fully sequenced genome. In this study, I transformed P. ostreatus using PEG with a plasmid containing the human insulin gene, a green fluorescent protein (GFP) reporter gene, and a selectable resistance gene. Transformed cells were selected using hygromycin, extracted, and regenerated on growth media. Confocal microscopy confirmed the presence of the GFP and presumably the human insulin gene. An ELISA for insulin and proinsulin will be used in the upcoming months to test for genetic expression, and to determine the efficacy of protein folding in the transgenic fungal cells. This has the potential to not only expand the market for diabetic treatment options, but it initiates a valuable conversation about the importance of diversifying production methods and costs in the treatment of diabetes.

Local processes in marine ecosystems, including coastal estuaries, modify ocean acidification caused by rising atmospheric CO2. Because ocean acidification poses a threat to shell-forming organisms, it is critical to understand how these processes affect acidification in specific regions. In the Snohomish River estuary, freshwater from river discharge deposits directly into Possession Sound, impacting the salinity and temperature of the area. River discharge in estuaries has been found to be slightly acidic, as well as a source of nutrients that fuel blooms of phytoplankton. Large phytoplankton blooms can lower the pH at depth because of the process of respiration, which releases CO2 and decreases dissolved oxygen levels. My research examines changes in pH with temperature, salinity, chlorophyll, and dissolved oxygen at different depths in Possession Sound, Washington, using data collected from January 2017 through January 2021 with a YSI EXO2 Sonde. I hypothesized that near-surface depths and sites located closer to the river would have lower temperatures and salinities correlating with lower pH. Additionally, lower dissolved oxygen at greater depths would correlate with greater amounts of chlorophyll and a decrease in pH at depth. Depths near the halocline were predicted to have alkaline pH values due to photosynthetic organisms. I analyzed data using Microsoft Excel and R Studio. Results found that with chlorophyll less than ~1.25 RFU, pH was greater than 8.0, while with lower dissolved oxygen, pH was less than 7.75. Temperatures less than 10°C corresponded with more pH values between 7.0 and 7.5, while salinity had no apparent trend. In most seasons, pH appeared to decrease slightly at greater depths. The exception to this was winter, when more acidic pH values were observed at near-surface depths. Overall these results indicate that local processes in the Snohomish River estuary are affecting changes in pH.