There are certain challenges that must be overcome in the field of synthetic biology, such as the relative instability of genetic devices when selective pressures are absent. This phenomenon simplified means that when we insert a genetic device within an organism, if the organism does not naturally produce this protein then it will remove or inhibit (make the device non-functional) due to the increased metabolic burden placed on the organism. Instability results from inherent mutations that accumulate, but also due to hotspots of long repeated sequences that can lead to an increase of mutations made by DNA polymerase. The accumulation of these mutations can in turn cause genetic devices to become nonfunctional through different mechanisms (i.e. polymerase slippage, homologous recombination). The Evolutionary Stability project has data that suggests repeat sequences as short as 8 nucleotides in length can lead to increased mutation rates for genetic devices. The project aims to examine how impactful these short repeats are, and if there is functional independence or positional independence in a Kanamycin antibiotic resistance gene. Recently, the project has completed trials and mutation rates for an eight base pair repeat (10 fold increase from background) , a six base pair repeat (similar to background), and 3 variations of ten base pair repeats (100 fold increase from background). Overall, we hope to utilize this data to optimize the Evolutionary Failure Mode calculator developed by the Barrick lab, which analyzes the stability of genetic devices but cannot yet detect these small repeat sequences.

Marine mussels (Mytilus) are common models for studying environmental stress on marine invertebrates and as a keystone species and filter feeder, they are essential for coastal community dynamics. However, most studies test a singular stressor while marine environments are composed of multiple stressors. Therefore, our project focused on investigating the effect of multiple stressors (hyposalinity and elevated heat) on Mytilus galloprovincialis clearance rate (CR), a proxy for feeding rate. We predicted that combinations of lower salinity (20 ppt) and elevated temperature (25ºC) will have a lower CR compared to the control conditions (17ºC, 34ppt). M. galloprovincialis (N = 95) were collected from Marina del Rey, CA and acclimated to control conditions without food in recirculating seawater tanks prior to experimentation. During experimentation, M. galloprovincialis were allowed to feed for 30 minutes in temperature and salinity treatment conditions (combinations of 17, 20 25 ºC and 34, 28, 20 ppt; total of 9 treatment combinations) and CR determined. We found that regardless of salinity mussels exposed to 20 or 25ºC showed a 50% decrease in CR compared to the control. For instance, at control conditions the mean CR 1.97 L/min*g while at 20 ppt and 25ºC the CR was 0.72 L/min*g. This study mimics the potential effects of multiple stressors due to climate change and gives an idea on how a foundational species of marine ecosystem may be affected.

Increasing antifungal resistance and limited classes of antifungal drugs pose severe threats for the treatment of fungal infections caused by Candida species. Candida albicans, the most common fungal pathogen, is a common gut and oral opportunistic pathogen of the human microbiome capable of large chromosomal rearrangements that confer drug resistance. One chromosome rearrangement includes an amplification of the left arm of chromosome 5 (Chr5) in an isochromosome structure which promotes drug resistance due to the additional copies of two genes (ERG11 and TAC1). Our experiments have identified that the centromere of Chr5 (CEN5) is flanked by inverted repeat sequences and the CEN5 sequence can become inverted during isochromosome formation. To determine if the CEN5 inversion is independent of the isochromosome structure, 30 single colonies were isolated from a strain that underwent CEN5 inversion and acquired the isochromosome. Single colonies were plated on YPAD and YPAD with 1 microgram fluconazole, and both were incubated overnight. Small and large colonies were observed on both media after 48 hours. Six colonies of each size, small and large, were isolated from drug and non-drug plates. An additional six large colonies were isolated from YPAD. Genomic DNA was extracted and quantified using nano-drop and Qubit. PCR analysis was performed on CEN5 to determine the orientation of the centromere followed by whole genome sequencing to the absence of the isochromosome and inversion of CEN5. Additional phenotypic analyses were performed, including growth curve analysis in the presence and absence of the antifungal drug.