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Extrachromosomal circular DNA (eccDNA) has been identified in a broad range of eukaryotes and have been shown to carry genes and regulatory sequences. Additionally, they can amplify within a cell by autonomous replication or reintegration into the genome, effectively influencing copy number in cells. This has significant implications for cancer, where oncogenes are frequently amplified on eccDNA. However, little is known about the exact molecular mechanisms governing eccDNA functionality. To this end, we constructed a fluorescent reporter at an eccDNA-prone locus of the yeast genome, CUP1. It is our hope that this reporter will contribute to a better understanding of eccDNA formation and amplification within a cell.
Protein and gene circuit level synthetic bioengineering can require years to develop a single target. Phage assisted continuous evolution (PACE) is a powerful new tool for rapidly engineering new genes and proteins, but the method requires an automated cell culture system, making it inaccessible to non industrial research programs. Complex protein functions, like specific binding, require similarly dynamic PACE selection that can be alternatively induced or suppressed, with heat labile chemicals like tetracycline. Selection conditions must be controlled continuously over days, with adjustments made every few minutes. To make PACE experiments accessible to the broader community, we designed dedicated cell culture hardware and integrated optogenetically controlled plasmids. The low cost and open source platform allows a user to conduct PACE with continuous monitoring and precise control of evolution using light.
Current advances in cellular models of neurodegenerative diseases overcome a variety of limitations possessed in animal and post-mortem human models. Human-induced pluripotent stem cells (hiPSCs) provide a platform with cells that can self-renew and differentiate into mature and functional cell types. HiPSCs are at the forefront of neurodegenerative disease research because of their ability to differentiate into neural cell types. Apolipoprotein E (ApoE) is a protein encoded by the APOE gene found on chromosome 19 of the human genome. There are three common polymorphisms in the APOE gene, resulting from a single amino acid change in the protein. The presence of these polymorphisms are studied as associated risk factors of developing AD. Different combinations of these alleles closely relate to the risk a patient has in developing Alzheimer’s disease. The risk associated effects of this gene are primarily investigated, however the protective effects are not examined to the same extent.
This research aims to overcome the existing limitations in cell differentiations and improve cell population purity that limits the variables present in the culture. To do this, this study optimized a differentiation protocol by separating and purifying neuronal cell populations to study the potential protective effects associated with ApoE, a risk factor seen in SAD. This platform aims to use a purified cell population to effectively analyze cell type specific affects of the ApoE risk factor, specifically in neurons.