Date of Award
Santa Clara : Santa Clara University, 2016.
Synthetic circuits provide novel ways for scientists to program and probe mammalian cell behavior. This allows for enhanced research tools and chemical sensors. Our project works with an engineered synthetic circuit and monitors the output of the circuit using green fluorescent protein( GFP) and luciferase. Expression of GFP is both quantifiable and observable over a time period in living cells. Luciferase can be detected by an assay in lysed cells to give data on overall expression of the circuit. By measuring and comparing output of this circuit over time, we can create a model to demonstrate the lack of expression, minimal expression, and maximum expression of the circuit. Our dual reporter system can be adapted to give detailed information on various promoter elements of synthetic circuits.
This dual reporter system was designed to monitor s ignal pathways within mammalian cells. Signal pathways are a complex and dynamic interconnected web of protein expression. Methods to monitor these pathways en vivo poses a challenge due to lack of noninvasive reporting systems in desired cell models. The use of a dualreporter system with expression of GFP and firefly luciferase allows for the assessment of signal transduction with resulting data sets of overall intensity and temporality in realtime. Neither light emitting marker could show these two properties alone. To demonstrate this system and establish a scale of expression, we transformed mammalian HEK cells with a vector coding for the dual reporter. The transformed cell lines were monitored via fluorescent microscopy and luciferase assays. Our findings show successful basal level expression of synthetic circuit dualreporter system both qualitatively and quantitatively in a way that neither reporter protein could achieve alone. Our system can successfully deliver this data type and can be an effective tool for cell signal pathway expression.
Jhutty, Darisha and Parker, Nicholas, "Design and Validate a Synthetic Circuit for Detecting Pathway Signaling in Mammalian Cells" (2016). Bioengineering Senior Theses. 42.