Team:BIT-China/Demonstrate
Our engineered yeast should finally have the function of monitoring antioxidants in real time. To demonstrate this, we integrated the two subsystems together into ∆yca1 yeast and had series of tests on it, in which the increasing of ROS in yeast can be effectively captured by our roGFP2-Orp1 protein.
In order to regulate endogenous ROS, we replaced yeast endogenous YNO1 promotor with a galactose-inducible promotor, GAL1-GAL10. We then constructed the plasmid pESC-TEF1p-roGFP2-Orp1-CYC1t, a shuttle vector marked with trp1. Cellular redox status were monitored by measuring the fluorescence ratio at 488 nm(reduced state) and 405 nm(oxidized state). Cells were cultured in SD medium supplemented with 1% galactose at 30 ℃, during which GAL1-GAL10 promotor was induced when galactose added. It suggests that overexpression of yno1yno1 gene will increase cells ROS level and decline the fluorescence ratio at 488 nm and 405 nm, which means there are more ROS in our modified strain.
As the results showing, our output subsystem can reflect the accumulation of endogenous ROS caused by regulator subsystem(figure). A simple decription.
Since we have proved the feasibility that the antioxidative ability of antioxidant can be measured by fluorescence probes(6.3.4). We can easily set up the relationship between our system and real antioxidative ability of antioxidant. However, due to time limitation, the final results will only be shown during the Giant Jamboree. Our presentation will on Saturday 3:15 PM - 3:45 PM, Room 312 and our poster will at Zone 4 - #235 and welcome to us to see our great work!
