Team:BIT-China/Results
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 yno1 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.
The red curve indicates the fluorescence ratio at 488 nm (reduced state) and 405 nm (oxidized state) of engineered yeast which overexpressed Yno1 and constructed the plasmid pESC-TEF1p-roGFP2-Orp1-CYC1t. And the blue curve indicates the fluorescence ratio at 488 nm (reduced state) and 405 nm (oxidized state) of engineered yeast which only overexpressed Yno1. The red curve decline over time but not the blue one, which means roGFP2-Orp1 protein is able to show the accumulation of ROS in our yeast.
Since we have proved the feasibility that the antioxidative ability of antioxidant can be measured by fluorescence probes. We can easily set up the relationship between our system and real antioxidative ability of antioxidant.
After the whole system construction, we need to test the function of our system and then evaluate the effect of antioxidants through our system.
To detect the antioxidant capacity of antioxidants, we have selected three antioxidants, Vitamin C, Quercetin and Catechol, as our samples, and cultured engineered strain (Δyca1-yno1-roGFP2-Orp1) until the period with high ROS accumulation (24-36 h). Then the antioxidants were added into yeast culture and incubated for 30 minutes. The control group was treated with aseptic water and ethanol. The fluorescence intensity ratio (I488/I408 or I405/I488) of the reduction peak (488nm) and oxidation peak (405nm) was measured by Microplate Reader, and the antioxidant activity of antioxidants was characterized by that ratio change rate. And the emission wavelength is 510nm. The results are showed in Fig. 2.
Low fluorescence ratio (I488/405) means low intracellular ROS level.
High fluorescence ratio (I488/405) means high antioxidant capacity.
As Fig.2~3 shown, in 30 mins, group Catechol and group Quercetin both show a decreasing in I405/I488 ratio (Fig.2), which means that Quercetin and Catechol have antioxidant activity. Besides, the Quercetin treated group shows a faster decreasing in I405/I488 ratio than Catechol under the same condition of time. According to our modeling, this indicates that the Quercetin have more antioxidant activity than Catechol, which is consistent with CAA assay result. (Wolfe K L, Liu R H et al.)[1]
But Vitamin C is less effective and had no obvious antioxidant effect in this assay. We thought this result may had two reasons. First, the Vitamin C shows a low direct-antioxidant activity, especially at low concentration (10 µM) compared to another two. Or the Vitamin C main principle of anti-oxidation is its indirect-antioxidant activity, like activation of cell's natural anti-oxidative enzymes, which is hard to reflect in fluorescence ratio level just in 30 mins.
In a word, our system gave a similar result to CAA assay in three selected antioxidant tests. This proves our system can detect the antioxidant in living cell. But we need more antioxidant test to verify our system's function and limitation, especially indirect-antioxidative antioxidant, for finding "Who can get an A?"
