Team:HIT-Harbin/Design

In order to make yeast be capable of detecting dioxins in the environment, we designed the circuit above. TEF is the constitutive promoter which can activate downstream sequences to express dual domain protein, namely, lexA-DBD/mDR83-805. mDR83-805 can express dioxin receptor hsp90. And after it combining intracellular dissociative dioxin, it will pass through karyotheca and combine with cyc1 promoter to activate the expression of green fluorescent protein downstream. Then we can know by observation whether there is dioxin in the environment or not.

Considering the amount of dioxin in the environment is in micro level or even trace level, when the concentration of dioxin decreases, those who can successfully combine with hsp90 and then nucleic DNA will be less. Thus, we added a feedback control to amplify the signal intensity.

So as to test the working state of promoter when there is no dioxin, we designed another circuit. We replaced TEF constitutive promoter by galactose induced promoter. mDR83-805 has also been modified. Its sequence of dioxin receptor was eliminated. When it is stimulated by galactose in the environment, the circuit will express the hsp90 without the ability to combine dioxin. This circuit has no integrated hsp90 which can unite with dioxin so that it will not be affected by dioxin. It is especially for testing whether LexA-DBD can still penetrate the nuclear membrane and combine with DNA and activate downstream sequences without signal of dioxin. It works as a contrast to correct the numbers.