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Team:HIT-Harbin/Wetlab
From 2014.igem.org
Control Group
So as to test the working state of promoter when there is no dioxin, we designed this control circuit showed above. We replaced mdr83-805 by mdr521-805. Its sequence of dioxin receptor was eliminated. When it is stimulated by galactose in the environment, the circuit will express the DNA binding sequence which is independent of dioxin. It is especially for testing whether mdr521-805 can trigger transmembrane transportation. What’s more, Its localization effect as well as zero correction function can be tested.
Experimental Group
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/mDR83-80. Comparing with other amplifiers, lexA can rapidly and efficiently induce the expression of downstream gene. At the meantime, we designed and applied the operator-lexop which possesses eight LEXAdbd binding sites, thus the catalytic effect of lexA operator is further improved. mDR83-805 can express mdr83-805（mouse dioxin receptor）. And after it combining intracellular dissociative dioxin, it will pass through karyotheca and combine with lexA operator to switch up downstream promoters and then 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 fusion protein lexadbd/mdr83-805 and then nucleic DNA will be less. Thus, we added a memory system to amplify the signal intensity by positive feedback regulation.
When there is a trace of dioxin, with the assistance of native intercellular hsp90 and arnt, the combination of fusion protein lexadbd/mdr83-805 and dioxin will activate the expression of florescent protein. Lex-A-DBD/mDR521-805 depicted in the picture is also dual domain protein but without binding site of dioxin hence cannot combine with dioxin. However, it can still pass through the nuclear membrane and combine with DNA to activate the expression of GFP. Such signal of the trace of dioxin can lead to the expression of a lot of GFP by amplification.
