Difference between revisions of "Team:BIT-China/Project/Detection"
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<p class="my-content-p">In order to find out whether the signal reporter works or not, we cultivate two kinds of haploid S.cerevisiae, Cen.PK2-1D(αtype) and Cen.PK2-1C(a type) together and observe them by fluorescence microscopy. The Cen.PK2-1D(a type) can excrete the a pheromone which can be detected by the pheromone receptor Ste2 positioning in the membrane of Cen.PK2-1C(a type). After detecting the signal, our reporter device will express RFP. Besides, we also use purified α pheromone to test our device’s function. </p> | <p class="my-content-p">In order to find out whether the signal reporter works or not, we cultivate two kinds of haploid S.cerevisiae, Cen.PK2-1D(αtype) and Cen.PK2-1C(a type) together and observe them by fluorescence microscopy. The Cen.PK2-1D(a type) can excrete the a pheromone which can be detected by the pheromone receptor Ste2 positioning in the membrane of Cen.PK2-1C(a type). After detecting the signal, our reporter device will express RFP. Besides, we also use purified α pheromone to test our device’s function. </p> | ||
| − | <p class="my-content- | + | <p class="my-content-li2">Group A: transformated CENPK2-1C(a type) and CENPK2-1D(a type)</p> |
| − | <p class="my-content- | + | <p class="my-content-li2">Group B: transformated CENPK2-1C(a type) alone</p> |
| − | <p class="my-content- | + | <p class="my-content-li2">Group C: CENPK2-1D(a type) alone</p> |
<div class="my-img-box"> | <div class="my-img-box"> | ||
Revision as of 14:39, 24 October 2017
Detection
The signal reporter device
To measure the sweetness of sweeteners, we design the signal reporter device. The device consists of a promoter pFUS, a reporter gene mRFP, and a terminator CYC1t.
When the human sweet receptor T1R2-T1R3 detects the sweeteners, the signal can be transmitted to this device through the MAP kinase pathway which exists in yeast naturally. And this pathway activates the promoter pFUS specificitly, thereby initiating the expression of the reporter gene.
In order to construct this device, first, we connect three parts (pFUS, mRFP, CYC1t) together by OE-PCR. But the result of this procedure is always fail. Then, we change to use Gibson assembly to connect this device with the linear plasmid pRS42K. pRS42K is a kind of shuttle vector using between E.coli and yeast. After finishing the construction in E.coli, we transformed the plasmid into competent cell Cen.PK2-1C, the mating a type haploid Saccharomyces cerevisiae.
In order to find out whether the signal reporter works or not, we cultivate two kinds of haploid S.cerevisiae, Cen.PK2-1D(αtype) and Cen.PK2-1C(a type) together and observe them by fluorescence microscopy. The Cen.PK2-1D(a type) can excrete the a pheromone which can be detected by the pheromone receptor Ste2 positioning in the membrane of Cen.PK2-1C(a type). After detecting the signal, our reporter device will express RFP. Besides, we also use purified α pheromone to test our device’s function.
Group A: transformated CENPK2-1C(a type) and CENPK2-1D(a type)
Group B: transformated CENPK2-1C(a type) alone
Group C: CENPK2-1D(a type) alone
After 9 hours, group A was fluorescent, and group B and group C didn’t fluoresce. The result means signal reporter device worked.
