Difference between revisions of "Team:BIT-China/Project/Detection"
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<p style="text-align: center">Fig.2 construction of detection circuit by Gibson assembly</p> | <p style="text-align: center">Fig.2 construction of detection circuit by Gibson assembly</p> | ||
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</div> | </div> | ||
| + | <p class="my-content-p">pRS42K is a kind of shuttle vector can be used both in <i>E.coli</i> and yeast. After finishing the construction in <i>E.coli</i>, we transformed the plasmid into competent cell </p> | ||
| + | <p class="my-content-p"><i>CEN.PK2-1C</i> by LiAc transformation.</p> | ||
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| + | <p class="my-content-p">In order to make sure the detection circuit works, we cultivated two types of haploids <i>S.cerevisiae</i>, <i>CEN.PK2-1D</i>(α type) and <i>CEN.PK2-1C</i>(a type) together and observed them by fluorescence microscopy. The <i>CEN.PK2-1D</i>(a type) can excrete a pheromone which can be detected by the pheromone receptor <i>Ste2</i> positioning on the membrane of <i>CEN.PK2-1C</i>(a type). After detecting the signal, our reporter device will express <i>mRFP</i>. Besides, we also used purified α pheromone to test our device’s function. </p> | ||
<p class="my-content-li2">Group A: transformated CENPK2-1C(a type) and CENPK2-1D(a type)</p> | <p class="my-content-li2">Group A: transformated CENPK2-1C(a type) and CENPK2-1D(a type)</p> | ||
<p class="my-content-li2">Group B: transformated CENPK2-1C(a type) alone</p> | <p class="my-content-li2">Group B: transformated CENPK2-1C(a type) alone</p> | ||
Revision as of 09:40, 27 October 2017
Detection
the detection circuit
To measure the sweetness of sweeteners, we designed the detection device consisting of the promoter Pfus, the reporter gene mRFP, and the terminator CYC1t.
Fig.1 The detection circuit
The upstream signal will be produced when the human sweet taste receptor T1R2-T1R3 detects sweeteners, the signal can be transmitted to detection device through the MAPK pathway which exists in yeast naturally. And this signal will activate the promoter Pfus specificitly, thereby initiating the expression of the mRFP reporter gene.
In order to construct the detection circuit, firstly, we connected the three parts Pfus , mRFP, and CYC1t by OE-PCR. However, the results were not as good as we expected. So we changed the method to Gibson assembly to connect this device with the linear plasmid pRS42K.
Fig.2 construction of detection circuit by Gibson assembly
pRS42K is a kind of shuttle vector can be used both in E.coli and yeast. After finishing the construction in E.coli, we transformed the plasmid into competent cell
CEN.PK2-1C by LiAc transformation.
In order to make sure the detection circuit works, we cultivated two types of haploids S.cerevisiae, CEN.PK2-1D(α type) and CEN.PK2-1C(a type) together and observed them by fluorescence microscopy. The CEN.PK2-1D(a type) can excrete a pheromone which can be detected by the pheromone receptor Ste2 positioning on the membrane of CEN.PK2-1C(a type). After detecting the signal, our reporter device will express mRFP. Besides, we also used 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.
