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.
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 specificity, 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.
pRS42K is a kind of shuttle vector which 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(α 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 the function of our device.
Group A: transformed CENPK2-1C(a type) and CENPK2-1D(α type)
Group B: transformed CENPK2-1C(a type) alone
Group C: CENPK2-1D(α 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.