Difference between revisions of "Team:BIT-China/Project/Sense"

Human sweet receptor T1R2-T1R3 heterologous expression

To synthesis the target gene, Human sweet receptor T1R2-T1R3, we searched gene from web site NCBI firstly, then we use the software Snapgene to optimize the sequence. The way we choose to synthesis the target gene is to use oligo design software OLIGO.

First we design 90 primers for each sweet receptor and each primer has 15bp overlap region with the adjacent one. Next we divided 90 primers into 3 groups，named block A、B and C，mix the primer of each group and made the final concentration to 10μm. We successfully get 6 desire fragments after PCR and purification.

To verify whether the heterodimer is successfully expressed and located at the certain position, we add different color protein as tags to each sweet receptor. After checking the part library，we get blue, yellow and green fluorescent tags. During the primer designing, we add another 20bp of N-terminal of each sweet receptor as the overlap region. So through OE-PCR we can link fluorescent tags to each sweet receptor.

Another way we design to verify whether the heterodimer is successfully expressed and located at the certain position is that to add different epitope tags instead the color protein to each sweet receptor. The reason we choose two different methods is that according to the protein translation process， add color protein on the N-terminal of the target tagging protein may not give color protein the enough time to fold as it’s functional state before it been secretion， thus a his tag along with antibody detection maybe a better solution.

In order to express the human receptor, we choose Saccharomyces cerevisiae strain Cen.PK2-1C as our host and the shuttle vector, pESC-Leu, to express T1R2-T1R3. We select two restrict sites for two receptor genes respectively. Then we use PCR to add BamHI and SalI to the T1R2 fragment. In the meantime, we add SpeI and NotI to T1R3 fragment. And the fragments with fluorescent tags or other tags also link to restrict sites through PCR.

After using T4 ligase for ligating the digested DNA fragments to the multi cloning sites of plasmid pESC-Leu, we transform the ligation production to the E.coli TOP 10 then select the positive colony using colony-PCR.

After finishing the construction of vector with sweetness receptor in E.coli, the recombination plasmid is transformed into yeast, Cen.PK2-1C with pFus-RFP-Cyc1t(G418 resistance vector)， through LiAc transformation. Because of the auxotrophic selection marker Ura in the pESC-Ura, the colony is chosen under the SD-Uracil defect medium with 0.3% G418. Then the positive cloning can survive in this medium. In this way, we can get the reconstruction Saccharomyces cerevisiae successfully.

Since the T1R2-T1R3 with fluorescent tags is designed to confirm the expression and location of T1R2-T1R3 in the Cen.PK2-1C, we test our yeast by fluorescence plate reader through the immunofluorescence technology. We use the minimal induction medium to induce the Gal 1/10 promoters and express their downstream gene then detect the fluorescence

Protocol for fluorescence test through fluorescence plate reader (Red fluorescence protein):