Difference between revisions of "Team:Scut-Champion-Park/Achievement/Results"

We design the primer by the software primer5.0, get the rtTA gene from pTet-On plasmid and add EcoR1 and Not1 sites at both ends respectively. The primer is as below:

To ensure the success of the experiment, we remodeled the Plasmid. We link the rtTA or rtTA-flag with Restriction Enzyme cutting site to the Plasmid pGAPZB. Construct the Plasmids pGAPZB-rtTA and pGAPZB-rtTA-flag.

The picture on the left shows the PCR result, and the right shows the identification result of double enzymes restriction. They indicate that pGAPZB-rtTA and pGAPZB-rtTA-flag are constructed correctly. The fragment size is right.

The two plasmids are led to the GS115 bacterial strain which are HIS auxotroph. Under The effect of GAP promoter, The bacterial can produce rtTA protein. In other word, we construct the regulation system in yeast and screen by resistant plate. The picture below is the positive transformation.

On the base of pTRE2, we determine the modification conditions of Tet-responsive promoter by correlated references. Use the CYC1TATA frame structure in yeast and construct the TRE-CYC1TATA promoter by fusion PCR. The promoter can sensitively regulate the Tet-On system in yeast. Link the Resistance selection markers such as HIS4 and Kanamycin by clone, we construct the pPICTC-EGFP Plasmid.

The E.coli positive transformation is as below:

Lead the pPICTC-EGFP Plasmid into the competent Yeast with pGAPZB-rtTA or pGAPZB-rtTA-flag to complete the construction of recombinant yeast.

Prepare recombinant yeast by chemical approach. Transform the yeast by electroporation transfection.
Transformation steps:
1) Prepare recombinant yeast by chemical approach, 80μL/tube, use it immediately or conserve it at -80℃.
2) Add 0.1~0.2mg linearization recombinant plasmid into the recombinant yeast cells, then take them in the 0.2cm electric shock cup in the ice-bath for 5min.
3) Electric shock it by pulse cell transfection system at 1.5Kv, 200Ω, 25mF, 5ms.
4) Add 1ml 1mol/L sorbitol taken from ice-bath into the mixed liquor after electric shock. Take it to 1.5ml centrifuge tube and wait for 1.5h at 30℃.
5) Take 200μl bacterium solution, smear it on the Resistance or auxotroph plate to screen.

Principle of electrotransformation. We lead the linearization plasmid into the inner of yeast cell. The linearization plasmid will occur homologous recombination with the genome of yeast to lead our target gene into the genome of yeast. We can screen the transformant by cultivate the cell on resistance or auxotroph plate.

The E.coli positive transformation is as below: