Overview

We connected the PPD sensor and reporter system by Tet-off system.

We transformed the newly constructed PPD sensor and the PPD reporter in to a and α haploid yeasts respectively.

We realized trans-activating the reporter part by yeast mating.

Figure 1. Overview of PPD switching system

Design

In order to link the sensor and reporter system by Tet-off system. We replace the mCherry in pTF4 by TetR-VP16. The expression of TetR-VP16 was under the control of cyc100 mini promoter. We constructed pTF5 plasmid.

Figure 2. pTF5

After demonstration of feasibility of pTF6, we created a reporter plasmid in pRS415. pRS415 is a single copy plasmid with centrosome used in leucine auxotrophic yeast. The plasmid fragment from TRE to CYC1 terminator was inserted in to pRS415. We got pTF7 plasmid.

Figure 3. pTF7

Discussion

Using the ADE2 KO strain and ADE2 gene we successfully tested that the tet-off system worked as expected. In order to rule out the possibility that the color change was the result of cyc1 mini promoter linkage, we can use two methods. We can add the Doxycycline into the plasmid or delete the tTA region. If the red color sustains, it will be verified that the system works as expected.