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

We can transformed the pTF5 into a type haploid yeast and pTF7 into α type haploid yeast. We let them mate and fuse into a diploid yeast. After AHL inducing, the tTA expressed by the pTF5 trans-activates the expression of ADE2 on pTF7, causing a color change of the yeast.

Discussion

The mating process is relatively efficient. Different combination of haploids is available using auxotrophic strains of yeast. However, it is still to be improved if we can shorten the time of mating. It will further increase the productivity in real application. Moreover, we plan to repeat the mating process in liquid condition, which is advantageous for more efficient mating. This will be tested by mixing haploid yeasts in YPD medium.