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Team:Tsinghua/Project-Switching-System
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Revision as of 19:37, 27 September 2013
PPD Switching System
We integrated the PPD sensor and the PPD reporter system by Tet-off system. AHL induction activates the expression of tTA in PPD sensor system, after which tTA trans-activates the downstream PPD reporter ADE2, realizing the connection between input AHL signal and output ADE2 signal.
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
