Team:Tsinghua/Project-Switching-System

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<h1>PPD Switching System</h1>
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<h2>Overview</h2>
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<p>
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  We reconstructed the PPD sensor and reporter system by linking the two system with Tet-off system.
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  </p>
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<p>
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  We transformed the newly constructed PPD sensor and the PPD reporter in to a and α haploid yeasts respectively.
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  </p>
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<p>
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  We realized transactivating the reporter part by yeast mating.
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  </p>
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<h2>PPD Transactivating by Yeast Mating</h2>
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<p>
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  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.
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<img class="center" src="https://static.igem.org/mediawiki/2013/3/37/Tsinghua-switch1.png"/>
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    Figure 1. pTF5
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    </p>
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<p>
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  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.
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<img class="center" src="https://static.igem.org/mediawiki/2013/a/ae/Tsinghua-switch2.png"/>
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    Figure 1. pTF7
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    </p>
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</div>
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<p>
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  Ideally, 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 transactivates the expression of ADE2 on pTF7, causing a color change of the yeast, which can be used as a reporter of the whole PPD system.
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Revision as of 04:11, 26 September 2013

PPD Switching System

Overview

We reconstructed the PPD sensor and reporter system by linking the two system with Tet-off system.

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

We realized transactivating the reporter part by yeast mating.

PPD Transactivating by Yeast Mating

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 1. 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 1. pTF7

Ideally, 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 transactivates the expression of ADE2 on pTF7, causing a color change of the yeast, which can be used as a reporter of the whole PPD system.

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