Team:Tsinghua/Project-Switching-System

From 2013.igem.org

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<h1>PPD Switching System</h1>
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<h1>PPD Switching System</h1>
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<h2>Overview</h2>
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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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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.
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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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  We realized transactivating the reporter part by yeast mating.
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Overview
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<h2>PPD Transactivating by Yeast Mating</h2>
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Design
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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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Discussion
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<img class="center" src="https://static.igem.org/mediawiki/2013/3/37/Tsinghua-switch1.png"/>
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<div class="section section1">
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<h2>Overview</h2>
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<p>
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We connected the PPD sensor and reporter system by 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 trans-activating the reporter part by yeast mating.
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</p>
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<div class="figure">
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<img class="center" src="https://static.igem.org/mediawiki/2013/5/5e/Tsinghua-switch-1.png"/>
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    Figure 1. pTF5
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Figure 1. Overview of PPD switching system
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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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<h2>Design</h2>
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<img class="center" src="https://static.igem.org/mediawiki/2013/a/ae/Tsinghua-switch2.png"/>
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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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</p>
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<div class="figure">
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<img class="center" src="https://static.igem.org/mediawiki/2013/d/dc/Tsinghua-switch-2.png"/>
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    Figure 1. pTF7
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Figure 2. pTF5
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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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</p>
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<div class="figure">
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<img class="center" src="https://static.igem.org/mediawiki/2013/0/09/Tsinghua-switch-3.png"/>
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Figure 3. pTF7
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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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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.
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<div class="section section3">
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<h2>Discussion</h2>
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<p>
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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.
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<h2>Reference</h2>
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[1] F.Sherman et al. Getting Started with Yeast By Fred Sherman, 2003, MethodsEnzymol. 350, 3-41.
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Revision as of 19:23, 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
  • Design
  • Discussion

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

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.

Reference

[1] F.Sherman et al. Getting Started with Yeast By Fred Sherman, 2003, MethodsEnzymol. 350, 3-41.

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