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 <b>PPD sensor</b> and the <b>PPD reporter</b> system by <b>Tet-off system</b>. <b>AHL</b> induction activates the expression of <b>tTA</b> in PPD sensor system, after which tTA trans-activates the downstream PPD reporter <b>ADE2</b>, 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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<h2>Overview</h2>
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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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<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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<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>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.</p>
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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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Latest revision as of 20:05, 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

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.

Reference

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

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