Team:Tsinghua/Project-Reporter

From 2013.igem.org

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<h1>PPD Reporter</h1>
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<h1>PPD Reporter</h1>
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<h2>Overview</h2>
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<div id="brief">
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<p>
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<p>
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  We created the output test part of portable pathogen detector using Tet-off system.
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We designed a PPD reporter system using anto-induced Tet-off system, in which tTA was constitutively expressed. Expressed tTA activated the expression of downstream reporter gene ADE2, which changed the yeast from red to white.
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</p>
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  We successfully demonstrated the effectiveness of transactivating function of tTA.  
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<ul>
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<p>
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<li class="section1">
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  We reconstituted the ADE2 gene expression in ADE2 KO yeast, switching from red to white color.
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Overview
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  </p>
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</li>
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<h2>Designed PPD reportor</h2>
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<li class="section2">
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<p>
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Design
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  We created a reporter as the output part of the PPD system. So we need a trans-activating system to realize releasing output signal. Among the available systems, we chose Tet-off system.
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</li>
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  </p>
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<li class="section3">
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<div class="figure">
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Experiment and Result
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<img class="center" src="https://static.igem.org/mediawiki/2013/0/0f/Tsinghua-reporter1.png"/>
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</li>
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<li class="section4">
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Discussion
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</ul>
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</div>
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<div class="section section1">
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<h2>Overview</h2>
 +
<p>
 +
We created the reporter part of portable pathogen detector using auto-induced Tet-off system.
 +
</p>
 +
<p>
 +
We successfully demonstrated the effectiveness of trans-activating function of tTA.
 +
</p>
 +
<p>
 +
We reconstituted the ADE2 gene expression in ADE2 knock out yeast.
 +
</p>
 +
<div class="figure">
 +
<img class="center" src="https://static.igem.org/mediawiki/2013/7/7d/Tsinghua-reporter-1.png"/>
<p class="legend">
<p class="legend">
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    Figure 1.
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Figure 1. Overview of PPD reporter
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    </p>
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</p>
</div>
</div>
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<p>
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</div>
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  At first, we design a test plasmid to determine the feasibility of applying Tet-off system in our system. After AHL inducing, a tetracycline-controlled transactivator (comprising a fusion of the tetracycline repressor TetR with the C-terminal activation domain of herpes simplex virus VP16) will be expressed theoretically. So we constitutively express tTA under the control of CMV enhancer and CMV promoter in pTF6.
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-
  </p>
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<div class="section section2">
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<p>
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<h2>Designed PPD reporter</h2>
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  In another ORF, we constructed a tetracycline response element followed by CYC1 region (TATA region), which controls the expression ADE2 gene. After ADE2 gene, a CYC1 terminator is used to terminate the transcription of mRNA.
+
<p>
-
  </p>
+
We created a reporter as the reporter of the PPD system. So we need a trans-activating system to achieve communication between the sensor and the reporter system. Among the available systems, we chose Tet-off system.
-
<p>
+
</p>
-
  ADE2 protein is phosphoribosylaminoimidazole carboxylase, which is required for adenine biosynthesis. When yeast loses ADE2 gene, the cells are in red color, because of deprivation of adenine, while the wild type yeast in in white. If the plasmid is transformed into ADE2 knock out yeast strain, TetR domain of tTA will bind to the TRE and VP16 will activate the expression of ADE2, which can reconstitute the phenotype of ADE2 knock out strain, turning the yeast from red to white.
+
<div class="figure">
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  </p>
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<img class="center" src="https://static.igem.org/mediawiki/2013/9/9b/Tsinghua-reporter-2.png"/>
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<h2>Result</h2>
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<p>
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  We constructed the reporter plasmid from cm184 plasmid. We insert ADE2 PCR product into cm184 vector, getting pTF6.  
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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/a/ad/Tsinghua-reporter2.png"/>
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<p class="legend">
<p class="legend">
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    Figure 2. pCM184
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Figure 2. Principle of Tet-off system
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    </p>
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</p>
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</div>
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<p>
 +
At first, we design a test plasmid to determine the feasibility of applying Tet-off system in our system. After AHL inducing, a tetracycline-controlled transactivator (comprising a fusion of the tetracycline repressor TetR with the C-terminal activation domain of herpes simplex virus VP16) will be expressed theoretically. So we constitutively express tTA under the control of CMV enhancer and CMV promoter in pTF6.
 +
</p>
 +
<p>
 +
In another ORF, we constructed a tetracycline response element followed by CYC1 region (TATA region), which controls the expression ADE2 gene. After ADE2 gene, a CYC1 terminator is used to terminate the transcription of mRNA.
 +
</p>
 +
<p>
 +
ADE2 protein is phosphoribosylaminoimidazole carboxylase, which is required for adenine biosynthesis. When yeast loses ADE2 gene, the cells are in red color, because of deprivation of adenine, while the wild type yeast is in white. If the plasmid is transformed into ADE2 knock out yeast strain, TetR domain of tTA will bind to the TRE and VP16 will activate the expression of ADE2, which can reconstitute the phenotype of ADE2 knock out strain, turning the yeast from red to white.
 +
</p>
 +
</div>
 +
 +
<div class="section section3">
 +
<h2>Experiments and Results</h2>
 +
<p>
 +
We constructed the reporter plasmid from cm184 plasmid. We insert ADE2 PCR product into cm184 vector, getting pTF6.
 +
</p>
 +
<div class="figure">
 +
<img class="center" src="https://static.igem.org/mediawiki/2013/1/14/Tsinghua-reporter-3.png"/>
 +
<p class="legend">
 +
Figure 3. pCM184
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</p>
</div>
</div>
<div class="figure">
<div class="figure">
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<img class="center" src="https://static.igem.org/mediawiki/2013/8/8d/Tsinghua-reporter3.png"/>
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<img class="center" src="https://static.igem.org/mediawiki/2013/8/80/Tsinghua-reporter-4.png"/>
<p class="legend">
<p class="legend">
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    Figure 3. pTF6
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Figure 4. pTF6
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    </p>
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</p>
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</div>
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<p>
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<p>
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  We tested the function of pTF6 in ADE2 knock out yeast strain. We observed an obvious color change. We used cm184 transformation group as control.
+
We tested the function of pTF6 in ADE2 knock out yeast strain. We observed an obvious color change. We used cm184 transformation group as control.
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  </p>
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</p>
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<div class="figure">
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<div class="figure">
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<img class="center" src="https://static.igem.org/mediawiki/2013/a/a1/Tsinghua-reporter4.png"/>
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<img class="center" src="https://static.igem.org/mediawiki/2013/4/43/Tsinghua-reporter-5.png"/>
<p class="legend">
<p class="legend">
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    Figure 4. PPD reporter gene ADE2 expression reconstitute the white phenotype of ADE2 KO yeast
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Figure 5. PPD reporter gene ADE2 expression reconstitute the white phenotype of ADE2 KO yeast
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    </p>
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</p>
</div>
</div>
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<p>
 +
The expression of ADE2 changed the color of yeast from red to white thoroughly, which indicated that expressed tTA could activate ADE2 expression. If we extend this system in our pathogen detector machine, the trans-activating efficiency of Tet-off system can be expected.
 +
</p>
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</div>
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<div class="section section4">
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</div>
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<div id="references">
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<h2>Reference</h2>
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[1] F. Sherman et al. Getting Started with Yeast By Fred Sherman, 2003, Methods Enzymol. 350, 3-41.
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Revision as of 19:15, 27 September 2013

PPD Reporter

We designed a PPD reporter system using anto-induced Tet-off system, in which tTA was constitutively expressed. Expressed tTA activated the expression of downstream reporter gene ADE2, which changed the yeast from red to white.

  • Overview
  • Design
  • Experiment and Result
  • Discussion

Overview

We created the reporter part of portable pathogen detector using auto-induced Tet-off system.

We successfully demonstrated the effectiveness of trans-activating function of tTA.

We reconstituted the ADE2 gene expression in ADE2 knock out yeast.

Figure 1. Overview of PPD reporter

Designed PPD reporter

We created a reporter as the reporter of the PPD system. So we need a trans-activating system to achieve communication between the sensor and the reporter system. Among the available systems, we chose Tet-off system.

Figure 2. Principle of Tet-off system

At first, we design a test plasmid to determine the feasibility of applying Tet-off system in our system. After AHL inducing, a tetracycline-controlled transactivator (comprising a fusion of the tetracycline repressor TetR with the C-terminal activation domain of herpes simplex virus VP16) will be expressed theoretically. So we constitutively express tTA under the control of CMV enhancer and CMV promoter in pTF6.

In another ORF, we constructed a tetracycline response element followed by CYC1 region (TATA region), which controls the expression ADE2 gene. After ADE2 gene, a CYC1 terminator is used to terminate the transcription of mRNA.

ADE2 protein is phosphoribosylaminoimidazole carboxylase, which is required for adenine biosynthesis. When yeast loses ADE2 gene, the cells are in red color, because of deprivation of adenine, while the wild type yeast is in white. If the plasmid is transformed into ADE2 knock out yeast strain, TetR domain of tTA will bind to the TRE and VP16 will activate the expression of ADE2, which can reconstitute the phenotype of ADE2 knock out strain, turning the yeast from red to white.

Experiments and Results

We constructed the reporter plasmid from cm184 plasmid. We insert ADE2 PCR product into cm184 vector, getting pTF6.

Figure 3. pCM184

Figure 4. pTF6

We tested the function of pTF6 in ADE2 knock out yeast strain. We observed an obvious color change. We used cm184 transformation group as control.

Figure 5. PPD reporter gene ADE2 expression reconstitute the white phenotype of ADE2 KO yeast

The expression of ADE2 changed the color of yeast from red to white thoroughly, which indicated that expressed tTA could activate ADE2 expression. If we extend this system in our pathogen detector machine, the trans-activating efficiency of Tet-off system can be expected.

Reference

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

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