Difference between revisions of "Team:ZJU-China/Demonstrate"

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{{ZJU-China}}
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<div class="column full_size judges-will-not-evaluate">
 
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<h3>★  ALERT! </h3>
 
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<p>This page is used by the judges to evaluate your team for the <a href="https://2017.igem.org/Judging/Medals">medal criterion</a> or <a href="https://2017.igem.org/Judging/Awards"> award listed above</a>. </p>
 
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<p> Delete this box in order to be evaluated for this medal criterion and/or award. See more information at <a href="https://2017.igem.org/Judging/Pages_for_Awards"> Instructions for Pages for awards</a>.</p>
 
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<h1>Demonstrate</h1>
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<h3>Gold Medal Criterion #4</h3>
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<p>
 
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Teams that can show their system working under real world conditions are usually good at impressing the judges in iGEM. To achieve gold medal criterion #4, convince the judges that your project works. There are many ways in which your project working could be demonstrated, so there is more than one way to meet this requirement. This gold medal criterion was introduced in 2016, so check our what 2016 teams did to achieve a their gold medals!
 
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Please see the <a href="https://2017.igem.org/Judging/Medals">2017 Medals Page</a> for more information.
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<h4> What should we do for our demonstration?</h4>
 
  
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<h5> Standard teams </h5>
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If you have built a proof of concept system, you can demonstrate it working under real world conditions. If you have built a biological device that is intended to be a sensor, can you show it detecting whatever it is intended to sense. If it is intended to work in the field, you can show how this might work using a simulated version in the lab, or a simulation of your device in the field.<strong> Please note biological materials must not be taken out of the lab</strong>.
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<h5> Special track teams </h5>
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Special track teams can achieve this medal criterion by bringing their work to the Jamboree and showcasing it in the track event. Art & Design, Measurement, Hardware and Software tracks will all have showcase events at the Giant Jamboree.<strong> Please note biological materials must not be taken out of the lab</strong>.
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                      <li class="m_nav_item dropdown">
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                          <a href="#" class="dropdown-toggle link" data-toggle="dropdown">Overview<b class="caret"></b></a>
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                          <ul class="dropdown-menu ">
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Overview">Description</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Demonstrate">Demonstrate</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Applied_Design">Applied Design</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Achievements">Achievements</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Improve">Improve Parts</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/InterLab">InterLab</a></li>
 +
 +
                          </ul>
 +
                      </li>
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                      <li class="m_nav_item dropdown">
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                          <a href="#" class="dropdown-toggle link" data-toggle="dropdown">Project<b class="caret"></b></a>
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                          <ul class="dropdown-menu ">
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Project/tp">Trichoderma Proof</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Project/voc">VOC sensors</a></li>
 +
                              <li><a style="font-size: 0.7em!important;" href="https://2017.igem.org/Team:ZJU-China/Project/st">Chemical Signal Transduction</a></li>
 +
                              <li><a style="font-size: 0.7em!important;" href="https://2017.igem.org/Team:ZJU-China/Project/mt">Medium Wave Transduction</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Project/Downstream">Downstream</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Project/conclusion">Conclusions</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Notebook">Notebook</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Protocols">Protocols</a></li>
 +
                          </ul>
 +
                      </li>
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        <h1 id="demonstrate" class="page-header ArticleHead GreenAH">Demonstrate</h1>
 +
 +
        <h2 id="overview" class="H2Head">Overview</h2>
 +
        <p class="PP">The general functions of our project:</p>
 +
        <p class="PP Retract"><strong>Detecting the phytopathogens or the unhealthy situation of the plant by Trichoderma atroviride or our device.</strong></p>
 +
        <p class="PP Retract"><strong>Signal transduction and amplification</strong></p>
 +
        <p class="PP Retract"><strong>Expression of downstream genes<br><br></strong></p>
 +
 +
        <p id="meetactivate" class="PP" style="border-top: 2px solid #00838F !important"><strong><br><br>When our engineered Trichoderma atroviride meets  phytopathogens, (take Phytophthora nicotianae as an example), some of report genes will be activated and give warning to our device.</strong></p>
 +
        <h3 id="how0" class="H3Head">How we prove it?</h3>
 +
        <p class="PP">☑︎Cloned the ech42 promoter (the promoter can be elicited when Trichoderma atroviride meets phytopathogens) from Trichoderma atroviride and performed confrontational coculture to test its phytopathogen sensitivity.</p>
 +
 +
        <div class="imgdiv"><img style="height: 370px !important; width: auto !important;"  src="https://static.igem.org/mediawiki/2017/3/36/ZJU_China_tp_ech42.jpg"></div>
 +
        <p class="capture">Fig.1 The relative fluorescent intensity of the hyphae contain the plasmids within Pech42<br></p>
 +
        <div class="imgdiv col-md-12"><img class="textimg" src="https://static.igem.org/mediawiki/2017/c/c7/ZJU_China_Project_TP_P3.png"></div>
 +
        <p class="capture">Fig.2 The fluorescence variation before and after activating the ech42 promoter
 +
            <br></p>
 +
 +
        <p class="PP" id="changedetect"><strong>Our device detects the change of VOC(Volatile Organic Compounds) released by plants and estimate whether our plants are infected.</strong></p>
 +
 +
        <h3 id="how1" class="H3Head">How to prove it?</h3>
 +
        <p class="PP"><strong>☑︎    </strong>We have constructed a classification model which can tell the health situation from the VOC they released.</p>
 +
 +
        <div style="text-align: center">
 +
            <a class="CuteButton YellowCB" href="https://2017.igem.org/Team:ZJU-China/Project/mt">More About Model...</a>
 +
        </div>
 +
        <br><br>
 +
 +
        <p class="PP" id="devicetransmit" style="border-top: 2px solid #00838F !important"><strong><br><br>Once the device can transmit the order to our engineered Trichoderma atroviride with chemical or electromagnetic signals.</strong></p>
 +
 +
        <h3 id="cs1" class="H3Head">Chemical signals：</h3>
 +
        <p class="PP"><strong>☑︎    </strong>Cloned the phlABCD cluster and carried out the bio-synthesis of DAPG in E.coli.</p>
 +
        <p class="PP"><strong>☑︎    </strong>Constructed the plasmids for DAPG bio-synthesis in Saccharomyces cerevisiae and Trichoderma atroviride.</p>
 +
        <p class="PP"><strong>☑︎    </strong>Constructed pho promoter and expressed phlF repressor.</p>
 +
 +
        <div class="imgdiv col-md-6"><img style="height: 200px !important; width: auto !important;"  src="https://static.igem.org/mediawiki/2017/4/4c/ZJU_China_best_composite_2.jpg"></div>
 +
        <div class="imgdiv col-md-6"><img style="height: 200px !important; width: auto !important;"  src="https://static.igem.org/mediawiki/2017/b/b0/ZJU_China_demonstrate_hh.png"></div>
 +
        <p class="capture">Fig.3 HPLC result of DAPG bio-synthesis in E.coli | Fig.4 Western blot of phlF protein<br></p>
 +
 +
        <h3 id="fw1" class="H3Head">Future work</h3>
 +
        <p class="PP"><strong>☐︎    </strong>Test pho-phlF system with DAPG</p>
 +
        <p class="PP"><strong>☐︎    </strong>Tested the function of phlABCD in Saccharomyces cerevisiae and Trichoderma atroviride and detect the bio-synthesis of DAPG.</p>
 +
 +
 +
        <h3 id="es1" class="H3Head">Electromagnetic signals</h3>
 +
        <p class="PP"><strong>☑︎    </strong>Expressed TRPV-Ferritin in Saccharomyces cerevisiae and tested the its function with heat shock and capsaicin.</p>
 +
        <p class="PP"><strong>☑︎    </strong>Constructed Pcdre-mRFP in Saccharomyces cerevisiae and measured the relative intracellular calcium content needed to activate CDRE promoter.</p>
 +
        <p class="PP"><strong>☑︎    </strong>Proved that the calcium influx induced by TRPV1 is strong enough to activate CDRE promoter.</p>
 +
 +
        <div class="imgdiv col-md-6"><img style="height: 300px !important; width: auto !important;"  src="https://static.igem.org/mediawiki/2017/3/3a/ZJU_China_MWF_fig9.jpeg"></div>
 +
        <div class="imgdiv col-md-6"><img style="height: 300px !important; width: auto !important;"  src="https://static.igem.org/mediawiki/2017/1/19/ZJU_China_MWF_Rplot05.jpeg"></div>
 +
        <p class="capture"> Fig.5 Relative calcium content of differet groups | Fig.6 Relative fluorescent intensity of two groups<br></p>
 +
 +
        <h3 id="fw2" class="H3Head">Future work</h3>
 +
        <p class="PP"><strong>☐︎    </strong>Test TRPV1-Ferritin system with medium radio frequencies in Saccharomyces cerevisiae.</p>
 +
        <p class="PP"><strong>☐︎    </strong>Construct TRPV1-Ferritin-CDRE system in Saccharomyces cerevisiae and Trichoderma atroviride.<br><br></p>
 +
 +
 +
        <p class="PP" id="receiveexpress" style="border-top: 2px solid #00838F !important"><br><br><strong>Once our Trichoderma atroviride has received the signal, the downstream gene will be expressed.</strong></p>
 +
 +
        <h3 id="how2" class="H3Head">How to prove it?</h3>
 +
        <p class="PP"><strong>☑︎    </strong>We managed to express a special Serine protases in Saccharomyces cerevisiae and tested its activity.</p>
 +
 +
        <div class="imgdiv"><img class="textimg" src="https://static.igem.org/mediawiki/2017/f/fb/ZJU_China_Design5.png"></div>
 +
        <p class="capture"> Fig.7 The values of OD253 increased with time<br></p>
 +
        <div class="imgdiv"><img class="textimg" style="width: 50% !important;" src="https://static.igem.org/mediawiki/2017/7/71/ZJU_China_Design6.png"></div>
 +
        <p class="capture">Fig.8 The values of OD253 increased with time and this solution did not add the PMSF.</p>
 +
        <p class="capture">These were the OD253 of the solutions after a period of reaction.The red one was the solution that did not add the PMSF; the blue one was the solution that added. Obviously, the OD253 of the former one is higher than the later one, so that, we could say that the yeast produced the serine protase successfully and effectively.</p>
 +
        <h3 id="fw3" class="H3Head">Future work</h3>
 +
        <p class="PP"><strong>☐︎    </strong>Express this serine protase in <em>T.atroviride</em>.</p>
 +
        <p class="PP"><strong>☐︎    </strong>Search and express more downstream genes to equip our <em>T.atroviride</em> with more functions.</p>
 +
 +
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Latest revision as of 16:23, 1 November 2017

"

Demonstrate

Overview

The general functions of our project:

Detecting the phytopathogens or the unhealthy situation of the plant by Trichoderma atroviride or our device.

Signal transduction and amplification

Expression of downstream genes



When our engineered Trichoderma atroviride meets phytopathogens, (take Phytophthora nicotianae as an example), some of report genes will be activated and give warning to our device.

How we prove it?

☑︎Cloned the ech42 promoter (the promoter can be elicited when Trichoderma atroviride meets phytopathogens) from Trichoderma atroviride and performed confrontational coculture to test its phytopathogen sensitivity.

Fig.1 The relative fluorescent intensity of the hyphae contain the plasmids within Pech42

Fig.2 The fluorescence variation before and after activating the ech42 promoter

Our device detects the change of VOC(Volatile Organic Compounds) released by plants and estimate whether our plants are infected.

How to prove it?

☑︎ We have constructed a classification model which can tell the health situation from the VOC they released.





Once the device can transmit the order to our engineered Trichoderma atroviride with chemical or electromagnetic signals.

Chemical signals：

☑︎ Cloned the phlABCD cluster and carried out the bio-synthesis of DAPG in E.coli.

☑︎ Constructed the plasmids for DAPG bio-synthesis in Saccharomyces cerevisiae and Trichoderma atroviride.

☑︎ Constructed pho promoter and expressed phlF repressor.

Fig.3 HPLC result of DAPG bio-synthesis in E.coli | Fig.4 Western blot of phlF protein

Future work

☐︎ Test pho-phlF system with DAPG

☐︎ Tested the function of phlABCD in Saccharomyces cerevisiae and Trichoderma atroviride and detect the bio-synthesis of DAPG.

Electromagnetic signals

☑︎ Expressed TRPV-Ferritin in Saccharomyces cerevisiae and tested the its function with heat shock and capsaicin.

☑︎ Constructed Pcdre-mRFP in Saccharomyces cerevisiae and measured the relative intracellular calcium content needed to activate CDRE promoter.

☑︎ Proved that the calcium influx induced by TRPV1 is strong enough to activate CDRE promoter.

Fig.5 Relative calcium content of differet groups | Fig.6 Relative fluorescent intensity of two groups

Future work

☐︎ Test TRPV1-Ferritin system with medium radio frequencies in Saccharomyces cerevisiae.

☐︎ Construct TRPV1-Ferritin-CDRE system in Saccharomyces cerevisiae and Trichoderma atroviride.



Once our Trichoderma atroviride has received the signal, the downstream gene will be expressed.

How to prove it?

☑︎ We managed to express a special Serine protases in Saccharomyces cerevisiae and tested its activity.

Fig.7 The values of OD253 increased with time

Fig.8 The values of OD253 increased with time and this solution did not add the PMSF.

These were the OD253 of the solutions after a period of reaction.The red one was the solution that did not add the PMSF; the blue one was the solution that added. Obviously, the OD253 of the former one is higher than the later one, so that, we could say that the yeast produced the serine protase successfully and effectively.

Future work

☐︎ Express this serine protase in T.atroviride.

☐︎ Search and express more downstream genes to equip our T.atroviride with more functions.


Copyright © 2017 ZJU-IGEM | Zhejiang University |