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

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{{ZJU-China}}
 
  
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<h1>Demonstrate</h1>
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  var _hmt = _hmt || [];
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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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                      <!-- Hidden li included to remove active class from about link when scrolled up past about section -->
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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">Project Description</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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 +
                          <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/st">Signal Transduction</a></li>
 +
                              <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 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">Conclusion</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Notebook">Notebook</a></li>
 +
                          </ul>
 +
                      </li>
 +
 
 +
                      <li class="m_nav_item dropdown" >
 +
                          <a href="#" class="dropdown-toggle link" data-toggle="dropdown">Model<b class="caret"></b></a>
 +
                          <ul class="dropdown-menu ">
 +
                              <!--<li><a href="https://2017.igem.org/Team:ZJU-China/Model">Summery</a></li>-->
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Model">VOC analysis</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Model/Coculture">Coculture</a></li>
 +
 
 +
                          </ul>
 +
                      </li>
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                      <li class="m_nav_item dropdown">
 +
                          <a href="#" class="dropdown-toggle link" data-toggle="dropdown">Parts<b class="caret"></b></a>
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 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Composite_Part">Composite Parts</a></li>
 +
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 +
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 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Parts">All Parts</a></li>
 +
 
 +
 
 +
                          </ul>
 +
                      </li>
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 +
                      <li><a href="https://2017.igem.org/Team:ZJU-China/Hardware">Hardware</a></li>
 +
 
 +
                      <li class="m_nav_item dropdown" >
 +
                          <a href="#" class="dropdown-toggle link" data-toggle="dropdown">Safety<b class="caret"></b></a>
 +
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                              <li><a href="https://2017.igem.org/Team:ZJU-China/HP/Silver">Silver</a></li>
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                              <li><a href="https://2017.igem.org/Team:ZJU-China/HP/Gold_Integrated">Gold</a></li>
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                              <li><a href="https://2017.igem.org/Team:ZJU-China/Team">Teammates</a></li>
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                              <li><a href="https://2017.igem.org/Team:ZJU-China/Attributions">Attribution</a></li>
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<p>
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          <h1 id="demonstrate" class="page-header ArticleHead GreenAH">Demonstrate</h1>
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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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</p>
+
  
 +
          <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</strong></p>
 +
          <p class="PP Retract"><strong>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>
  
 +
          三张图
 +
          <div class="imgdiv"><img class="textimg" src="https://static.igem.org/mediawiki/2017/2/20/ZJU_China_Project_TP_P2.png"></div>
 +
          <div class="imgdiv"><img class="textimg" src="https://static.igem.org/mediawiki/2017/3/36/ZJU_China_tp_ech42.jpg"></div>
 +
          <div class="imgdiv"><img class="textimg" src="https://static.igem.org/mediawiki/2017/c/c7/ZJU_China_Project_TP_P3.png"></div>
 +
 +
 +
          <p class="PP"><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.More details(方哥分类模型的超链接）</p>
 +
 +
          <p class="PP"><strong>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"><img class="textimg" src="https://static.igem.org/mediawiki/2017/4/4c/ZJU_China_best_composite_2.jpg"></div>
 +
          还有一张phlF western blot的图 在老姜那里
 +
 +
 +
          <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"><img class="textimg" src="https://static.igem.org/mediawiki/2017/3/3a/ZJU_China_MWF_fig9.jpeg"></div>
 +
          <div class="imgdiv"><img class="textimg" src="https://static.igem.org/mediawiki/2017/1/19/ZJU_China_MWF_Rplot05.jpeg"></div>
 +
 +
 +
          <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.</p>
 +
 +
 +
          <p class="PP"><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>
 +
          <div class="imgdiv"><img class="textimg" src="https://static.igem.org/mediawiki/2017/7/71/ZJU_China_Design6.png"></div>
 +
 +
          <h3 id="fw3" class="H3Head">Future work</h3>
 +
          <p class="PP"><strong>☐︎    </strong>Express this serine protase in T.atroviride.</p>
 +
          <p class="PP"><strong>☐︎    </strong>Search and express more downstream genes to equip our T.atroviride with more functions.</p>
 +
 +
 +
 +
 +
          <!--<h2 id="introduction" class="H2Head">Introduction</h2>-->
 +
                  <!--<p class="PP">When our <em>T.atroviride</em> is activated by the signals which have been described above, they will produce the corresponding effects to save our little plants. Those can be zwittermycin A, chitinase, serine protease and anything you need to protect your lovely plants. Zwittermycin A is an antibiotic which can inhibite the growth of <em>P. nicotianae</em>. Chitinase is a lytic enzyme that breaks down fungal cell walls. Serine protases plays an important role in hydrolyzing the eggshell of root-knot nematodes. With these fungal growth inhibitors our engineered <em>T.atroviride</em> will be able to better protect our plants.</p>-->
 +
                  <!--<div class="imgdiv"><img class="textimg" src="https://static.igem.org/mediawiki/2017/8/82/ZJU_China_Downstream_1.png"></div>-->
 +
                  <!--<p class="capture">Fig.1 Genetic circuit of the downstream</p>-->
 +
 +
              <!--<h3 id="za" class="H3Head">Zwittermicin A</h3>-->
 +
                  <!--<p class="PP">Zwittermicin A is an antibiotic that  has the potential to suppress plant disease due to its broad spectrum activity against certain gram positive and gram negative prokaryotic micro-organisms. Since <em>T.atroviride</em> does not produce Zwittermycin A by itself, a gene cluster obtained from  Bacillus cereus UW85 was introduced into <em>T.atroviride</em>. The genes responsible for the production of zwittermicin A are located on a 16 kb cluster containing nine orfs, from orf1 to orf9, and a self resistant gene zmaR, a gene that encodes an acylation enzyme that deactivate zwittermicin A.[1]</p>-->
 +
                  <!--<div class="imgdiv"><img class="textimg" src="https://static.igem.org/mediawiki/2017/4/4f/ZJU_China_Downstream_2.png"></div>-->
 +
                  <!--<p class="capture">Fig.2A Gene organization of the Zwittermicin A biosynthetic cluster[2].</p>-->
 +
                  <!--<p class="capture">  </p>-->
 +
                  <!--<div class="imgdiv"><img class="textimg" src="https://static.igem.org/mediawiki/2017/0/0b/ZJU_China_Design3.png"></div>-->
 +
                  <!--<p class="capture">Fig.2B Units used in Zwittermicin A Production.[2]</p>-->
 +
 +
 +
              <!--<h3 id="sp" class="H3Head">Serine protease</h3>-->
 +
                <!--<p class="PP">Root-knot nematodes (Meloidogyne spp.), which are one of the most destructive nematodes, cause the loss of crop about 10%, serious as high as 75%. [3] In the present, the egg-parasitic fungus P.lilacinum is the main biocontrol material of root-knot nematodes.P.lilacinum. secretes protease and chitinase to hydrolyze the nematode eggshell, so that the root knot nematodes cannot grow normally.[4] However, because P.lilacinum can live in the cornea, the usage of P.lilacinum is still dangerous. Therefore, in this part, the purpose of our project is to give our harmless <em>T.atroviride</em> the ability to kill the root-knot nematodes by overexpressing serine protease which plays an important role in hydrolyzing the eggshell of root-knot nematodes.</p>-->
 +
              <!--<h3 id="ch" class="H3Head">Chitinase</h3>-->
 +
                <!--<p class="PP">Chitinase is a hydrolytic enzyme that breaks down hydrolytic bonds in chitin.  As chitin is a component of the cell walls of fungi and exoskeletal elements of some animals (including worms and arthropods), chitinase has been shown to be useful in biological control against fungi.[5] Therefore, in order to inhibit fungl growth, our <em>T.atroviride</em> can produce chitinase when the plants are infested by fungi and the signal conversion systems work well.</p>-->
 +
              <!--<h3 id="anythingelse" class="H3Head">Anything else</h3>-->
 +
                <!--<p class="PP">Your lovely plants will face many challenges in the complex and dangerous soil condition, so that, the little plants must be protected by the strong <em>T.atroviride</em> which can product corresponding effects to inhibite the fungi. The following table can help you choose the right inhibitor to help your plants.</p>-->
 +
 +
 +
          <!--<h2 id="result" class="H2Head">Result</h2>-->
 +
                    <!--<p class="PP">Because of lacking of time, we only did the experience of serine protease. We structured two plasimads: one could work in the yeast and the other could work in <em>T.atroviride</em>.</p>-->
 +
 +
              <!--<h3 id="yeast" class="H3Head">Yeast</h3>-->
 +
                  <!--<p class="PP">The gene which can express serine protease in yeast was synthesized by Genscript. Before synthesizing this gene, we did codon optimization based on the codon preference of yeast and added a flag-tag to the N-terminal of the serine protease.After extracting the whole proteins of the yeast which transferred plasmid successfully, we performed western-blot and checked the serine protein was expressed in the yeast. (Result is as follow) The band was very shallow, in other words, the concentration of the serine protease was very low.</p>-->
 +
                  <!--<div class="imgdiv"><img class="textimg" src="https://static.igem.org/mediawiki/2017/d/dd/ZJU_China_Design4.png"></div>-->
 +
                  <!--<p class="capture">Fig.3 Western-blot result</p>-->
 +
                  <!--<p class="PP">The band with red circle was the band of the serine protease and the marker was a protein marker of aidlab.</p>-->
 +
                  <!--<p class="PP">In order to test whether the serine protease could work normally in the yeast, we performed the enzyme activity detection using BAEE solution. We did two sets of experiments: one added PMSF, which was a inhibitor of serine protease, and the other did not. And then, reading the OD253 of these two solutions.(You can know more details about the detection from the protocol) Obviously, the OD253 of the former one is higher than the later one and the values of OD253 increased with time in a period of time ; therefore, we made the conclusion that the yeast produced the serine protase successfully and effectively.</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.4 The values of OD253 increased with time</p>-->
 +
                  <!--<div class="imgdiv"><img class="textimg" src="https://static.igem.org/mediawiki/2017/7/71/ZJU_China_Design6.png"></div>-->
 +
                  <!--<p class="capture">Fig.5 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="ta" class="H3Head"><em>T.atroviride</em></h3>-->
 +
                  <!--<p class="PP">By contrast to the yeast, the serine protease that worked in the <em>T.atroviride</em> was cloned from the genome of P.lilacinum for they have high homology. Because of lacking of time to extract protein from <em>T.atroviride</em>, we putted the EGFP gene behind the serine protease gene, so that, we could test the serine protease by detecting the fluorescent. From the picture below, mycelium was fluorescent and the <em>T.atroviride</em> expressed the protease successfully. In the future, we will extract the serine protease and detect the activity of it.</p>-->
 +
                  <!--<div class="imgdiv"><img class="textimg" src="https://static.igem.org/mediawiki/2017/b/ba/ZJU_China_Design7.png"></div>-->
 +
                  <!--<p class="capture">Fig.6 Fluorescence of mycelium</p>-->
 +
 +
          <!--<h2 id="ref" class="H2Head">Reference</h2>-->
 +
              <!--<p class="ref">[1] Stohl E A, Milner J L, Handelsman J. Zwittermicin A biosynthetic cluster[J]. Gene, 1999, 237(2):403-411.</p>-->
 +
              <!--<p class="ref">[2] Zwittermicin A, https://en.wikipedia.org/wiki/Zwittermicin_A  ,7 June 2016(24/10/2017)</p>-->
 +
              <!--<p class="ref">[3] Wang J P, Wang J X, Liu F, et al. Enhancing the virulence of Paecilomyces lilacinus against Meloidogyne incognita eggs by overexpression of a serine protease. Biotechnology Letters, 32, 1159-1166[J]. Biotechnology Letters, 2010, 32(8):1159-1166.</p>-->
 +
              <!--<p class="ref">[4] Brand D, Roussos S, Pandey A, et al. Development of a bionematicide with Paecilomyces lilacinus to control Meloidogyne incognita.[J]. Applied Biochemistry & Biotechnology, 2004, 118(1-3):81-88.</p>-->
 +
              <!--<p class="ref">[5] Sámi L, Pusztahelyi T, Emri T, et al. Autolysis and aging of Penicillium chrysogenum cultures under carbon starvation: Chitinase production and antifungal effect of allosamidin.[J]. Journal of General & Applied Microbiology, 2001, 47(4):201.</p>-->
 +
 +
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Revision as of 00:46, 28 October 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.

三张图

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.More details(方哥分类模型的超链接）

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.

还有一张phlF western blot的图 在老姜那里

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.

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.

Future work

☐︎ Express this serine protase in T.atroviride.

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


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