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

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           <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>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>Signal transduction and amplification</strong></p>
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           <p class="PP Retract"><strong>Expression of downstream genes</strong></p>
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           <p class="PP Retract"><strong>Expression of downstream genes<br><br></strong></p>
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           <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>
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           <p id="meet&activate" 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>
 
           <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>
 
           <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>
  
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           三张图
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           <div class="col-md-12 col-sm-12">
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          <div class="imgdiv"><img class="textimg" src="https://static.igem.org/mediawiki/2017/2/20/ZJU_China_Project_TP_P2.png"></div>
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              <div class="imgdiv col-md-6"><img style="height: 300px !important; width: auto !important;" src="https://static.igem.org/mediawiki/2017/2/20/ZJU_China_Project_TP_P2.png"></div>
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          <div class="imgdiv"><img class="textimg" src="https://static.igem.org/mediawiki/2017/3/36/ZJU_China_tp_ech42.jpg"></div>
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              <div class="imgdiv col-md-6"><img style="height: 270px !important; width: auto !important;" src="https://static.igem.org/mediawiki/2017/3/36/ZJU_China_tp_ech42.jpg"></div>
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           <div class="imgdiv"><img class="textimg" src="https://static.igem.org/mediawiki/2017/c/c7/ZJU_China_Project_TP_P3.png"></div>
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           </div>
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              <p class="capture"> Fig.1 Write something here | Fig.2 Write something here<br></p>
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              <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.3 Write something here<br></p>
  
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           <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>
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           <p class="PP" id="change&detect"><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>
 
           <h3 id="how1" class="H3Head">How to prove it?</h3>
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           <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>
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           <p class="PP"><strong>☑︎    </strong>We have constructed a classification model which can tell the health situation from the VOC they released.</p>
  
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           <p class="PP"><strong>Once the device can transmit the order to our engineered Trichoderma atroviride with chemical or electromagnetic signals.</strong></p>
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          <div style="text-align: center">
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                  <a class="CuteButton YellowCB" href="https://2017.igem.org/Team:ZJU-China/Project/mt">More About Model...</a>
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              </div>
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          <br><br>
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           <p class="PP" id="device&transmit" 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>
 
           <h3 id="cs1" class="H3Head">Chemical signals：</h3>
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           <p class="PP"><strong>☑︎    </strong>Constructed pho promoter and expressed phlF repressor.</p>
 
           <p class="PP"><strong>☑︎    </strong>Constructed pho promoter and expressed phlF repressor.</p>
  
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           <div class="imgdiv"><img class="textimg" src="https://static.igem.org/mediawiki/2017/4/4c/ZJU_China_best_composite_2.jpg"></div>
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           <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>
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           还有一张phlF western blot的图 在老姜那里
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           <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>
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          <p class="capture"> Fig.4 Write something here | Fig.5 Write something here<br></p>
  
 
           <h3 id="fw1" class="H3Head">Future work</h3>
 
           <h3 id="fw1" class="H3Head">Future work</h3>
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           <h3 id="es1" class="H3Head">Electromagnetic signals：</h3>
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           <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>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>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>
 
           <p class="PP"><strong>☑︎    </strong>Proved that the calcium influx induced by TRPV1 is strong enough to activate CDRE promoter.</p>
  
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           <div class="imgdiv"><img class="textimg" src="https://static.igem.org/mediawiki/2017/3/3a/ZJU_China_MWF_fig9.jpeg"></div>
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           <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>
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           <div class="imgdiv"><img class="textimg" src="https://static.igem.org/mediawiki/2017/1/19/ZJU_China_MWF_Rplot05.jpeg"></div>
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           <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>
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          <p class="capture"> Fig.6 Write something here | Fig.7 Write something here<br></p>
  
 
           <h3 id="fw2" class="H3Head">Future work</h3>
 
           <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>Test TRPV1-Ferritin system with medium radio frequencies in Saccharomyces cerevisiae.</p>
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           <p class="PP"><strong>☐︎    </strong>Construct TRPV1-Ferritin-CDRE system in Saccharomyces cerevisiae and Trichoderma atroviride.</p>
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           <p class="PP"><strong>☐︎    </strong>Construct TRPV1-Ferritin-CDRE system in Saccharomyces cerevisiae and Trichoderma atroviride.<br><br></p>
  
  
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           <p class="PP"><strong>Once our Trichoderma atroviride has received the signal, the downstream gene will be expressed.</strong></p>
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           <p class="PP" id="receive&express" 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>
 
           <h3 id="how2" class="H3Head">How to prove it?</h3>
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           <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/f/fb/ZJU_China_Design5.png"></div>
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          <p class="capture"> Fig.8 Write something here<br></p>
 
           <div class="imgdiv"><img class="textimg" src="https://static.igem.org/mediawiki/2017/7/71/ZJU_China_Design6.png"></div>
 
           <div class="imgdiv"><img class="textimg" src="https://static.igem.org/mediawiki/2017/7/71/ZJU_China_Design6.png"></div>
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          <p class="capture"> Fig.9 Write something here<br></p>
  
 
           <h3 id="fw3" class="H3Head">Future work</h3>
 
           <h3 id="fw3" class="H3Head">Future work</h3>
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           <p class="PP"><strong>☐︎    </strong>Express this serine protase in T.atroviride.</p>
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           <p class="PP"><strong>☐︎    </strong>Express this serine protase in <em>T.atroviride</em>.</p>
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           <p class="PP"><strong>☐︎    </strong>Search and express more downstream genes to equip our T.atroviride with more functions.</p>
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           <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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          <!--<h2 id="introduction" class="H2Head">Introduction</h2>-->
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                  <!--<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>-->
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                  <!--<div class="imgdiv"><img class="textimg" src="https://static.igem.org/mediawiki/2017/8/82/ZJU_China_Downstream_1.png"></div>-->
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                  <!--<p class="capture">Fig.1 Genetic circuit of the downstream</p>-->
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              <!--<h3 id="za" class="H3Head">Zwittermicin A</h3>-->
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                  <!--<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>-->
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                  <!--<div class="imgdiv"><img class="textimg" src="https://static.igem.org/mediawiki/2017/4/4f/ZJU_China_Downstream_2.png"></div>-->
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                  <!--<p class="capture">Fig.2A Gene organization of the Zwittermicin A biosynthetic cluster[2].</p>-->
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                  <!--<p class="capture">  </p>-->
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                  <!--<div class="imgdiv"><img class="textimg" src="https://static.igem.org/mediawiki/2017/0/0b/ZJU_China_Design3.png"></div>-->
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                  <!--<p class="capture">Fig.2B Units used in Zwittermicin A Production.[2]</p>-->
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+
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              <!--<h3 id="sp" class="H3Head">Serine protease</h3>-->
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                <!--<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>-->
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              <!--<h3 id="ch" class="H3Head">Chitinase</h3>-->
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                <!--<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>-->
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              <!--<h3 id="anythingelse" class="H3Head">Anything else</h3>-->
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                <!--<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>-->
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          <!--<h2 id="result" class="H2Head">Result</h2>-->
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                    <!--<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>-->
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              <!--<h3 id="yeast" class="H3Head">Yeast</h3>-->
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                  <!--<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>-->
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                  <!--<div class="imgdiv"><img class="textimg" src="https://static.igem.org/mediawiki/2017/d/dd/ZJU_China_Design4.png"></div>-->
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                  <!--<p class="capture">Fig.3 Western-blot result</p>-->
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                  <!--<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>-->
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                  <!--<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>-->
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                  <!--<div class="imgdiv"><img class="textimg" src="https://static.igem.org/mediawiki/2017/f/fb/ZJU_China_Design5.png"></div>-->
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                  <!--<p class="capture">Fig.4 The values of OD253 increased with time</p>-->
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                  <!--<div class="imgdiv"><img class="textimg" src="https://static.igem.org/mediawiki/2017/7/71/ZJU_China_Design6.png"></div>-->
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                  <!--<p class="capture">Fig.5 The values of OD253 increased with time and this solution did not add the PMSF. </p>-->
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                  <!--<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>-->
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              <!--<h3 id="ta" class="H3Head"><em>T.atroviride</em></h3>-->
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                  <!--<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>-->
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                  <!--<div class="imgdiv"><img class="textimg" src="https://static.igem.org/mediawiki/2017/b/ba/ZJU_China_Design7.png"></div>-->
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                  <!--<p class="capture">Fig.6 Fluorescence of mycelium</p>-->
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          <!--<h2 id="ref" class="H2Head">Reference</h2>-->
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              <!--<p class="ref">[1] Stohl E A, Milner J L, Handelsman J. Zwittermicin A biosynthetic cluster[J]. Gene, 1999, 237(2):403-411.</p>-->
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              <!--<p class="ref">[2] Zwittermicin A, https://en.wikipedia.org/wiki/Zwittermicin_A  ,7 June 2016(24/10/2017)</p>-->
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              <!--<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>-->
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              <!--<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>-->
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              <!--<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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   <nav  style="position: fixed; top: 100px ; left:50px; " class="bs-docs-sidebar hidden-print hidden-xs hidden-sm">
 
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        <li><a href="#overview">Overview</a></li>
 
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         <a href="#introduction">Introduction</a>
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         <a href="#meet&activate">Meet & Activate</a>
 
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             <li><a href="#za">Zwittermicin A</a></li>
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             <li><a href="#how0">How to prove?</a></li>
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            <li><a href="#sp">Serine protease</a></li>
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            <li><a href="#ch">Chitinase</a></li>
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            <li><a href="#anythingelse">Anything else</a></li>
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         <li>
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             <a href="#result">Result</a>
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             <a href="#change&detect">Change & Detect</a>
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            <ul class="nav">
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                <li><a href="#how1">How to prove?</a></li>
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            </ul>
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            <a href="#device&transmit">Device & Transmit</a>
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                <li><a href="#cs1">Chemical Signals</a></li>
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                <li><a href="#fw1">Future work</a></li>
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                <li><a href="#es1">Electromagnetic signals</a></li>
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                <li><a href="#fw2">Future work</a></li>
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            </ul>
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            <a href="#receive&express">Receive & Express</a>
 
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                 <li><a href="#yeast">Yeast</a></li>
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                 <li><a href="#how2">How to prove?</a></li>
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                 <li><a href="#ta">T.atroviride</a></li>
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                 <li><a href="#fw3">Future work</a></li>
 
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        <li><a href="#ref">Reference</a></li>
 
 
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Revision as of 01:36, 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.

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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.

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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.6 Write something here | Fig.7 Write something here

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.

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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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