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

Line 502: Line 502:
 
     <div class="col-md-9" role="main">
 
     <div class="col-md-9" role="main">
 
         <p class="bs-docs-section">
 
         <p class="bs-docs-section">
 +
        <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>
  
−
         <h1 id="humanpractice" class="ArticleHead GreenAH">Best Composite Part</h1>
+
         <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>
−
         <h2 class="H2Head" id="overview">Overview</h2>
+
         <h3 id="how0" class="H3Head">How we prove it?</h3>
−
         <p class="PP">This year, we aim to construct a diseases-monitoring system for automatic agriculture (AA) in which biological, physical and chemical elements can act as a whole. DAPG plays an essential role in operation of the system including serving as signal molecule or effector. Thanks to BBa_K2207013, we successfully made <em>E.coli</em> synthesize the DAPG and achieved our goal.</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>
  
−
         <h2 class="H2Head" id="introduction">Introduction</h2>
+
         <div class="col-md-12 col-sm-12">
−
        <p class="PP">2,4-DAPG (DAPG), full name 2,4-diacetylphloroglucinol, is a natural phenol found in specific strains of Gram-negative bacterium <em>Pseudomonas fluorescens</em>. This compound is found to be anti-phytopathogenic and plays an important role in the biocontrol of many plant pathogens<sup>[1]</sup>. Meanwhile, 2,4-DAPG has also shown to induce systematic resistance in plants and enhance the plant’s resistance<sup>[2]</sup>. What’s more, DAPG can also serve as signal molecule to construct a transcriptional switch that is similar to Tet on/off system<sup>[3]</sup>. What’s also worth mentioning is that previous studies confirmed that DAPG almost has no harmful impact on human health under the effective concentration, and can be degraded easily in nature. <sup>[4]</sup></p>
+
            <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>
 +
            <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>
 +
        </div>
 +
        <p class="capture"> Fig.1 Write something here | Fig.2 Write something here<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.3 Write something here<br></p>
  
−
         <p class="PP"></p>
+
         <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>
−
        <h2 class="H2Head" id="design">Design</h2>
+
−
        <p class="PP">As has mentioned before, DAPG is initially found in specific strain of <em>Pseudomonas fluorescens</em>. And it has been demonstrated that a cluster named phl responsible for the biosynthesis of DAPG, which contains eight genes, from phlA to phlH. Further studies shows that phlABCD are the DAPG synthesis genes. <sup>[5][6][7]</sup></p>
+
−
        <div class="imgdiv"><img class="textimg" src="https://static.igem.org/mediawiki/2017/2/23/ZJU_China_best_composite_1.jpg"></div>
+
−
        <p style="color: #363636; text-align: center;" class="capture">Fig.1 2,4-DAPG synthesis pathway</p>
+
−
        <p class="PP">We successfully cloned the phlABCD via colony PCR from <em>Pseudomonas fluorescens</em> 2P24. We put these four genes, phlABCD, under the control of a strong promoter which is present in the plasmid backbone we used. This backbone(BBa_K525998) was obtained from the part registry. We call this new biobrick <strong>2,4-DAPG PhlABCD Cluster</strong> (BBa_K2207013). Upon transformation of this biobrick into BL21 E. coli cells and induced with IPTG，our engineered bacteria will synthesize DAPG.</p>
+
  
−
         <h2 class="H2Head" id="result">Result</h2>
+
         <h3 id="how1" class="H3Head">How to prove it?</h3>
−
        <p class="PP">We utilize the High-performance liquid chromatography (HPLC) method to detect the biosynthesized DAPG. Luckily, we finally detected the DAPG from <em>E.coli</em> cells as Fig. 2. shows.</p>
+
         <p class="PP"><strong>☑︎    </strong>We have constructed a classification model which can tell the health situation from the VOC they released.</p>
−
        <div class="imgdiv"><img class="textimg" src="https://static.igem.org/mediawiki/2017/4/4c/ZJU_China_best_composite_2.jpg"></div>
+
−
         <p class="PP">In this way, we successfully confirmed that this biobrick can work in <em>E.coli</em>.</p>
+
  
−
         <h2 class="H2Head" id="prospect">Application Prospect</h2>
+
         <div style="text-align: center">
−
        <p class="PP">We have confirmed that the biobrick can work smoothly in <em>E.coli</em>, and the amount of biosynthesized DAPG is considerable. Therefore, we believe this part has promising application prospect.</p>
+
            <a class="CuteButton YellowCB" href="https://2017.igem.org/Team:ZJU-China/Project/mt">More About Model...</a>
−
        <p class="PP Retract">DAPG can act as signal molecules to affect the structure and function of the relative protein (phlF). Therefore, we can make use this biobrick to construct a new type of transcriptional switch together with phlF protein and phlO operator. We will have other choice other than tet system. Meanwhile, it is demonstrated that the transcriptional regulation is rapid, so this set of system has strong potential to be applied to several signal transduction pathways that requires higher sensitivity.</p>
+
         </div>
−
         <p class="PP Retract">DAPG can act as antibiotic, and be toxic to certain organisms. So we can use this biobrick to construct a type of kill switch for some organisms together with phlE(BBa_ K2207031), which is codes for an efflux pump<sup>[8]</sup>.</p>
+
         <br><br>
−
         <p class="PP Retract">DAPG can act as induction factors of ISR. The plants' autoimmune mechanism can be activated under the presence of the DAPG, which will lead the plants to their prime state. Thus, the plants will have stronger resistance to the following diseases and invasion.</p>
+
  
 +
        <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>
  
−
         <h2 class="H2Head" id="ref">Reference<hr></h2>
+
         <h3 id="cs1" class="H3Head">Chemical signals：</h3>
−
         <p class="PP ref">[1] https://en.wikipedia.org/wiki/DAPG</p>
+
         <p class="PP"><strong>☑︎    </strong>Cloned the phlABCD cluster and carried out the bio-synthesis of DAPG in E.coli.</p>
−
        <p class="PP ref">[2] Rezzonico F, Zala M, Keel C, et al. Is the ability of biocontrol fluorescent pseudomonads to produce the antifungal metabolite 2,4‐diacetylphloroglucinol really synonymous with higher plant protection?[J]. New Phytologist, 2007, 173(4):861-872.</p>
+
         <p class="PP"><strong>☑︎    </strong>Constructed the plasmids for DAPG bio-synthesis in Saccharomyces cerevisiae and Trichoderma atroviride.</p>
−
         <p class="PP ref">[3] Ikushima S, Boeke J D. New Orthogonal Transcriptional Switches Derived from Tet Repressor Homologues for Saccharomyces cerevisiae Regulated by 2, 4-Diacetylphloroglucinol and Other Ligands[J]. ACS Synthetic Biology, 2016.</p>
+
         <p class="PP"><strong>☑︎    </strong>Constructed pho promoter and expressed phlF repressor.</p>
−
        <p class="PP ref">[4] Gao Y, Chi J, eta. Effects of 2,4-diacetylphloroglucinol on two plant diseases[J]. Shandong Science, 2006, 19(4):36-39.</p>
+
−
        <p class="PP ref">[5] Yang F, Cao Y. Biosynthesis of phloroglucinol compounds in microorganisms—review[J]. Applied Microbiology and Biotechnology, 2012, 93(2):487-495.</p>
+
−
         <p class="PP ref">[6] Bangera M G, Thomashow L S. Characterization of a genomic locus required for synthesis of the antibiotic 2,4-diacetylphloroglucinol by the biological control agent Pseudomonas fluorescens Q2-87[J]. Molecular plant-microbe interactions : MPMI, 1996, 9(2):83.</p>
+
−
        <p class="PP ref">[7] Bangera M G, Thomashow L S. Identification and Characterization of a Gene Cluster for Synthesis, of the Polyketide Antibiotic 2,4-Diacetylphloroglucinol, from Pseudomonas fluorescens Q2-87[J]. Journal of Bacteriology, 1999, 181(10):3155.</p>
+
−
        <p class="PP ref">[8] Bangera M G, Thomashow L S. Identification and Characterization of a Gene Cluster for Synthesis, of the Polyketide Antibiotic 2,4-Diacetylphloroglucinol, from Pseudomonas fluorescens Q2-87[J]. Journal of Bacteriology, 1999, 181(10):3155.<br><br><br></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.4 Write something here | Fig.5 Write something here<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.6 Write something here | Fig.7 Write something here<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="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>
 +
        <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.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>
 +
        <p class="capture"> Fig.9 Write something here<br></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>
  
 
     </div>
 
     </div>
Line 549: Line 584:
  
 
                 <li><a href="#overview">Overview</a></li>
 
                 <li><a href="#overview">Overview</a></li>
−
                 <li><a href="#introduction">Introduction</a></li>
+
                 <li><a href="#fw2">Introduction</a></li>
 
                 <li><a href="#design">Design</a></li>
 
                 <li><a href="#design">Design</a></li>
 
                 <li><a href="#prospect">Application Prospect</a></li>
 
                 <li><a href="#prospect">Application Prospect</a></li>

Revision as of 01:48, 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.

Fig.1 Write something here | Fig.2 Write something here

Fig.3 Write something here

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.4 Write something here | Fig.5 Write something here

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.

Fig.8 Write something here

Fig.9 Write something here

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 | "


"