Team:Toulouse/Project/Fungicides

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   <!--Short description : à changer!!!-->
   <!--Short description : à changer!!!-->
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<img style="width:800px; " src="https://static.igem.org/mediawiki/2014/0/0c/Recap_fungicides.jpg">
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<center><img style="width:700px; " src="https://static.igem.org/mediawiki/2014/0/0c/Recap_fungicides.jpg">
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<br>
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<p class="legend">Figure 1: Schema of the fungicide module</p></center>
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         <p class="textesimple">The main objective of SubtiTree is to ensure the <b> destruction of the pathogenic fungi </b> inside the tree. In order to achieve this goal, we built a genetic module to produce three different peptides with antifungal activities. </p> <br>
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         <p class="textesimple">The main objective of SubtiTree is to ensure the <b> destruction of the pathogenic fungi </b> inside the tree.
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In order to achieve this goal, we built a genetic module to produce three different peptides with antifungal activities. This triple therapy provides
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the advantage to minimize the resistance phenomenon.</p> <br>
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  <p class="textesimple"> Originated from plants, these peptides have different target to maximize the lethality on C. platani.  
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  <p class="textesimple">Originally from plants, these peptides have different targets thus increasing the lethality on <i>Ceratocystis platani</i>.</p>
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<p class="textesimple">
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<br></br>
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- <b>D4E1</b> is a synthetic peptide anlogo to Cecropin B AMPs (antimicrobial peptides) made of 17 amino acids which has been shown to have an antifungal activity by complexing with a sterol present in the conidia’s wall of numerous fungi. </p>
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<ul>
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<p  class="textesimple">
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<li class="tree"><p class="texte"><b>D4E1</b> is a synthetic peptide analog to Cecropin B AMPs (AntiMicrobial Peptides) made of 17 amino acids
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- <b>GAFP-1 </b>(<i>Gastrodia</i> Anti Fungal Protein 1), also known as gastrodianin, is a mannose and chitin binding lectin originating from the Asiatic orchid Gastrodia elata, a traditional Chinese medicinal herb cultured for thousands of years. GAFP1 accumulates in nutritive corms where the fungal infection takes place, and in vitro assays demonstrated it can inhibit the growth of ascomycete and basidiomycete fungal plant pathogens.</p>
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which has been shown to have an antifungal activity by complexing with a sterol present in the conidia’s wall of numerous fungi.</p></li>
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<p class="textesimple">
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<li class="tree"><p class="texte"><b>GAFP-1 </b>(<i>Gastrodia</i> Anti Fungal Protein 1), also known as gastrodianin, is a mannose and chitin binding lectin
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- <b>EcAMP-1 </b>(<i>Echinochloa crus-galli </i> Anti Microbial Peptide) consists in 37 amino acids inhibiting hyphae elongation. EcAMP1 is the first example of AMP with a novel disulfide-stabilized-α helical hairpin fold. It is isolated from kernels of barnyard grass. EcAMP1 exhibits high activity against fungi of the genus Fusarium.</p>
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originating from the Asiatic orchid <i>Gastrodia elata</i>, a traditional Chinese medicinal herb cultured for thousands of years.  
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GAFP-1 accumulates in nutritive corms where the fungal infection takes place, and <i>in vitro</i> assays demonstrated it can inhibit the growth of
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ascomycete and basidiomycete fungal plant pathogens.</p></li>
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<li class="tree"><p class="texte"><b>EcAMP-1 </b>(<i>Echinochloa crus-galli</i> AntiMicrobial Peptide) consists in 37 amino acids inhibiting hyphae elongation
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EcAMP-1 is the first example of AMP with a novel disulfide-stabilized-α helical hairpin fold. It is isolated from kernels of barnyard grass.
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EcAMP-1 exhibits high activity against fungi of the genus <i>Fusarium</i>.</p></li>
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</ul>
</p>
</p>
<br>
<br>
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<p class="title1" style="margin-top:30px;"><b>More information on this module </p></b> <br>
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<p class="title1" style="margin-top:30px;">More information about this module </p>
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<p  class="texte">Final constructions: (see Parts to have more details on the intermediate parts)
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<p  class="texte">
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We built different genetic constructions to test each fungicide separately and to test them all together on the same operon where the 3 genes are placed under the control of a constitutive promoter in Bacillus subtilis: Pveg. </p>
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We built different genetic constructions to test each fungicide separately and to test them all together on the same operon. The three genes coding for the
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antifungal peptides are placed under the control of the constitutive promoter P<sub>veg</sub> in <i>Bacillus subtilis</i>.</p>
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<img style="width:930px; float:left; margin: 30px 0;" src="https://static.igem.org/mediawiki/parts/d/d0/Fungicideprod.jpg">  
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<center><img style="width:930px; float:left; margin: 30px 0 45px;" src="https://static.igem.org/mediawiki/parts/d/d0/Fungicideprod.jpg">
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<p class="legend">Figure 2: Fungicide operon</p></center>
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<p  class="texte">EcAMP-1 was already present in the Registry, added by the Utah State 2013 iGEM team (BB_K1162001).  We added D4E1 and GAFP-1 to the Registry of Standard Biological Parts (see parts). These new BioBricks were designed in order to be expressed and secreted with Bacillus subtilis.   
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<p class="title2">Added parts</p>
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</p>
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<p class="title3">EcAMP-1</p>
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<p  class="texte">EcAMP-1 was already present in the Registry, added by the Utah State 2013 iGEM team  
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(<a href="http://parts.igem.org/Part:BBa_K1162001"_blank">BBa_K1162001</a>). This part has been modified and improved by our team
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(<a href="http://parts.igem.org/Part:BBa_K1364019"_blank">BBa_K1364019</a>) with the addition of a STOP codon after the coding sequence</p>
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<p class="title3">D4E1 and GAFP-1</p>
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<p class="texte">We added D4E1 and GAFP-1 to the Registry of Standard Biological Parts  
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(See <a href="https://2014.igem.org/Team:Toulouse/Result/parts/Submitted_parts"_blank">Submitted parts</a>).
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<br> These new BioBricks were designed in order to be expressed and secreted with <i>B. subtilis</i>.  </p>
<br>
<br>
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<p  class="title1"><b>Secretion </b> </p>
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<p  class="title2">Secretion</p>
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<p  class="texte">In order to export the peptides outside the bacteria, the coding sequence of D4E1 and GAFP-1 was flanked on the N-terminal end with a signal peptide (amyE signal peptide) followed by a pro peptide, cleaved during the secretion process. </p> <br>
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<p  class="texte">In order to export the peptides outside the bacteria, the coding sequences of D4E1 and GAFP-1 were flanked on the N-terminal end with
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a signal peptide (amyE signal peptide) followed by a pro peptide, cleaved during the secretion process.</p><br>
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<img style="width:400px; " src="https://static.igem.org/mediawiki/2014/2/2e/Secretion.jpg">
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<center><img style="width:400px; " src="https://static.igem.org/mediawiki/2014/2/2e/Secretion.jpg">
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<img style="width:400px; " src="https://static.igem.org/mediawiki/2014/d/d7/Fongpep.jpg">
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<img style="width:500px; " src="https://static.igem.org/mediawiki/2014/d/d7/Fongpep.jpg">
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<br><p class="legend">Figure 3: Design of GAFP-1 and D4E1</p></center>
<br>
<br>
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<br>
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<center><a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results"> <img src="https://static.igem.org/mediawiki/parts/f/fe/Jump.jpg"> </a></center>
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</P>  
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<p  class="title1"><b>References</b> </p>
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<p  class="texte">
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- A.J De Lucca, J.M Bland, C. Grimm, T.J Jacks.<b> Fungicidal properties, sterol binding, and proteolytic resistance of the synthetic peptide D4E1 </b>. Canadian Journal of Microbiology. 1998, Vol. 44:514-520. <br>
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<p  class="title1">References</p>
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Kanniah Rajasekaran, Kurt D. Stromberg, Jeffrey W. Cary, and Thomas E. Cleveland.<b> Broad-Spectrum Antimicrobial Activity in vitro of the Synthetic Peptide D4E1</b>. J. Agric. Food Chem. 2001, Vol. 49, 2799-2803.<br>
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-
-M. Visser, D. Stephan, J.M. Jaynes and J.T. Burger.<b> A transient expression assay for the in planta efficacy screening of an antimicrobial peptide against grapevine bacterial pathogens</b>. Letters in Applied Microbiology. 2012, Vol. 54, 543–551.<br>
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<ul>
-
-K. D. Cox, D. R. Layne, R. Scorza, G Schnabel. <b>Gastrodia anti-fungal protein from the orchid Gastrodia elata confers disease resistance to root pathogens in transgenic tobacco</b>. Planta. 2006, Vol. 224:1373–1383<br>
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<li class="tree"><p class="texte">A. J De Lucca, J.M Bland, C. Grimm, and T.J Jacks.<b> Fungicidal properties, sterol binding, and proteolytic resistance of the synthetic peptide D4E1 </b>. Canadian Journal of Microbiology. 1998, Vol. 44:514-520. </p></li>
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-Xiaochen Wang, Guy Bauw, Els J.M. Van Damme, Willy J. Peumans, Zhang-Liang Chen, Marc Van Montagu and Willy Dillen. <b>Gastrodianin-like mannose-binding proteins: a novel class of plant proteins with antifungal properties</b>. The Plant Journal. 2001, Vol. 25(6), 651±661<br>
+
<li class="tree"><p class="texte">Kanniah Rajasekaran, Kurt D. Stromberg, Jeffrey W. Cary, and Thomas E. Cleveland.<b> Broad-Spectrum Antimicrobial Activity in vitro of the Synthetic Peptide D4E1</b>. J. Agric. Food Chem. 2001, Vol. 49, 2799-2803.</p></li>
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-Svetlana B. Nolde, Alexander A. Vassilevski, Eugene A. Rogozhin, Nikolay A. Barinov, Tamara A. Balashova, Olga V. Samsonova, Yuri V. Baranov, Alexey S. Arseniev and Eugene V. Grishin. <b>Disulfide-stabilized Helical Hairpin Structure and Activity of a Novel Antifungal Peptide EcAMP1 from Seeds of Barnyard Grass (Echinochloa crus-galli)</b>. The journal of Biological Chemistry. 2011, Vol. 286, 25145–25153<br>
+
<li class="tree"><p class="texte">M. Visser, D. Stephan, J.M. Jaynes, and J.T. Burger.<b> A transient expression assay for the in planta efficacy screening of an antimicrobial peptide against grapevine bacterial pathogens</b>. Letters in Applied Microbiology. 2012, Vol. 54, 543–551.</p></li>
-
</p>
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<li class="tree"><p class="texte">K. D. Cox, D. R. Layne, R. Scorza,and G Schnabel. <b>Gastrodia anti-fungal protein from the orchid <i>Gastrodia elata</i> confers disease resistance to root pathogens in transgenic tobacco</b>. Planta. 2006, Vol. 224:1373–1383.</p></li>
 +
<li class="tree"><p class="texte">Xiaochen Wang, Guy Bauw, Els J.M. Van Damme, Willy J. Peumans, Zhang-Liang Chen, Marc Van Montagu, and Willy Dillen. <b>Gastrodianin-like mannose-binding proteins: a novel class of plant proteins with antifungal properties</b>. The Plant Journal. 2001, Vol. 25(6), 651±661.</p></li>
 +
<li class="tree"><p class="texte">Svetlana B. Nolde, Alexander A. Vassilevski, Eugene A. Rogozhin, Nikolay A. Barinov, Tamara A. Balashova, Olga V. Samsonova, Yuri V. Baranov, Alexey S. Arseniev and, Eugene V. Grishin. <b>Disulfide-stabilized Helical Hairpin Structure and Activity of a Novel Antifungal Peptide EcAMP1 from Seeds of Barnyard Grass (<i>Echinochloa crus-galli</i>)</b>. The journal of Biological Chemistry. 2011, Vol. 286, 25145–25153.</p></li>
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</ul>
      
      

Latest revision as of 02:58, 18 October 2014