Team:Toulouse/Project/Fungicides

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       <h2>Fungicides</h2>
       <h2>Fungicides</h2>
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SCHEMA DESCRIPTIF
 
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        <p>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> Originated from plants, these peptides have different target to maximize the lethality on C. platani.  
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<center><img style="width:700px; " src="http://2014.igem.org/wiki/images/0/0c/Recap_fungicides.jpg">
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<br>
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- <b>D4E1</b> is a synthetic peptide 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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<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.
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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">Originally from plants, these peptides have different targets thus increasing the lethality on <i>Ceratocystis platani</i>.</p>
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- <b>GAFP-1 </b>(Gastrodia Anti Fungal Protein 1) is a lectin binding to mannose thus inhibiting the growth of a variety of fungi. </p>
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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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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>
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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>(Echinochloa crus-galli Anti Microbial Peptide) consists in 37 amino acids inhibiting hyphae elongation. </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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<p><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>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:940px; float:left;" src="http://2014.igem.org/wiki/images/b/b3/BBa_K1364014.png"> (image à changer : mauvais RBS)
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<center><img style="width:930px; float:left; margin: 30px 0 45px;" src="http://parts.igem.org/wiki/images/d/d0/Fungicideprod.jpg">
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<p class="legend">Figure 2: Fungicide operon</p></center>
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<p>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="http://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>
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<p><b>Secretion : </p></b>
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<p class="title2">Secretion</p>
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<p>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:500px; " src="http://2014.igem.org/wiki/images/2/2e/Secretion.jpg">
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<center><img style="width:400px; " src="http://2014.igem.org/wiki/images/2/2e/Secretion.jpg">
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<br><p class="legend">Figure 3: Design of GAFP-1 and D4E1</p></center>
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<center><a href="http://2014.igem.org/Team:Toulouse/Result/experimental-results"> <img src="http://parts.igem.org/wiki/images/f/fe/Jump.jpg"> </a></center>
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<p class="title1">References</p>
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<ul>
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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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<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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<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>
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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>
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<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>
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<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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Latest revision as of 02:58, 18 October 2014