http://2014.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=50&target=Jourdan2014.igem.org - User contributions [en]2024-03-28T22:38:53ZFrom 2014.igem.orgMediaWiki 1.16.5http://2014.igem.org/Team:Toulouse/Result/achievementTeam:Toulouse/Result/achievement2014-12-12T19:07:45Z<p>Jourdan: </p>
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<h2>Achievements</h2><br />
<p>Bronze, Silver or Gold Medal? That is the question!</p><br />
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<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Results&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Achievements</p> <br />
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<p class="title1" style="text-align:left;"> 2014 Giant Jamboree Results !</p><br />
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The 2014 Toulouse iGEM team had a lot of fun during the AMAZING Giant Jamboree in Boston ... And all the work done was rewarded by a GOLD medal and the Best Prize "Innovation in measurement" ! </p><br />
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<p class="title1" style="text-align:left;">Let's sum up what we did during this summer!</p><br />
<p class="title2">First step...</p><br />
<p class="texte"> <br />
After a summer of hard work, our team is proud of the achieved results. The first step of our project was to have a <b>proof of concept</b> of the three functions requiered to fight against canker. The verifications of our genetic constructions were successful (PCR, Migration on an agarose gel and Sequencing). We could then proceed to specific test of the modules: capillary test, chitin beads test or fungicide tests.</p><br />
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<p class="title2">Second step...</p><br />
<p class="texte"> <br />
Once these results obtained, we decided to move on to the next step: in order to get closer to our final objective, the three genetic modules have to be assembled and to be tested in <b>model plants</b>. In the short time we had, these tests were only performed with the antifungal module, but the results were very encouraging (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">3. In planta tests with SubtiTree</a> in the Fungicides module). <br />
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<p class="title2">Third step...</p><br />
<p class="texte"> <br />
So encouraging that we moved on to the third step of our project and think about the <b>real injection of SubtiTree in diseased trees</b>. We then considered the <a href="https://2014.igem.org/Team:Toulouse/Project/Spreading"><b>spreading</b></a> problems and proposed different strategies to avoid this major issue. <a href="https://2014.igem.org/Team:Toulouse/Modelling"><b>Modeling</b></a> enables us to predict the growth and the lifespan of SubtiTree in Plane Trees. <br />
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<p class="title2">And what comes next?</p><br />
<p class="texte"> With the support of Governmental institutions, scientific organizations and the popular enthusiasm raised by our project, we are still looking for the best way to continue and achieve our goal: save the plane trees and preserve the beauty of the Canal du Midi.<br><br />
However, SubtiTree's spreading must be controlled, fungicide production regulated, specifity improved, tests of the whole system performed. In a nutshell, we still have a lot of things to do even if we did our best during the whole summer!</p><br />
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<p class="title2">iGEM Community</p><br />
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Well aware that we are part of the <b>iGEM Community</b>, our team also worked for this community by improving the iGEMer's Bible: <a href="http://parts.igem.org/Catalog">the Registry of Standard Biological Parts</a>. <br><br />
We improved the EcAMP-1 BioBrick by adding a promotor, a RBS, but mainly by the addition of a STOP codon (<a href="http://parts.igem.org/Part:BBa_K1364019">BBa_K1364019</a>).<br><br />
As <i>Bacillus subtilis</i> appears to be for us an ideal chassis, we also developed new genetic tools to facilitate its utilization. Thanks to numerous interactions with other iGEM teams, we didn't see the time flying by during this <b>amazing summer</b> !</p><br />
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<p class="title2">It is better to have "well-made rather than a well-filled head"... (Michel de Montaigne)</p><br />
<p class="texte">We developed a deep <a href="https://2014.igem.org/Team:Toulouse/ethics">ethical questioning</a>, trying to understand the relationship between Science and the protection of the Beauty. We strongly believe that neglecting the ethical aspect in a scientific project is a mistake. All the team was involved in this part and the reflexions made were very profitable to us.</p><br />
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<p class="texte">We filled the <a href="https://igem.org/2014_Judging_Form?id=1364">2014 Judging Form</a> thanks to the following accomplishments:</p><br />
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<td style="border-bottom:4px solid #e5e6e6; border-top-left-radius:9px;"><center><br>Bronze Medal <img src="https://static.igem.org/mediawiki/2013/8/86/Medal_bronze.png" class="imgcontenttable"/></center><br></td><br />
<td style="border-bottom:4px solid #e5e6e6;"><center><br>Silver Medal <img src="https://static.igem.org/mediawiki/2013/d/dd/Medal_silver.png" class="imgcontenttable"/></center><br></td><br />
<td style="border-bottom:4px solid #e5e6e6; border-top-right-radius:9px;"><center><br>Gold Medal<img src="https://static.igem.org/mediawiki/2013/b/bd/Medal_gold_3.png" class="imgcontenttable"/></center><br></td><br />
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<td style="border-right:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/>Register the team, have a great summer, and plan to have fun at the Jamboree</td><br />
<td style="border-right:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/>Experimentally validate that at least three new BioBrick Part or Device of your own design and construction work as expected.</td><br />
<td><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Improve the function of an existing BioBrick Part or Device and improve the <i>Bacillus subtilis</i> genetic tools, enter this information in the Registry : <a href="http://parts.igem.org/Part:BBa_K1364019"target="_blank">BBa_K1364019</a>, <a href="http://parts.igem.org/Part:BBa_K1364016"target="_blank">BBa_K1364016</a>, <a href="http://parts.igem.org/Part:BBa_K1364017"target="_blank">BBa_K1364017</a>, <a href="http://parts.igem.org/Part:BBa_K1364021"target="_blank">BBa_K1364021</a>, </td><br />
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<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Successfully complete and submit this iGEM Judging form </td><br />
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/>Document the characterization of these three parts in the 'Main Page' of that Part's/Device's Registry entry<br />
Submit this new part to the iGEM Parts Registry:<br> <a href="http://parts.igem.org/Part:BBa_K1364005"target="_blank">BBa_K1364005</a>, <a href="http://parts.igem.org/Part:BBa_K1364009"target="_blank">BBa_K1364009</a>, <a href="http://parts.igem.org/Part:BBa_K1364013"target="_blank">BBa_K1364013.</a></td><br />
<td style="border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Your project may have implications for the environment, security, safety and ethics and/or ownership and sharing. Describe a novel approach that your team has used to help you and others consider these aspects of the design and outcomes of synthetic biology efforts. → <a href="https://2014.igem.org/Team:Toulouse/ethics" target="_blank">Ethic Part</a> </td><br />
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<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Create and share a Description of the team’s project using the iGEM wiki and the team’s parts using the Registry Biological Parts </td><br />
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Submit these new part to the iGEM Parts Registry (submissions must adhere to the iGEM Registry guidelines)</td><br />
<td style="border-top:1px solid #e5e6e6;"> </td><br />
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<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Plan to present a Poster and Talk at the iGEM Jamboree </td><br />
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Your project may have implications for the environment, security, safety and ethics and/or ownership and sharing. Describe one or more ways in which these or other broader implications have been taken into consideration in the design and execution of your project. → <a href="https://2014.igem.org/Team:Toulouse/ethics" target="_blank"> Ethical Page</a></td><br />
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<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Document at least one new standard BioBrick Part or Device used in your project/central to your project and submit this part to the iGEM Registry : <a href="http://parts.igem.org/Part:BBa_K1364000" target="_blank">BBa_K1364000</a>, <a href="http://parts.igem.org/Part:BBa_K1364002"target="_blank">BBa_K1364002</a>, <a href="http://parts.igem.org/Part:BBa_K1364003"target="_blank">BBa_K1364003</a>, <a href="http://parts.igem.org/Part:BBa_K1364005"target="_blank">BBa_K1364005</a>. </td><br />
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{{:Team:Toulouse/template/footer}}</div>Jourdanhttp://2014.igem.org/Team:Toulouse/Result/parts/Submitted_partsTeam:Toulouse/Result/parts/Submitted parts2014-10-18T01:24:29Z<p>Jourdan: </p>
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<h2>Parts</h2><br />
<p>What did we send to the Registry?</p><br />
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<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Results</a>&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;<a href="https://2014.igem.org/Team:Toulouse/Result/parts">Parts</a>&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Submitted parts</p> <br />
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We deposited 16 new BioBrick parts in the Registry. <br />
Most of them were cloned into the standard plasmid pSB1C3, tested and sequenced.<br><br />
All our Biobrick are resumed in <a href="http://parts.igem.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2014&group=Toulouse&Done=1">Parts Sandbox</a>.<br />
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<p class="title1">Chemotaxis</p><br />
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<a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</a></p></td><br />
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<div class="technology2">Strong RBS - N-acetylglucosamine based chemotaxis for <i>Bacillus subtilis</i> - Double terminator</div><br />
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<p class="texte">This part is designed to enable a N-acetylglucosamine <br />
based chemotaxis in <i>B. subtilis</i>.</p><br />
<p class="title4">Design</p><br />
<p class="texte">The encoded protein is a chimera of two proteins:<br><br />
- the methyl accepting Chemotaxis protein (McpA from <i>B. subtilis</i> which is required for taxis towards glucose.<br><br />
- the N-acetylglucosamine regulated methyl-accepting chemotaxis protein from <i>Vibrio cholerae</i> (VCD).<br><br />
<br><br />
The chimeric protein contains the intracellular domains of McpA from <i>Bacillus subtilis</i> to enable the transduction of the signal: <br />
these domains correspond to the amino acids 1-37 and 282-610. <br />
The extracellular domain of VCD is inserted between the intracellular regions of McpA <br />
to sense N-acetylglucosamine and corresponds to the amino acids 31-310.</p><br />
<img src="https://static.igem.org/mediawiki/2014/a/a2/BBa_K1364000.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Translation unit</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part is not tested yet.</p><br />
<p class="title4">References</p><br />
<p class="texte">Uniprot:<br><br />
- <a href="http://www.uniprot.org/uniprot/P39214">McpA</a><br><br />
- <a href="http://www.uniprot.org/uniprot/C3NYT2">N-acetylglucosamine regulated methyl-accepting chemotaxis protein</a></p><br />
<!--à compléter--><br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology2', 0); return false">Collapse</a></p><br />
</div></td></table><br />
<br />
<table><br />
<td valign="top"; align="center";><br />
<p class="title2";><br />
<a href="http://parts.igem.org/Part:BBa_K1364004">BBa_K1364004</a></p></td><br />
<td valign="top"; align="center";><br />
<div class="technology2">P<sub>veg</sub> - Strong RBS - N-acetylglucosamine based chemotaxis for <i>Bacillus subtilis</i> - Double terminator</div><br />
<div class="thelanguage2"><br />
<p class="texte">This part is designed to enable a N-acetylglucosamine based chemotaxis in <i>Bacillus subtilis</i>.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This expression cassette is designed for the expression of an antifungal peptide, <br />
D4E1 and for its secretion in <i>B. subtilis</i>.<br />
It is composed of the strong, constitutive promoter of <i>B. subtilis</i> <br />
P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">BBa_K823003</a>),<br />
strong RBS for <i>B. subtilis</i> (<a href="http://parts.igem.org/Part:BBa_K780002">BBa_K780002</a>),<br />
the open reading frame of D4E1<br />
and a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">BBa_B0015</a>)</p><br />
<img src="https://static.igem.org/mediawiki/2014/3/35/BBa_K1364004.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was not tested yet.</p><br />
<p class="title4">References</p><br />
<p class="texte">Uniprot:<br><br />
- <a href="http://www.uniprot.org/uniprot/P39214">McpA</a><br><br />
- <a href="http://www.uniprot.org/uniprot/C3NYT2">N-acetylglucosamine regulated methyl-accepting chemotaxis protein</a></p><br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology2', 1); return false">Collapse</a></p><br />
</div></td></table><br />
<br />
<br></br><br />
<br />
<p class="title1">Binding</p><br />
<table><br />
<td valign="top"; align="center";><br />
<p class="title2";><br />
<a href="http://parts.igem.org/Part:BBa_K1364005">BBa_K1364005</a></p></td><br />
<td valign="top"; align="center";><br />
<div class="technology2"></a>P<sub>veg</sub> - RBS consensus - Chitin Binding protein - Double terminator</div><br />
<div class="thelanguage2"><br />
<br />
<p class="texte">This part is designed to enable the binding of <i>Bacillus subtilis</i> to the fungi wall made of chitin.<p/><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a Cell Wall Binding (CWB) sequence <br />
and a Chitin Binding Domain (CBD) linked with a 6 amino acids linker.<br><br />
<br><br />
The CWB domain has been extracted from LytC (<a href="http://parts.igem.org/Part:BBa_K316030">BBa_K316030</a>) of the iGEM 2010 Imperial College team.<br><br />
The CBD is composed of the Domain 4 of the GbpA protein of <i>Vibrio cholerae</i>,<br />
a protein reported to mediate bacterial attachment to chitin. <br />
It has been shown that the Domain 4 was essential to bind chitin.</p><br />
<img src="https://static.igem.org/mediawiki/2014/f/f5/BBa_K1364005.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested with chitin beads. (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Binding module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)<br><br />
- The <i>Vibrio cholerae</i> Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373<br />
</p><br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology2', 2); return false">Collapse</a></p><br />
</div></td></table><br />
<br />
<br></br><br />
<br />
<p class="title1">Fungicides</p><br />
<p class="title3">GAFP-1</p><br />
<br />
<br><br />
<table><br />
<td valign="top"; align="center";><br />
<p class="title2";><br />
<a href="http://parts.igem.org/Part:BBa_K1364002">BBa_K1364002</a></p></td><br />
<td valign="top"; align="center";><br />
<div class="technology2"> Strong RBS - Antifungal GAFP-1</div><br />
<div class="thelanguage2"><br />
<br />
<p class="texte">The <i>Gastrodia</i> anti-fungal protein(GAFP-1), also known as gastrodianin, is a mannose and chitin binding lectin<br />
originating from the Asiatic orchid <i>Gastrodia elata</i>, a traditional Chinese medicinal herb cultured for thousands of years.<br />
GAFP-1 is composed of 15 amino acids LDSLSFSYNNFEEDD and is able to inhibit the growth of multiples species of plant pathogenic fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a Strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">BBa_K780002</a>) and<br />
the open reading frame of the <i>Gastrodia</i> anti-fungal protein 1 (GAFP-1)<br />
optimized for its expression and its secretion in <i>Bacillus subtilis</i>.<br />
The codon optimization was made thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/5/5f/BBa_K1364002.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Translational unit</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)<br><br />
- The <i>Vibrio cholerae</i> Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373</p><br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology2', 3); return false">Collapse</a></p><br />
</div></td></table><br />
<br />
<table><br />
<td valign="top"; align="center";><br />
<p class="title2";><br />
<a href="http://parts.igem.org/Part:BBa_K1364007">BBa_K1364007</a></p></td><br />
<td valign="top"; align="center";><br />
<div class="technology2">Strong RBS - Antifungal GAFP-1 + Double terminator</div><br />
<div class="thelanguage2"><br />
<p class="texte">The <i>Gastrodia</i> anti-fungal protein(GAFP-1), also known as gastrodianin, is a mannose and chitin binding lectin<br />
originating from the Asiatic orchid <i>Gastrodia elata</i>, a traditional Chinese medicinal herb cultured for thousands of years.<br />
GAFP-1 is composed of 15 amino acids LDSLSFSYNNFEEDD and is able to inhibit the growth of multiples species of plant pathogenic fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a Strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">BBa_K780002</a>),<br />
the open reading frame of the <i>Gastrodia</i> anti-fungal protein 1 (GAFP-1) <br />
and a double terminator <a href="http://parts.igem.org/Part:BBa_B0015">BBa_B0015</a>.<br />
optimized for its expression and its secretion in <i>Bacillus subtilis</i>.<br />
The codon optimization was made thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/4/42/BBa_K1364007.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Composite part</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)<br><br />
- The <i>Vibrio cholerae</i> Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373</p><br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology2', 4); return false">Collapse</a></p><br />
</div></td></table><br />
<br />
<table><br />
<td valign="top";align="center";><br />
<p class="title2";><br />
<a href="http://parts.igem.org/Part:BBa_K1364008">BBa_K1364008</a></p></td><br />
<td valign="top"; align="center";><br />
<div class="technology2"> P<sub>veg</sub> - Strong RBS - Antifungal GAFP-1 - Double terminator</div><br />
<div class="thelanguage2"><br />
<p class="texte">This expression cassette is designed for the expression and secretion of the <i>Gastrodia</i> anti-fungal protein(GAFP-1), also known as gastrodianin. GAFP-1 is a mannose and chitin binding lectin<br />
originating from the Asiatic orchid <i>Gastrodia elata</i>, a traditional Chinese medicinal herb cultured for thousands of years.<br />
GAFP-1 is composed of 15 amino acids LDSLSFSYNNFEEDD and is able to inhibit the growth of multiples species of plant pathogenic fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of the strong, constitutive promoter of <i>Bacillus subtilis</i> P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">BBa_K823003</a>),<br />
strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">BBa_K780002</a>),<br />
the open reading frame of the <i>Gastrodia</i> anti-fungal protein 1 (GAFP-1) <br />
and a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>).<br />
optimized for its expression and its secretion in <i>B. subtilis</i>.<br />
The codon optimization was made thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/parts/4/47/1364008a.jpg"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)<br><br />
- The <i>Vibrio cholerae</i> Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373<br />
</p><br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology2', 5); return false">Collapse</a></p><br />
</div></td></table><br />
<br />
<br></br><br />
<br />
<p class="title3">D4E1</p><br />
<br />
<br><br />
<table><br />
<td valign="top"; align="center";><br />
<p class="title2";><br />
<a href="http://parts.igem.org/Part:BBa_K1364003">BBa_K1364003</a></p></td><br />
<td valign="top"; align="center";><br />
<div class="technology2">Strong RBS - Antifungal D4E1 - Double terminator</div><br />
<div class="thelanguage2"><br />
<p class="texte">D4E1 is a linear synthetic peptide of 17 amino acids <br />
which has shown to have antifungal activities <br />
by complexing with a sterol present in conodial wall of a varety of fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a Strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">BBa_K780002</a>),<br />
the open reading frame of D4E1 and <br />
a Double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">BBa_B0015</a>)<br />
optimized for its expression and its secretion in <i>Bacillus subtilis</i>.<br />
This part was optimized for the expression and its secretion in <i>B. subtilis </i><br />
thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/b/bb/BBa_K1364003.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Translational unit</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- De Lucca AJ, Bland JM, Grimm C, Jacks TJ, Cary JW, Jaynes JM, Cleveland TE, Walsh TJ. Fungicidal properties, sterol binding, and proteolytic resistance of the synthetic peptide D4E1.<br><br />
- Can J Microbiol. 1998 Jun;44(6):514-20.</p><br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology2', 6); return false">Collapse</a></p><br />
</div></td></table><br />
<br />
<table><br />
<td valign="top"; align="center";><br />
<p class="title2";><br />
<a href="http://parts.igem.org/Part:BBa_K1364009">BBa_K1364009</a></p></td><br />
<td valign="top"; align="center";><br />
<div class="technology2">P<sub>veg</sub> - Strong RBS - Antifungal D4E1 - Double Terminator</div><br />
<div class="thelanguage2"><br />
<p class="texte"><br />
D4E1 is a linear synthetic peptide of 17 amino acids <br />
which has shown to have antifungal activities <br />
by complexing with a sterol present in conodial wall of a varety of fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of the strong, constitutive promoter of <i>Bacillus subtilis</i> <br />
P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">BBa_K823003</a>),<br />
a strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">BBa_K780002</a>),<br />
the open reading frame of D4E1 and <br />
a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">BBa_B0015</a>).<br />
This part was optimized for the expression and its secretion in <i>B.subtilis </i><br />
thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/4/47/BBa_K1364009.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Translational unit</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- De Lucca AJ, Bland JM, Grimm C, Jacks TJ, Cary JW, Jaynes JM, Cleveland TE, Walsh TJ. Fungicidal properties, sterol binding, and proteolytic resistance of the synthetic peptide D4E1.<br><br />
- Can J Microbiol. 1998 Jun;44(6):514-20.</p><br />
<br />
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</div></td></table><br />
<br />
<br></br><br />
<br />
<p class="title3">EcAMP-1</p><br />
<br />
<table><br />
<td valign="top"; align="center";><br />
<p class="title2";><br />
<a href="http://parts.igem.org/Part:BBa_K1364010">BBa_K1364010</a></p></td><br />
<td valign="top"; align="center";><br />
<div class="technology2">P<sub>veg</sub> - RBS SpoVG - Antifungal EcAMP-1</div><br />
<div class="thelanguage2"><br />
<p class="texte"><br />
EcAMP-1 is an antimicrobial peptide of 37 amino acids <br />
originated from the specie <i>Echinochloa crus-galli</i>, <br />
a type of wild grass. <br />
This peptide has a particular structure : it is helical because of two disulfide bonds.<br><br />
EcAMP-1 has shown to have antifungal activities. Its mode of action may be the prevention of hyphae elongation.<br><br />
This part was added to the Registry by the iGEM Utah State team in 2013.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of the constitutive promoter P<sub>veg</sub> and<br />
strong RBS for <i>Bacillus subtilis</i><br />
(<a href="http://parts.igem.org/Part:BBa_K733013">BBa_K733013</a>) and<br />
the open reading frame of EcAMP-1.<br><br />
The EcAMP-1 part was codon optimized for <i>E. coli</i> by the iGEM Utah State team<br />
and thanks to the Life Technologies GeneArt software program. </p><br />
<img src="https://static.igem.org/mediawiki/2014/5/5f/BBa_K1364010.png"><br />
<p class="title4">Type</p><br />
<p class="texte"> Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part is not tested yet.</p><br />
<p class="title4">References</p><br />
<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><br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology2', 8); return false">Collapse</a></p><br />
</div></td></table><br />
<br />
<table><br />
<td valign="top"; align="center";><br />
<p class="title2";><br />
<a href="http://parts.igem.org/Part:BBa_K1364011">BBa_K1364011</a></p></td><br />
<td valign="top"; align="center";><br />
<div class="technology2"> P<sub>veg</sub> - RBS SpoVG - Antifungal EcAMP-1 - Double terminator</div><br />
<div class="thelanguage2"><br />
<br />
<p class="texte" id="select10"><br />
EcAMP-1 is an antimicrobial peptide of 37 amino acids <br />
originated from the specie <i>Echinochloa crus-galli</i>, <br />
a type of wild grass. <br />
This peptide has a particular structure : it is helical because of two disulfide bonds.<br><br />
EcAMP-1 has shown to have antifungal activities. Its mode of action may be the prevention of hyphae elongation.<br><br />
This part was added to the Registry by the iGEM Utah State team in 2013.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of the constitutive promoter P<sub>veg</sub> and<br />
strong RBS for <i>Bacillus subtilis</i><br />
(<a href="http://parts.igem.org/Part:BBa_K733013">BBa_K733013</a>),<br />
the open reading frame of EcAMP-1 and <br />
a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">BBa_B0015</a>).<br><br />
The EcAMP-1 part was codon optimized for <i>Escherichia coli</i> by the iGEM Utah State team<br />
and thanks to the Life Technologies GeneArt software program. </p><br />
<img src="https://static.igem.org/mediawiki/2014/7/74/BBa_K1364011.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part is not tested yet.</p><br />
<p class="title4">References</p><br />
<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><br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology2', 9); return false">Collapse</a></p><br />
</div></td></table><br />
<br />
<table><br />
<td valign="top"; align="center";><br />
<p class="title2";><br />
<a href="http://parts.igem.org/Part:BBa_K1364019">BBa_K1364019</a></p></td><br />
<td valign="top"; align="center";><br />
<div class="technology2">P<sub>veg</sub> - RBS - Antifungal EcAMP-1 (revised with a stop codon) - Double terminator</div><br />
<div class="thelanguage2"><br />
<br />
<p class="texte">EcAMP-1 is an antimicrobial peptide of 37 amino acids <br />
originated from the specie <i>Echinochloa crus-galli</i>, <br />
a type of wild grass. <br />
This peptide has a particular structure : it is helical because of two disulfide bonds.<br><br />
EcAMP-1 has shown to have antifungal activities. Its mode of action may be the prevention of hyphae elongation.<br><br />
This part was added to the Registry by the iGEM Utah State team in 2013.</br><br />
We observed the presence of the stop codon in the suffix. When we have digested this Biobrick, it disappeared. So we added this codon in upstream of the suffix and now, we can reuse this Biobrick.<br />
</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of the constitutive promoter P<sub>veg</sub> and<br />
strong RBS for <i>Bacillus subtilis</i><br />
(<a href="http://parts.igem.org/Part:BBa_K733013">BBa_K733013</a>),<br />
<b>the revised open reading frame of EcAMP-1 (with a stop codon) </b> and <br />
a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">BBa_B0015</a>).<br><br />
The EcAMP-1 part was codon optimized for <i>Escherichia coli</i> by the iGEM Utah State team<br />
and thanks to the Life Technologies GeneArt software program. </p><br />
<img src="https://static.igem.org/mediawiki/2014/7/74/BBa_K1364011.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Composite</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was not tested yet</p><br />
<p class="title4">References</p><br />
<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><br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology2', 13); return false">Collapse</a></p><br />
</div></td></table><br />
<br />
<br></br><br />
<br />
<p class="title3">Fungicide operons</p><br />
<br />
<table><br />
<td valign="top"; align="center";><br />
<p class="title2";><br />
<a href="http://parts.igem.org/Part:BBa_K1364013">BBa_K1364013</a></p></td><br />
<td valign="top"; align="center";><br />
<div class="technology2">P<sub>veg</sub> - Strong RBS - Antifungal GAFP-1 - Strong RBS - Antifungal D4E1 - Double terminator</div><br />
<div class="thelanguage2"><br />
<p class="texte"><br />
<p class="title4">Design</p><br />
<p class="texte">This part is designed for the co-expression of two different peptides <br />
with anti-fungal activities : D4E1 and GAFP-1. <br />
It is composed of the strong, constitutive promoter of <i>Bacillus subtilis</i> <br />
P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">BBa_K823003</a>) <br />
and the translation unit with GAFP-1 and D4E1 (BBa_K1364012).</p><br />
<img src="https://static.igem.org/mediawiki/2014/4/40/BBa_K1364013.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
See <a href="http://parts.igem.org/Part:BBa_K1364002">BBa_K1364002</a> and <a href="http://parts.igem.org/Part:BBa_K1364003">BBa_K1364003</a>.</p><br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology2', 10); return false">Collapse</a></p><br />
</div></td></table><br />
<br />
<br></br><br />
<br />
<p class="title1">Basic tools</p><br />
<br />
<table><br />
<td valign="top"; align="center";><br />
<p class="title2";><br />
<a href="http://parts.igem.org/Part:BBa_K1364015">BBa_K1364015</a></p></td><br />
<td valign="top"; align="center";><br />
<div class="technology2">P<sub>veg</sub> - SpoVG RBS - RFP - Double terminator </div><br />
<div class="thelanguage2"><br />
<br />
<p class="texte"><!--Compléter--><br />
This part is designed to enable the expression of a Red Fluorescent Protein in <i>Bacillus subtilis</i> under the control of a constitutive promoter P<sub>veg</sub>. This construction has been checked by sequencing and has shown to work also in <i>Escherichia coli</i><br />
</p><br />
<img src="https://static.igem.org/mediawiki/parts/6/69/Pveg%2BRFP.jpg"><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a constitutive promoter P<sub>veg</sub> (BBa_K823003), spoVG RBS (BBa_KK143021), the coding sequence of the RFP (E1010 from BBa_K606013) and a double terminator (BBa_KB0015) </p><br />
<p class="title4">Type</p><br />
<p class="texte">Reporter</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested in both <i>E. coli</i> and <i>B. subtilis</i>. The sequences was verified by sequencing. </p><br />
<br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology2', 10); return false">Collapse</a></p><br />
</div></td></table><br />
<br />
<table><br />
<td valign="top"; align="center";><br />
<p class="title2";><br />
<a href="http://parts.igem.org/Part:BBa_K1364016">BBa_K1364016</a></p></td><br />
<td valign="top"; align="center";><br />
<div class="technology2"> P<sub>lepA</sub> - SpoVG RBS - RFP - Double terminator </div><br />
<div class="thelanguage2"><br />
<br />
<p class="texte"><!--Compléter--><br />
This part is designed to enable the expression of a Red Fluorescent Protein in <i>Bacillus subtilis</i> under the control of a constitutive promoter P<sub>lepA</sub>. This construction has been checked by sequencing and has shown to work also in <i>Escherichia coli</i><br />
</p><br />
<img src="https://static.igem.org/mediawiki/parts/1/12/PlepA%2BRFP.jpg"><br />
<br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a constitutive promoter P<sub>lepA</sub> (BBa_K823002), spoVG RBS (BBa_K143021), the coding sequence of the RFP (E1010 from BBa_K606013) and a double terminator (BBa_B0015) </p><br />
<p class="title4">Type</p><br />
<p class="texte">Reporter</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested in both <i>E. coli</i> and <i>B. subtilis</i>. The sequences was verified by sequencing.</p><br />
<br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology2', 11); return false">Collapse</a></p><br />
</div></td></table><br />
<br />
<table><br />
<td valign="top"; align="center";><br />
<p class="title2";><br />
<a href="http://parts.igem.org/Part:BBa_K1364017">BBa_K1364017</a></p></td><br />
<td valign="top"; align="center";><br />
<div class="technology2">P<sub>lepA</sub> - SpoVG RBS</div><br />
<div class="thelanguage2"><br />
<br />
<p class="texte"><!--Compléter--><br />
P<sub>lepA</sub> is a constitutive promoter in <i>Bacillus subtilis</i> (BBa_K823002) coupled with a RBS spoVG (BBa_K143021). To get the highest level of translation from this Promoter-RBS combination it must be connected to a coding region preceded by a coding region prefix. A standard prefix will increase the distance between the RBS and the start codon, reducing translational efficiency. <br />
This construction is also working with <i>Escherichia coli</i> and has been verified by sequencing.<br />
</p><br />
<img src="https://static.igem.org/mediawiki/parts/d/da/PlepA%2BSpoVG.jpg"><br />
<p class="title4">Type</p><br />
<p class="texte">Composite</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was checked by sequencing</p><br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology2', 12); return false">Collapse</a></p><br />
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<br />
<table><br />
<td valign="top"; align="center";><br />
<p class="title2";><br />
<a href="http://parts.igem.org/Part:BBa_K1364021">BBa_K1364021</a></p></td><br />
<td valign="top"; align="center";><br />
<div class="technology2">Integrative plasmid for <i>Bacillus subtilis</i> (pSB<sub>bs</sub>4E)</div><br />
<div class="thelanguage2"><br />
<br />
<p class="texte">This part is an empty backbone vector for the usage in <i>Bacillus subtilis</i>. It is on biobrick version of pKL190 which integrates in the thrC locus and can be selected with Erythromycin . It has an Ampicillin resistance for cloning in <i>Escherichia coli</i>. The backbone contains an RFP in the BioBrick site (BBa_J04450) to facilitate the cloning in <i>E.coli</i>. <br><br />
The handling of this type of vector is described <a href="https://2014.igem.org/Team:Toulouse/Notebook/Protocols#select6">here</a>.<br />
<br />
</p><br />
<p class="title4">Design</p><br />
<p class="texte">We digested pKL190 plasmid and PCR products of BBa_J04450 with BamHI and EcoRI. We proceded to ligation.</p><br />
<img src="https://static.igem.org/mediawiki/2014/archive/6/6d/20141017182537!PKL190_map.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Backbone</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested by transforming <i>B. subtilis</i>. Clones obtained were Threonine-dependant and resistant to Erythromycin (10µg/ml).</p><br />
<p class="title4">References</p><br />
<p class="texte"><br />
R. Bernard, K.A. Marquis, and D.Z. Rudner. <b>Nucleoid occlusion prevents cell division during replication fork arrest in <i>Bacillus subtilis</i></b>Mol Microbiol. Nov 2010; 78(4): 866–882.</p><br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology2', 13); return false">Collapse</a></p><br />
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{{:Team:Toulouse/template/footer}}</div>Jourdanhttp://2014.igem.org/Team:Toulouse/Result/parts/Submitted_partsTeam:Toulouse/Result/parts/Submitted parts2014-10-18T01:22:38Z<p>Jourdan: </p>
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<h2>Parts</h2><br />
<p>What did we send to the Registry?</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Results</a>&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;<a href="https://2014.igem.org/Team:Toulouse/Result/parts">Parts</a>&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Submitted parts</p> <br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 20px; padding-bottom:40px;"><br />
<br />
<br />
<p class="texte"><br />
We deposited 16 new BioBrick parts in the Registry. <br />
Most of them were cloned into the standard plasmid pSB1C3, tested and sequenced.<br><br />
All our Biobrick are resumed in <a href="http://parts.igem.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2014&group=Toulouse&Done=1">Parts Sandbox</a>.<br />
<!--à vérifier et confirmer--><br />
</p><br />
<br />
<p class="title1">Chemotaxis</p><br />
<br />
<table><br />
<td valign="top"; align="center";><br />
<p class="title2";><br />
<a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</a></p></td><br />
<td valign="top"; align="center";><br />
<div class="technology2">Strong RBS - N-acetylglucosamine based chemotaxis for <i>Bacillus subtilis</i> - Double terminator</div><br />
<div class="thelanguage2"><br />
<p class="texte">This part is designed to enable a N-acetylglucosamine <br />
based chemotaxis in <i>B. subtilis</i>.</p><br />
<p class="title4">Design</p><br />
<p class="texte">The encoded protein is a chimera of two proteins:<br><br />
- the methyl accepting Chemotaxis protein (McpA from <i>B. subtilis</i> which is required for taxis towards glucose.<br><br />
- the N-acetylglucosamine regulated methyl-accepting chemotaxis protein from <i>Vibrio cholerae</i> (VCD).<br><br />
<br><br />
The chimeric protein contains the intracellular domains of McpA from <i>Bacillus subtilis</i> to enable the transduction of the signal: <br />
these domains correspond to the amino acids 1-37 and 282-610. <br />
The extracellular domain of VCD is inserted between the intracellular regions of McpA <br />
to sense N-acetylglucosamine and corresponds to the amino acids 31-310.</p><br />
<img src="https://static.igem.org/mediawiki/2014/a/a2/BBa_K1364000.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Translation unit</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part is not tested yet.</p><br />
<p class="title4">References</p><br />
<p class="texte">Uniprot:<br><br />
- <a href="http://www.uniprot.org/uniprot/P39214">McpA</a><br><br />
- <a href="http://www.uniprot.org/uniprot/C3NYT2">N-acetylglucosamine regulated methyl-accepting chemotaxis protein</a></p><br />
<!--à compléter--><br />
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</div></td></table><br />
<br />
<table><br />
<td valign="top"; align="center";><br />
<p class="title2";><br />
<a href="http://parts.igem.org/Part:BBa_K1364004">BBa_K1364004</a></p></td><br />
<td valign="top"; align="center";><br />
<div class="technology2">P<sub>veg</sub> - Strong RBS - N-acetylglucosamine based chemotaxis for <i>Bacillus subtilis</i> - Double terminator</div><br />
<div class="thelanguage2"><br />
<p class="texte">This part is designed to enable a N-acetylglucosamine based chemotaxis in <i>Bacillus subtilis</i>.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This expression cassette is designed for the expression of an antifungal peptide, <br />
D4E1 and for its secretion in <i>B. subtilis</i>.<br />
It is composed of the strong, constitutive promoter of <i>B. subtilis</i> <br />
P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">BBa_K823003</a>),<br />
strong RBS for <i>B. subtilis</i> (<a href="http://parts.igem.org/Part:BBa_K780002">BBa_K780002</a>),<br />
the open reading frame of D4E1<br />
and a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">BBa_B0015</a>)</p><br />
<img src="https://static.igem.org/mediawiki/2014/3/35/BBa_K1364004.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was not tested yet.</p><br />
<p class="title4">References</p><br />
<p class="texte">Uniprot:<br><br />
- <a href="http://www.uniprot.org/uniprot/P39214">McpA</a><br><br />
- <a href="http://www.uniprot.org/uniprot/C3NYT2">N-acetylglucosamine regulated methyl-accepting chemotaxis protein</a></p><br />
<br />
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<br></br><br />
<br />
<p class="title1">Binding</p><br />
<table><br />
<td valign="top"; align="center";><br />
<p class="title2";><br />
<a href="http://parts.igem.org/Part:BBa_K1364005">BBa_K1364005</a></p></td><br />
<td valign="top"; align="center";><br />
<div class="technology2"></a>P<sub>veg</sub> - RBS consensus - Chitin Binding protein - Double terminator</div><br />
<div class="thelanguage2"><br />
<br />
<p class="texte">This part is designed to enable the binding of <i>Bacillus subtilis</i> to the fungi wall made of chitin.<p/><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a Cell Wall Binding (CWB) sequence <br />
and a Chitin Binding Domain (CBD) linked with a 6 amino acids linker.<br><br />
<br><br />
The CWB domain has been extracted from LytC (<a href="http://parts.igem.org/Part:BBa_K316030">BBa_K316030</a>) of the iGEM 2010 Imperial College team.<br><br />
The CBD is composed of the Domain 4 of the GbpA protein of <i>Vibrio cholerae</i>,<br />
a protein reported to mediate bacterial attachment to chitin. <br />
It has been shown that the Domain 4 was essential to bind chitin.</p><br />
<img src="https://static.igem.org/mediawiki/2014/f/f5/BBa_K1364005.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested with chitin beads. (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Binding module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)<br><br />
- The <i>Vibrio cholerae</i> Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373<br />
</p><br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology2', 2); return false">Collapse</a></p><br />
</div></td></table><br />
<br />
<br></br><br />
<br />
<p class="title1">Fungicides</p><br />
<p class="title3">GAFP-1</p><br />
<br />
<br><br />
<table><br />
<td valign="top"; align="center";><br />
<p class="title2";><br />
<a href="http://parts.igem.org/Part:BBa_K1364002">BBa_K1364002</a></p></td><br />
<td valign="top"; align="center";><br />
<div class="technology2"> Strong RBS - Antifungal GAFP-1</div><br />
<div class="thelanguage2"><br />
<br />
<p class="texte">The <i>Gastrodia</i> anti-fungal protein(GAFP-1), also known as gastrodianin, is a mannose and chitin binding lectin<br />
originating from the Asiatic orchid <i>Gastrodia elata</i>, a traditional Chinese medicinal herb cultured for thousands of years.<br />
GAFP-1 is composed of 15 amino acids LDSLSFSYNNFEEDD and is able to inhibit the growth of multiples species of plant pathogenic fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a Strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">BBa_K780002</a>) and<br />
the open reading frame of the <i>Gastrodia</i> anti-fungal protein 1 (GAFP-1)<br />
optimized for its expression and its secretion in <i>Bacillus subtilis</i>.<br />
The codon optimization was made thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/parts/5/55/BBa_K1364002.jpg"><br />
<p class="title4">Type</p><br />
<p class="texte">Translational unit</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)<br><br />
- The <i>Vibrio cholerae</i> Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373</p><br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology2', 3); return false">Collapse</a></p><br />
</div></td></table><br />
<br />
<table><br />
<td valign="top"; align="center";><br />
<p class="title2";><br />
<a href="http://parts.igem.org/Part:BBa_K1364007">BBa_K1364007</a></p></td><br />
<td valign="top"; align="center";><br />
<div class="technology2">Strong RBS - Antifungal GAFP-1 + Double terminator</div><br />
<div class="thelanguage2"><br />
<p class="texte">The <i>Gastrodia</i> anti-fungal protein(GAFP-1), also known as gastrodianin, is a mannose and chitin binding lectin<br />
originating from the Asiatic orchid <i>Gastrodia elata</i>, a traditional Chinese medicinal herb cultured for thousands of years.<br />
GAFP-1 is composed of 15 amino acids LDSLSFSYNNFEEDD and is able to inhibit the growth of multiples species of plant pathogenic fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a Strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">BBa_K780002</a>),<br />
the open reading frame of the <i>Gastrodia</i> anti-fungal protein 1 (GAFP-1) <br />
and a double terminator <a href="http://parts.igem.org/Part:BBa_B0015">BBa_B0015</a>.<br />
optimized for its expression and its secretion in <i>Bacillus subtilis</i>.<br />
The codon optimization was made thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/parts/4/47/1364008a.jpg"><br />
<p class="title4">Type</p><br />
<p class="texte">Composite part</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)<br><br />
- The <i>Vibrio cholerae</i> Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373</p><br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology2', 4); return false">Collapse</a></p><br />
</div></td></table><br />
<br />
<table><br />
<td valign="top";align="center";><br />
<p class="title2";><br />
<a href="http://parts.igem.org/Part:BBa_K1364008">BBa_K1364008</a></p></td><br />
<td valign="top"; align="center";><br />
<div class="technology2"> P<sub>veg</sub> - Strong RBS - Antifungal GAFP-1 - Double terminator</div><br />
<div class="thelanguage2"><br />
<p class="texte">This expression cassette is designed for the expression and secretion of the <i>Gastrodia</i> anti-fungal protein(GAFP-1), also known as gastrodianin. GAFP-1 is a mannose and chitin binding lectin<br />
originating from the Asiatic orchid <i>Gastrodia elata</i>, a traditional Chinese medicinal herb cultured for thousands of years.<br />
GAFP-1 is composed of 15 amino acids LDSLSFSYNNFEEDD and is able to inhibit the growth of multiples species of plant pathogenic fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of the strong, constitutive promoter of <i>Bacillus subtilis</i> P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">BBa_K823003</a>),<br />
strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">BBa_K780002</a>),<br />
the open reading frame of the <i>Gastrodia</i> anti-fungal protein 1 (GAFP-1) <br />
and a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>).<br />
optimized for its expression and its secretion in <i>B. subtilis</i>.<br />
The codon optimization was made thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/parts/4/47/1364008a.jpg"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)<br><br />
- The <i>Vibrio cholerae</i> Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373<br />
</p><br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology2', 5); return false">Collapse</a></p><br />
</div></td></table><br />
<br />
<br></br><br />
<br />
<p class="title3">D4E1</p><br />
<br />
<br><br />
<table><br />
<td valign="top"; align="center";><br />
<p class="title2";><br />
<a href="http://parts.igem.org/Part:BBa_K1364003">BBa_K1364003</a></p></td><br />
<td valign="top"; align="center";><br />
<div class="technology2">Strong RBS - Antifungal D4E1 - Double terminator</div><br />
<div class="thelanguage2"><br />
<p class="texte">D4E1 is a linear synthetic peptide of 17 amino acids <br />
which has shown to have antifungal activities <br />
by complexing with a sterol present in conodial wall of a varety of fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a Strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">BBa_K780002</a>),<br />
the open reading frame of D4E1 and <br />
a Double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">BBa_B0015</a>)<br />
optimized for its expression and its secretion in <i>Bacillus subtilis</i>.<br />
This part was optimized for the expression and its secretion in <i>B. subtilis </i><br />
thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/parts/a/a8/K1364003a.jpg"><br />
<p class="title4">Type</p><br />
<p class="texte">Translational unit</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- De Lucca AJ, Bland JM, Grimm C, Jacks TJ, Cary JW, Jaynes JM, Cleveland TE, Walsh TJ. Fungicidal properties, sterol binding, and proteolytic resistance of the synthetic peptide D4E1.<br><br />
- Can J Microbiol. 1998 Jun;44(6):514-20.</p><br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology2', 6); return false">Collapse</a></p><br />
</div></td></table><br />
<br />
<table><br />
<td valign="top"; align="center";><br />
<p class="title2";><br />
<a href="http://parts.igem.org/Part:BBa_K1364009">BBa_K1364009</a></p></td><br />
<td valign="top"; align="center";><br />
<div class="technology2">P<sub>veg</sub> - Strong RBS - Antifungal D4E1 - Double Terminator</div><br />
<div class="thelanguage2"><br />
<p class="texte"><br />
D4E1 is a linear synthetic peptide of 17 amino acids <br />
which has shown to have antifungal activities <br />
by complexing with a sterol present in conodial wall of a varety of fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of the strong, constitutive promoter of <i>Bacillus subtilis</i> <br />
P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">BBa_K823003</a>),<br />
a strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">BBa_K780002</a>),<br />
the open reading frame of D4E1 and <br />
a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">BBa_B0015</a>).<br />
This part was optimized for the expression and its secretion in <i>B.subtilis </i><br />
thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/4/47/BBa_K1364009.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Translational unit</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- De Lucca AJ, Bland JM, Grimm C, Jacks TJ, Cary JW, Jaynes JM, Cleveland TE, Walsh TJ. Fungicidal properties, sterol binding, and proteolytic resistance of the synthetic peptide D4E1.<br><br />
- Can J Microbiol. 1998 Jun;44(6):514-20.</p><br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology2', 7); return false">Collapse</a></p><br />
</div></td></table><br />
<br />
<br></br><br />
<br />
<p class="title3">EcAMP-1</p><br />
<br />
<table><br />
<td valign="top"; align="center";><br />
<p class="title2";><br />
<a href="http://parts.igem.org/Part:BBa_K1364010">BBa_K1364010</a></p></td><br />
<td valign="top"; align="center";><br />
<div class="technology2">P<sub>veg</sub> - RBS SpoVG - Antifungal EcAMP-1</div><br />
<div class="thelanguage2"><br />
<p class="texte"><br />
EcAMP-1 is an antimicrobial peptide of 37 amino acids <br />
originated from the specie <i>Echinochloa crus-galli</i>, <br />
a type of wild grass. <br />
This peptide has a particular structure : it is helical because of two disulfide bonds.<br><br />
EcAMP-1 has shown to have antifungal activities. Its mode of action may be the prevention of hyphae elongation.<br><br />
This part was added to the Registry by the iGEM Utah State team in 2013.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of the constitutive promoter P<sub>veg</sub> and<br />
strong RBS for <i>Bacillus subtilis</i><br />
(<a href="http://parts.igem.org/Part:BBa_K733013">BBa_K733013</a>) and<br />
the open reading frame of EcAMP-1.<br><br />
The EcAMP-1 part was codon optimized for <i>E. coli</i> by the iGEM Utah State team<br />
and thanks to the Life Technologies GeneArt software program. </p><br />
<img src="https://static.igem.org/mediawiki/2014/5/5f/BBa_K1364010.png"><br />
<p class="title4">Type</p><br />
<p class="texte"> Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part is not tested yet.</p><br />
<p class="title4">References</p><br />
<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><br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology2', 8); return false">Collapse</a></p><br />
</div></td></table><br />
<br />
<table><br />
<td valign="top"; align="center";><br />
<p class="title2";><br />
<a href="http://parts.igem.org/Part:BBa_K1364011">BBa_K1364011</a></p></td><br />
<td valign="top"; align="center";><br />
<div class="technology2"> P<sub>veg</sub> - RBS SpoVG - Antifungal EcAMP-1 - Double terminator</div><br />
<div class="thelanguage2"><br />
<br />
<p class="texte" id="select10"><br />
EcAMP-1 is an antimicrobial peptide of 37 amino acids <br />
originated from the specie <i>Echinochloa crus-galli</i>, <br />
a type of wild grass. <br />
This peptide has a particular structure : it is helical because of two disulfide bonds.<br><br />
EcAMP-1 has shown to have antifungal activities. Its mode of action may be the prevention of hyphae elongation.<br><br />
This part was added to the Registry by the iGEM Utah State team in 2013.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of the constitutive promoter P<sub>veg</sub> and<br />
strong RBS for <i>Bacillus subtilis</i><br />
(<a href="http://parts.igem.org/Part:BBa_K733013">BBa_K733013</a>),<br />
the open reading frame of EcAMP-1 and <br />
a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">BBa_B0015</a>).<br><br />
The EcAMP-1 part was codon optimized for <i>Escherichia coli</i> by the iGEM Utah State team<br />
and thanks to the Life Technologies GeneArt software program. </p><br />
<img src="https://static.igem.org/mediawiki/2014/7/74/BBa_K1364011.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part is not tested yet.</p><br />
<p class="title4">References</p><br />
<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><br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology2', 9); return false">Collapse</a></p><br />
</div></td></table><br />
<br />
<table><br />
<td valign="top"; align="center";><br />
<p class="title2";><br />
<a href="http://parts.igem.org/Part:BBa_K1364019">BBa_K1364019</a></p></td><br />
<td valign="top"; align="center";><br />
<div class="technology2">P<sub>veg</sub> - RBS - Antifungal EcAMP-1 (revised with a stop codon) - Double terminator</div><br />
<div class="thelanguage2"><br />
<br />
<p class="texte">EcAMP-1 is an antimicrobial peptide of 37 amino acids <br />
originated from the specie <i>Echinochloa crus-galli</i>, <br />
a type of wild grass. <br />
This peptide has a particular structure : it is helical because of two disulfide bonds.<br><br />
EcAMP-1 has shown to have antifungal activities. Its mode of action may be the prevention of hyphae elongation.<br><br />
This part was added to the Registry by the iGEM Utah State team in 2013.</br><br />
We observed the presence of the stop codon in the suffix. When we have digested this Biobrick, it disappeared. So we added this codon in upstream of the suffix and now, we can reuse this Biobrick.<br />
</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of the constitutive promoter P<sub>veg</sub> and<br />
strong RBS for <i>Bacillus subtilis</i><br />
(<a href="http://parts.igem.org/Part:BBa_K733013">BBa_K733013</a>),<br />
<b>the revised open reading frame of EcAMP-1 (with a stop codon) </b> and <br />
a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">BBa_B0015</a>).<br><br />
The EcAMP-1 part was codon optimized for <i>Escherichia coli</i> by the iGEM Utah State team<br />
and thanks to the Life Technologies GeneArt software program. </p><br />
<img src="https://static.igem.org/mediawiki/2014/7/74/BBa_K1364011.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Composite</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was not tested yet</p><br />
<p class="title4">References</p><br />
<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><br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology2', 13); return false">Collapse</a></p><br />
</div></td></table><br />
<br />
<br></br><br />
<br />
<p class="title3">Fungicide operons</p><br />
<br />
<table><br />
<td valign="top"; align="center";><br />
<p class="title2";><br />
<a href="http://parts.igem.org/Part:BBa_K1364013">BBa_K1364013</a></p></td><br />
<td valign="top"; align="center";><br />
<div class="technology2">P<sub>veg</sub> - Strong RBS - Antifungal GAFP-1 - Strong RBS - Antifungal D4E1 - Double terminator</div><br />
<div class="thelanguage2"><br />
<p class="texte"><br />
<p class="title4">Design</p><br />
<p class="texte">This part is designed for the co-expression of two different peptides <br />
with anti-fungal activities : D4E1 and GAFP-1. <br />
It is composed of the strong, constitutive promoter of <i>Bacillus subtilis</i> <br />
P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">BBa_K823003</a>) <br />
and the translation unit with GAFP-1 and D4E1 (BBa_K1364012).</p><br />
<img src="https://static.igem.org/mediawiki/parts/e/e1/BBa_K1364013.jpg"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
See <a href="http://parts.igem.org/Part:BBa_K1364002">BBa_K1364002</a> and <a href="http://parts.igem.org/Part:BBa_K1364003">BBa_K1364003</a>.</p><br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology2', 10); return false">Collapse</a></p><br />
</div></td></table><br />
<br />
<br></br><br />
<br />
<p class="title1">Basic tools</p><br />
<br />
<table><br />
<td valign="top"; align="center";><br />
<p class="title2";><br />
<a href="http://parts.igem.org/Part:BBa_K1364015">BBa_K1364015</a></p></td><br />
<td valign="top"; align="center";><br />
<div class="technology2">P<sub>veg</sub> - SpoVG RBS - RFP - Double terminator </div><br />
<div class="thelanguage2"><br />
<br />
<p class="texte"><!--Compléter--><br />
This part is designed to enable the expression of a Red Fluorescent Protein in <i>Bacillus subtilis</i> under the control of a constitutive promoter P<sub>veg</sub>. This construction has been checked by sequencing and has shown to work also in <i>Escherichia coli</i><br />
</p><br />
<img src="https://static.igem.org/mediawiki/parts/6/69/Pveg%2BRFP.jpg"><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a constitutive promoter P<sub>veg</sub> (BBa_K823003), spoVG RBS (BBa_KK143021), the coding sequence of the RFP (E1010 from BBa_K606013) and a double terminator (BBa_KB0015) </p><br />
<p class="title4">Type</p><br />
<p class="texte">Reporter</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested in both <i>E. coli</i> and <i>B. subtilis</i>. The sequences was verified by sequencing. </p><br />
<br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology2', 10); return false">Collapse</a></p><br />
</div></td></table><br />
<br />
<table><br />
<td valign="top"; align="center";><br />
<p class="title2";><br />
<a href="http://parts.igem.org/Part:BBa_K1364016">BBa_K1364016</a></p></td><br />
<td valign="top"; align="center";><br />
<div class="technology2"> P<sub>lepA</sub> - SpoVG RBS - RFP - Double terminator </div><br />
<div class="thelanguage2"><br />
<br />
<p class="texte"><!--Compléter--><br />
This part is designed to enable the expression of a Red Fluorescent Protein in <i>Bacillus subtilis</i> under the control of a constitutive promoter P<sub>lepA</sub>. This construction has been checked by sequencing and has shown to work also in <i>Escherichia coli</i><br />
</p><br />
<img src="https://static.igem.org/mediawiki/parts/1/12/PlepA%2BRFP.jpg"><br />
<br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a constitutive promoter P<sub>lepA</sub> (BBa_K823002), spoVG RBS (BBa_K143021), the coding sequence of the RFP (E1010 from BBa_K606013) and a double terminator (BBa_B0015) </p><br />
<p class="title4">Type</p><br />
<p class="texte">Reporter</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested in both <i>E. coli</i> and <i>B. subtilis</i>. The sequences was verified by sequencing.</p><br />
<br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology2', 11); return false">Collapse</a></p><br />
</div></td></table><br />
<br />
<table><br />
<td valign="top"; align="center";><br />
<p class="title2";><br />
<a href="http://parts.igem.org/Part:BBa_K1364017">BBa_K1364017</a></p></td><br />
<td valign="top"; align="center";><br />
<div class="technology2">P<sub>lepA</sub> - SpoVG RBS</div><br />
<div class="thelanguage2"><br />
<br />
<p class="texte"><!--Compléter--><br />
P<sub>lepA</sub> is a constitutive promoter in <i>Bacillus subtilis</i> (BBa_K823002) coupled with a RBS spoVG (BBa_K143021). To get the highest level of translation from this Promoter-RBS combination it must be connected to a coding region preceded by a coding region prefix. A standard prefix will increase the distance between the RBS and the start codon, reducing translational efficiency. <br />
This construction is also working with <i>Escherichia coli</i> and has been verified by sequencing.<br />
</p><br />
<img src="https://static.igem.org/mediawiki/parts/d/da/PlepA%2BSpoVG.jpg"><br />
<p class="title4">Type</p><br />
<p class="texte">Composite</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was checked by sequencing</p><br />
<br />
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<p class="title2";><br />
<a href="http://parts.igem.org/Part:BBa_K1364021">BBa_K1364021</a></p></td><br />
<td valign="top"; align="center";><br />
<div class="technology2">Integrative plasmid for <i>Bacillus subtilis</i> (pSB<sub>bs</sub>4E)</div><br />
<div class="thelanguage2"><br />
<br />
<p class="texte">This part is an empty backbone vector for the usage in <i>Bacillus subtilis</i>. It is on biobrick version of pKL190 which integrates in the thrC locus and can be selected with Erythromycin . It has an Ampicillin resistance for cloning in <i>Escherichia coli</i>. The backbone contains an RFP in the BioBrick site (BBa_J04450) to facilitate the cloning in <i>E.coli</i>. <br><br />
The handling of this type of vector is described <a href="https://2014.igem.org/Team:Toulouse/Notebook/Protocols#select6">here</a>.<br />
<br />
</p><br />
<p class="title4">Design</p><br />
<p class="texte">We digested pKL190 plasmid and PCR products of BBa_J04450 with BamHI and EcoRI. We proceded to ligation.</p><br />
<img src="https://static.igem.org/mediawiki/2014/archive/6/6d/20141017182537!PKL190_map.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Backbone</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested by transforming <i>B. subtilis</i>. Clones obtained were Threonine-dependant and resistant to Erythromycin (10µg/ml).</p><br />
<p class="title4">References</p><br />
<p class="texte"><br />
R. Bernard, K.A. Marquis, and D.Z. Rudner. <b>Nucleoid occlusion prevents cell division during replication fork arrest in <i>Bacillus subtilis</i></b>Mol Microbiol. Nov 2010; 78(4): 866–882.</p><br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology2', 13); return false">Collapse</a></p><br />
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{{:Team:Toulouse/template/footer}}</div>Jourdanhttp://2014.igem.org/Team:Toulouse/Result/parts/Submitted_partsTeam:Toulouse/Result/parts/Submitted parts2014-10-18T01:09:52Z<p>Jourdan: </p>
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<div class="banniere-content"><br />
<h2>Parts</h2><br />
<p>What did we send to the Registry?</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Results</a>&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;<a href="https://2014.igem.org/Team:Toulouse/Result/parts">Parts</a>&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Submitted parts</p> <br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 20px; padding-bottom:40px;"><br />
<br />
<br />
<p class="texte"><br />
We deposited 16 new BioBrick parts in the Registry. <br />
Most of them were cloned into the standard plasmid pSB1C3, tested and sequenced.<br><br />
All our Biobrick are resumed in <a href="http://parts.igem.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2014&group=Toulouse&Done=1">Parts Sandbox</a>.<br />
<!--à vérifier et confirmer--><br />
</p><br />
<br />
<p class="title1">Chemotaxis</p><br />
<br />
<table><br />
<td valign="top"; align="center";><br />
<p class="title2";><br />
<a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</a></p></td><br />
<td valign="top"; align="center";><br />
<div class="technology2">Strong RBS - N-acetylglucosamine based chemotaxis for <i>Bacillus subtilis</i> - Double terminator</div><br />
<div class="thelanguage2"><br />
<p class="texte">This part is designed to enable a N-acetylglucosamine <br />
based chemotaxis in <i>B. subtilis</i>.</p><br />
<p class="title4">Design</p><br />
<p class="texte">The encoded protein is a chimera of two proteins:<br><br />
- the methyl accepting Chemotaxis protein (McpA from <i>B. subtilis</i> which is required for taxis towards glucose.<br><br />
- the N-acetylglucosamine regulated methyl-accepting chemotaxis protein from <i>Vibrio cholerae</i> (VCD).<br><br />
<br><br />
The chimeric protein contains the intracellular domains of McpA from <i>Bacillus subtilis</i> to enable the transduction of the signal: <br />
these domains correspond to the amino acids 1-37 and 282-610. <br />
The extracellular domain of VCD is inserted between the intracellular regions of McpA <br />
to sense N-acetylglucosamine and corresponds to the amino acids 31-310.</p><br />
<img src="https://static.igem.org/mediawiki/2014/a/a2/BBa_K1364000.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Translation unit</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part is not tested yet.</p><br />
<p class="title4">References</p><br />
<p class="texte">Uniprot:<br><br />
- <a href="http://www.uniprot.org/uniprot/P39214">McpA</a><br><br />
- <a href="http://www.uniprot.org/uniprot/C3NYT2">N-acetylglucosamine regulated methyl-accepting chemotaxis protein</a></p><br />
<!--à compléter--><br />
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</div></td></table><br />
<br />
<table><br />
<td valign="top"; align="center";><br />
<p class="title2";><br />
<a href="http://parts.igem.org/Part:BBa_K1364004">BBa_K1364004</a></p></td><br />
<td valign="top"; align="center";><br />
<div class="technology2">P<sub>veg</sub> - Strong RBS - N-acetylglucosamine based chemotaxis for <i>Bacillus subtilis</i> - Double terminator</div><br />
<div class="thelanguage2"><br />
<p class="texte">This part is designed to enable a N-acetylglucosamine based chemotaxis in <i>Bacillus subtilis</i>.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This expression cassette is designed for the expression of an antifungal peptide, <br />
D4E1 and for its secretion in <i>B. subtilis</i>.<br />
It is composed of the strong, constitutive promoter of <i>B. subtilis</i> <br />
P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">BBa_K823003</a>),<br />
strong RBS for <i>B. subtilis</i> (<a href="http://parts.igem.org/Part:BBa_K780002">BBa_K780002</a>),<br />
the open reading frame of D4E1<br />
and a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">BBa_B0015</a>)</p><br />
<img src="https://static.igem.org/mediawiki/2014/3/35/BBa_K1364004.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was not tested yet.</p><br />
<p class="title4">References</p><br />
<p class="texte">Uniprot:<br><br />
- <a href="http://www.uniprot.org/uniprot/P39214">McpA</a><br><br />
- <a href="http://www.uniprot.org/uniprot/C3NYT2">N-acetylglucosamine regulated methyl-accepting chemotaxis protein</a></p><br />
<br />
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<br />
<br></br><br />
<br />
<p class="title1">Binding</p><br />
<table><br />
<td valign="top"; align="center";><br />
<p class="title2";><br />
<a href="http://parts.igem.org/Part:BBa_K1364005">BBa_K1364005</a></p></td><br />
<td valign="top"; align="center";><br />
<div class="technology2"></a>P<sub>veg</sub> - RBS consensus - Chitin Binding protein - Double terminator</div><br />
<div class="thelanguage2"><br />
<br />
<p class="texte">This part is designed to enable the binding of <i>Bacillus subtilis</i> to the fungi wall made of chitin.<p/><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a Cell Wall Binding (CWB) sequence <br />
and a Chitin Binding Domain (CBD) linked with a 6 amino acids linker.<br><br />
<br><br />
The CWB domain has been extracted from LytC (<a href="http://parts.igem.org/Part:BBa_K316030">BBa_K316030</a>) of the iGEM 2010 Imperial College team.<br><br />
The CBD is composed of the Domain 4 of the GbpA protein of <i>Vibrio cholerae</i>,<br />
a protein reported to mediate bacterial attachment to chitin. <br />
It has been shown that the Domain 4 was essential to bind chitin.</p><br />
<img src="https://static.igem.org/mediawiki/2014/f/f5/BBa_K1364005.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested with chitin beads. (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Binding module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)<br><br />
- The <i>Vibrio cholerae</i> Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373<br />
</p><br />
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<br />
<br></br><br />
<br />
<p class="title1">Fungicides</p><br />
<p class="title3">GAFP-1</p><br />
<br />
<br><br />
<table><br />
<td valign="top"; align="center";><br />
<p class="title2";><br />
<a href="http://parts.igem.org/Part:BBa_K1364002">BBa_K1364002</a></p></td><br />
<td valign="top"; align="center";><br />
<div class="technology2"> Strong RBS - Antifungal GAFP-1</div><br />
<div class="thelanguage2"><br />
<br />
<p class="texte">The <i>Gastrodia</i> anti-fungal protein(GAFP-1), also known as gastrodianin, is a mannose and chitin binding lectin<br />
originating from the Asiatic orchid <i>Gastrodia elata</i>, a traditional Chinese medicinal herb cultured for thousands of years.<br />
GAFP-1 is composed of 15 amino acids LDSLSFSYNNFEEDD and is able to inhibit the growth of multiples species of plant pathogenic fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a Strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">BBa_K780002</a>) and<br />
the open reading frame of the <i>Gastrodia</i> anti-fungal protein 1 (GAFP-1)<br />
optimized for its expression and its secretion in <i>Bacillus subtilis</i>.<br />
The codon optimization was made thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/5/5f/BBa_K1364002.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Translational unit</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)<br><br />
- The <i>Vibrio cholerae</i> Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373</p><br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology2', 3); return false">Collapse</a></p><br />
</div></td></table><br />
<br />
<table><br />
<td valign="top"; align="center";><br />
<p class="title2";><br />
<a href="http://parts.igem.org/Part:BBa_K1364007">BBa_K1364007</a></p></td><br />
<td valign="top"; align="center";><br />
<div class="technology2">Strong RBS - Antifungal GAFP-1 + Double terminator</div><br />
<div class="thelanguage2"><br />
<p class="texte">The <i>Gastrodia</i> anti-fungal protein(GAFP-1), also known as gastrodianin, is a mannose and chitin binding lectin<br />
originating from the Asiatic orchid <i>Gastrodia elata</i>, a traditional Chinese medicinal herb cultured for thousands of years.<br />
GAFP-1 is composed of 15 amino acids LDSLSFSYNNFEEDD and is able to inhibit the growth of multiples species of plant pathogenic fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a Strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">BBa_K780002</a>),<br />
the open reading frame of the <i>Gastrodia</i> anti-fungal protein 1 (GAFP-1) <br />
and a double terminator <a href="http://parts.igem.org/Part:BBa_B0015">BBa_B0015</a>.<br />
optimized for its expression and its secretion in <i>Bacillus subtilis</i>.<br />
The codon optimization was made thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/4/42/BBa_K1364007.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Composite part</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)<br><br />
- The <i>Vibrio cholerae</i> Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373</p><br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology2', 4); return false">Collapse</a></p><br />
</div></td></table><br />
<br />
<table><br />
<td valign="top";align="center";><br />
<p class="title2";><br />
<a href="http://parts.igem.org/Part:BBa_K1364008">BBa_K1364008</a></p></td><br />
<td valign="top"; align="center";><br />
<div class="technology2"> P<sub>veg</sub> - Strong RBS - Antifungal GAFP-1 - Double terminator</div><br />
<div class="thelanguage2"><br />
<p class="texte">This expression cassette is designed for the expression and secretion of the <i>Gastrodia</i> anti-fungal protein(GAFP-1), also known as gastrodianin. GAFP-1 is a mannose and chitin binding lectin<br />
originating from the Asiatic orchid <i>Gastrodia elata</i>, a traditional Chinese medicinal herb cultured for thousands of years.<br />
GAFP-1 is composed of 15 amino acids LDSLSFSYNNFEEDD and is able to inhibit the growth of multiples species of plant pathogenic fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of the strong, constitutive promoter of <i>Bacillus subtilis</i> P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">BBa_K823003</a>),<br />
strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">BBa_K780002</a>),<br />
the open reading frame of the <i>Gastrodia</i> anti-fungal protein 1 (GAFP-1) <br />
and a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>).<br />
optimized for its expression and its secretion in <i>B. subtilis</i>.<br />
The codon optimization was made thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/parts/4/47/1364008a.jpg"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)<br><br />
- The <i>Vibrio cholerae</i> Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373<br />
</p><br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology2', 5); return false">Collapse</a></p><br />
</div></td></table><br />
<br />
<br></br><br />
<br />
<p class="title3">D4E1</p><br />
<br />
<br><br />
<table><br />
<td valign="top"; align="center";><br />
<p class="title2";><br />
<a href="http://parts.igem.org/Part:BBa_K1364003">BBa_K1364003</a></p></td><br />
<td valign="top"; align="center";><br />
<div class="technology2">Strong RBS - Antifungal D4E1 - Double terminator</div><br />
<div class="thelanguage2"><br />
<p class="texte">D4E1 is a linear synthetic peptide of 17 amino acids <br />
which has shown to have antifungal activities <br />
by complexing with a sterol present in conodial wall of a varety of fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a Strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">BBa_K780002</a>),<br />
the open reading frame of D4E1 and <br />
a Double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">BBa_B0015</a>)<br />
optimized for its expression and its secretion in <i>Bacillus subtilis</i>.<br />
This part was optimized for the expression and its secretion in <i>B. subtilis </i><br />
thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/b/bb/BBa_K1364003.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Translational unit</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- De Lucca AJ, Bland JM, Grimm C, Jacks TJ, Cary JW, Jaynes JM, Cleveland TE, Walsh TJ. Fungicidal properties, sterol binding, and proteolytic resistance of the synthetic peptide D4E1.<br><br />
- Can J Microbiol. 1998 Jun;44(6):514-20.</p><br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology2', 6); return false">Collapse</a></p><br />
</div></td></table><br />
<br />
<table><br />
<td valign="top"; align="center";><br />
<p class="title2";><br />
<a href="http://parts.igem.org/Part:BBa_K1364009">BBa_K1364009</a></p></td><br />
<td valign="top"; align="center";><br />
<div class="technology2">P<sub>veg</sub> - Strong RBS - Antifungal D4E1 - Double Terminator</div><br />
<div class="thelanguage2"><br />
<p class="texte"><br />
D4E1 is a linear synthetic peptide of 17 amino acids <br />
which has shown to have antifungal activities <br />
by complexing with a sterol present in conodial wall of a varety of fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of the strong, constitutive promoter of <i>Bacillus subtilis</i> <br />
P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">BBa_K823003</a>),<br />
a strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">BBa_K780002</a>),<br />
the open reading frame of D4E1 and <br />
a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">BBa_B0015</a>).<br />
This part was optimized for the expression and its secretion in <i>B.subtilis </i><br />
thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/4/47/BBa_K1364009.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Translational unit</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- De Lucca AJ, Bland JM, Grimm C, Jacks TJ, Cary JW, Jaynes JM, Cleveland TE, Walsh TJ. Fungicidal properties, sterol binding, and proteolytic resistance of the synthetic peptide D4E1.<br><br />
- Can J Microbiol. 1998 Jun;44(6):514-20.</p><br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology2', 7); return false">Collapse</a></p><br />
</div></td></table><br />
<br />
<br></br><br />
<br />
<p class="title3">EcAMP-1</p><br />
<br />
<table><br />
<td valign="top"; align="center";><br />
<p class="title2";><br />
<a href="http://parts.igem.org/Part:BBa_K1364010">BBa_K1364010</a></p></td><br />
<td valign="top"; align="center";><br />
<div class="technology2">P<sub>veg</sub> - RBS SpoVG - Antifungal EcAMP-1</div><br />
<div class="thelanguage2"><br />
<p class="texte"><br />
EcAMP-1 is an antimicrobial peptide of 37 amino acids <br />
originated from the specie <i>Echinochloa crus-galli</i>, <br />
a type of wild grass. <br />
This peptide has a particular structure : it is helical because of two disulfide bonds.<br><br />
EcAMP-1 has shown to have antifungal activities. Its mode of action may be the prevention of hyphae elongation.<br><br />
This part was added to the Registry by the iGEM Utah State team in 2013.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of the constitutive promoter P<sub>veg</sub> and<br />
strong RBS for <i>Bacillus subtilis</i><br />
(<a href="http://parts.igem.org/Part:BBa_K733013">BBa_K733013</a>) and<br />
the open reading frame of EcAMP-1.<br><br />
The EcAMP-1 part was codon optimized for <i>E. coli</i> by the iGEM Utah State team<br />
and thanks to the Life Technologies GeneArt software program. </p><br />
<img src="https://static.igem.org/mediawiki/2014/5/5f/BBa_K1364010.png"><br />
<p class="title4">Type</p><br />
<p class="texte"> Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part is not tested yet.</p><br />
<p class="title4">References</p><br />
<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><br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology2', 8); return false">Collapse</a></p><br />
</div></td></table><br />
<br />
<table><br />
<td valign="top"; align="center";><br />
<p class="title2";><br />
<a href="http://parts.igem.org/Part:BBa_K1364011">BBa_K1364011</a></p></td><br />
<td valign="top"; align="center";><br />
<div class="technology2"> P<sub>veg</sub> - RBS SpoVG - Antifungal EcAMP-1 - Double terminator</div><br />
<div class="thelanguage2"><br />
<br />
<p class="texte" id="select10"><br />
EcAMP-1 is an antimicrobial peptide of 37 amino acids <br />
originated from the specie <i>Echinochloa crus-galli</i>, <br />
a type of wild grass. <br />
This peptide has a particular structure : it is helical because of two disulfide bonds.<br><br />
EcAMP-1 has shown to have antifungal activities. Its mode of action may be the prevention of hyphae elongation.<br><br />
This part was added to the Registry by the iGEM Utah State team in 2013.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of the constitutive promoter P<sub>veg</sub> and<br />
strong RBS for <i>Bacillus subtilis</i><br />
(<a href="http://parts.igem.org/Part:BBa_K733013">BBa_K733013</a>),<br />
the open reading frame of EcAMP-1 and <br />
a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">BBa_B0015</a>).<br><br />
The EcAMP-1 part was codon optimized for <i>Escherichia coli</i> by the iGEM Utah State team<br />
and thanks to the Life Technologies GeneArt software program. </p><br />
<img src="https://static.igem.org/mediawiki/2014/7/74/BBa_K1364011.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part is not tested yet.</p><br />
<p class="title4">References</p><br />
<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><br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology2', 9); return false">Collapse</a></p><br />
</div></td></table><br />
<br />
<table><br />
<td valign="top"; align="center";><br />
<p class="title2";><br />
<a href="http://parts.igem.org/Part:BBa_K1364019">BBa_K1364019</a></p></td><br />
<td valign="top"; align="center";><br />
<div class="technology2">P<sub>veg</sub> - RBS - Antifungal EcAMP-1 (revised with a stop codon) - Double terminator</div><br />
<div class="thelanguage2"><br />
<br />
<p class="texte">EcAMP-1 is an antimicrobial peptide of 37 amino acids <br />
originated from the specie <i>Echinochloa crus-galli</i>, <br />
a type of wild grass. <br />
This peptide has a particular structure : it is helical because of two disulfide bonds.<br><br />
EcAMP-1 has shown to have antifungal activities. Its mode of action may be the prevention of hyphae elongation.<br><br />
This part was added to the Registry by the iGEM Utah State team in 2013.</br><br />
We observed the presence of the stop codon in the suffix. When we have digested this Biobrick, it disappeared. So we added this codon in upstream of the suffix and now, we can reuse this Biobrick.<br />
</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of the constitutive promoter P<sub>veg</sub> and<br />
strong RBS for <i>Bacillus subtilis</i><br />
(<a href="http://parts.igem.org/Part:BBa_K733013">BBa_K733013</a>),<br />
<b>the revised open reading frame of EcAMP-1 (with a stop codon) </b> and <br />
a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">BBa_B0015</a>).<br><br />
The EcAMP-1 part was codon optimized for <i>Escherichia coli</i> by the iGEM Utah State team<br />
and thanks to the Life Technologies GeneArt software program. </p><br />
<img src="https://static.igem.org/mediawiki/2014/7/74/BBa_K1364011.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Composite</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was not tested yet</p><br />
<p class="title4">References</p><br />
<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><br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology2', 13); return false">Collapse</a></p><br />
</div></td></table><br />
<br />
<br></br><br />
<br />
<p class="title3">Fungicide operons</p><br />
<br />
<table><br />
<td valign="top"; align="center";><br />
<p class="title2";><br />
<a href="http://parts.igem.org/Part:BBa_K1364013">BBa_K1364013</a></p></td><br />
<td valign="top"; align="center";><br />
<div class="technology2">P<sub>veg</sub> - Strong RBS - Antifungal GAFP-1 - Strong RBS - Antifungal D4E1 - Double terminator</div><br />
<div class="thelanguage2"><br />
<p class="texte"><br />
<p class="title4">Design</p><br />
<p class="texte">This part is designed for the co-expression of two different peptides <br />
with anti-fungal activities : D4E1 and GAFP-1. <br />
It is composed of the strong, constitutive promoter of <i>Bacillus subtilis</i> <br />
P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">BBa_K823003</a>) <br />
and the translation unit with GAFP-1 and D4E1 (BBa_K1364012).</p><br />
<img src="https://static.igem.org/mediawiki/2014/4/40/BBa_K1364013.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
See <a href="http://parts.igem.org/Part:BBa_K1364002">BBa_K1364002</a> and <a href="http://parts.igem.org/Part:BBa_K1364003">BBa_K1364003</a>.</p><br />
<br />
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</div></td></table><br />
<br />
<br></br><br />
<br />
<p class="title1">Basic tools</p><br />
<br />
<table><br />
<td valign="top"; align="center";><br />
<p class="title2";><br />
<a href="http://parts.igem.org/Part:BBa_K1364015">BBa_K1364015</a></p></td><br />
<td valign="top"; align="center";><br />
<div class="technology2">P<sub>veg</sub> - SpoVG RBS - RFP - Double terminator </div><br />
<div class="thelanguage2"><br />
<br />
<p class="texte"><!--Compléter--><br />
This part is designed to enable the expression of a Red Fluorescent Protein in <i>Bacillus subtilis</i> under the control of a constitutive promoter P<sub>veg</sub>. This construction has been checked by sequencing and has shown to work also in <i>Escherichia coli</i><br />
</p><br />
<img src="https://static.igem.org/mediawiki/parts/6/69/Pveg%2BRFP.jpg"><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a constitutive promoter P<sub>veg</sub> (BBa_K823003), spoVG RBS (BBa_KK143021), the coding sequence of the RFP (E1010 from BBa_K606013) and a double terminator (BBa_KB0015) </p><br />
<p class="title4">Type</p><br />
<p class="texte">Reporter</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested in both <i>E. coli</i> and <i>B. subtilis</i>. The sequences was verified by sequencing. </p><br />
<br />
<br />
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</div></td></table><br />
<br />
<table><br />
<td valign="top"; align="center";><br />
<p class="title2";><br />
<a href="http://parts.igem.org/Part:BBa_K1364016">BBa_K1364016</a></p></td><br />
<td valign="top"; align="center";><br />
<div class="technology2"> P<sub>lepA</sub> - SpoVG RBS - RFP - Double terminator </div><br />
<div class="thelanguage2"><br />
<br />
<p class="texte"><!--Compléter--><br />
This part is designed to enable the expression of a Red Fluorescent Protein in <i>Bacillus subtilis</i> under the control of a constitutive promoter P<sub>lepA</sub>. This construction has been checked by sequencing and has shown to work also in <i>Escherichia coli</i><br />
</p><br />
<img src="https://static.igem.org/mediawiki/parts/1/12/PlepA%2BRFP.jpg"><br />
<br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a constitutive promoter P<sub>lepA</sub> (BBa_K823002), spoVG RBS (BBa_K143021), the coding sequence of the RFP (E1010 from BBa_K606013) and a double terminator (BBa_B0015) </p><br />
<p class="title4">Type</p><br />
<p class="texte">Reporter</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested in both <i>E. coli</i> and <i>B. subtilis</i>. The sequences was verified by sequencing.</p><br />
<br />
<br />
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</div></td></table><br />
<br />
<table><br />
<td valign="top"; align="center";><br />
<p class="title2";><br />
<a href="http://parts.igem.org/Part:BBa_K1364017">BBa_K1364017</a></p></td><br />
<td valign="top"; align="center";><br />
<div class="technology2">P<sub>lepA</sub> - SpoVG RBS</div><br />
<div class="thelanguage2"><br />
<br />
<p class="texte"><!--Compléter--><br />
P<sub>lepA</sub> is a constitutive promoter in <i>Bacillus subtilis</i> (BBa_K823002) coupled with a RBS spoVG (BBa_K143021). To get the highest level of translation from this Promoter-RBS combination it must be connected to a coding region preceded by a coding region prefix. A standard prefix will increase the distance between the RBS and the start codon, reducing translational efficiency. <br />
This construction is also working with <i>Escherichia coli</i> and has been verified by sequencing.<br />
</p><br />
<img src="https://static.igem.org/mediawiki/parts/d/da/PlepA%2BSpoVG.jpg"><br />
<p class="title4">Type</p><br />
<p class="texte">Composite</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was checked by sequencing</p><br />
<br />
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</div></td></table><br />
<br />
<table><br />
<td valign="top"; align="center";><br />
<p class="title2";><br />
<a href="http://parts.igem.org/Part:BBa_K1364021">BBa_K1364021</a></p></td><br />
<td valign="top"; align="center";><br />
<div class="technology2">Integrative plasmid for <i>Bacillus subtilis</i> (pSB<sub>bs</sub>4E)</div><br />
<div class="thelanguage2"><br />
<br />
<p class="texte">This part is an empty backbone vector for the usage in <i>Bacillus subtilis</i>. It is on biobrick version of pKL190 which integrates in the thrC locus and can be selected with Erythromycin . It has an Ampicillin resistance for cloning in <i>Escherichia coli</i>. The backbone contains an RFP in the BioBrick site (BBa_J04450) to facilitate the cloning in <i>E.coli</i>. <br><br />
The handling of this type of vector is described <a href="https://2014.igem.org/Team:Toulouse/Notebook/Protocols#select6">here</a>.<br />
<br />
</p><br />
<p class="title4">Design</p><br />
<p class="texte">We digested pKL190 plasmid and PCR products of BBa_J04450 with BamHI and EcoRI. We proceded to ligation.</p><br />
<img src="https://static.igem.org/mediawiki/2014/archive/6/6d/20141017182537!PKL190_map.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Backbone</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested by transforming <i>B. subtilis</i>. Clones obtained were Threonine-dependant and resistant to Erythromycin (10µg/ml).</p><br />
<p class="title4">References</p><br />
<p class="texte"><br />
R. Bernard, K.A. Marquis, and D.Z. Rudner. <b>Nucleoid occlusion prevents cell division during replication fork arrest in <i>Bacillus subtilis</i></b>Mol Microbiol. Nov 2010; 78(4): 866–882.</p><br />
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{{:Team:Toulouse/template/footer}}</div>Jourdanhttp://2014.igem.org/Team:Toulouse/Result/partsTeam:Toulouse/Result/parts2014-10-18T00:50:50Z<p>Jourdan: </p>
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<h2>Parts</h2><br />
<p>What did we send to the Registry?</p><br />
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<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Results&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Parts</p> <br />
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<p class="texte">As the chassis <i>B. subtilis</i> is not as used as <i>E. coli</i>, <br />
it was sometimes hard for us to get or find desired parts.<br><br />
However, in collaboration with other iGEM teams and with a synthesis company, we managed to obtain or make the following parts:<br><br />
</p><br />
<ul><br />
<li class="tree"><p class="title1"><a href="https://2014.igem.org/Team:Toulouse/Result/parts/Submitted_parts"><font color="green">Submitted parts</p></a></li><br />
<li class="tree"><p class="title1"><a href="https://2014.igem.org/Team:Toulouse/Result/parts/Used_parts"><font color="green">Used parts</p></a></li><br />
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{{:Team:Toulouse/template/footer}}</div>Jourdanhttp://2014.igem.org/Team:Toulouse/Result/achievementTeam:Toulouse/Result/achievement2014-10-18T00:49:53Z<p>Jourdan: </p>
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<h2>Achievements</h2><br />
<p>Bronze, Silver or Gold Medal? That is the question!</p><br />
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<p class="title1" style="text-align:left;">Let's sum up what we did during this summer!</p><br />
<p class="title2">First step...</p><br />
<p class="texte"> <br />
After a summer of hard work, our team is proud of the achieved results. The first step of our project was to have a <b>proof of concept</b> of the three functions requiered to fight against canker. The verifications of our genetic constructions were successful (PCR, Migration on an agarose gel and Sequencing). We could then proceed to specific test of the modules: capillary test, chitin beads test or fungicide tests.</p><br />
<br />
<p class="title2">Second step...</p><br />
<p class="texte"> <br />
Once these results obtained, we decided to move on to the next step: in order to get closer to our final objective, the three genetic modules have to be assembled and to be tested in <b>model plants</b>. In the short time we had, these tests were only performed with the antifungal module, but the results were very encouraging (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">3. In planta tests with SubtiTree</a> in the Fungicides module). <br />
<br />
<p class="title2">Third step...</p><br />
<p class="texte"> <br />
So encouraging that we moved on to the third step of our project and think about the <b>real injection of SubtiTree in diseased trees</b>. We then considered the <a href="https://2014.igem.org/Team:Toulouse/Project/Spreading"><b>spreading</b></a> problems and proposed different strategies to avoid this major issue. <a href="https://2014.igem.org/Team:Toulouse/Modelling"><b>Modeling</b></a> enables us to predict the growth and the lifespan of SubtiTree in Plane Trees. <br />
<br />
<p class="title2">And what comes next?</p><br />
<p class="texte"> With the support of Governmental institutions, scientific organizations and the popular enthusiasm raised by our project, we are still looking for the best way to continue and achieve our goal: save the plane trees and preserve the beauty of the Canal du Midi.<br><br />
However, SubtiTree's spreading must be controlled, fungicide production regulated, specifity improved, tests of the whole system performed. In a nutshell, we still have a lot of things to do even if we did our best during the whole summer!</p><br />
<br />
<p class="title2">iGEM Community</p><br />
<p class="texte"><br />
Well aware that we are part of the <b>iGEM Community</b>, our team also worked for this community by improving the iGEMer's Bible: <a href="http://parts.igem.org/Catalog">the Registry of Standard Biological Parts</a>. <br><br />
We improved the EcAMP BioBrick by adding a promotor, a RBS, but mainly by the addition of a STOP codon (<a href="http://parts.igem.org/Part:BBa_K1364019">BBa_K1364019</a>).<br><br />
As <i>Bacillus subtilis</i> appears to be for us an ideal chassis, we also developed new genetic tools to facilitate its utilization. Thanks to numerous interactions with other iGEM teams, we didn't see the time flying by during this <b>amazing summer</b> !</p><br />
<br />
<center><img style="width:550px; " src="https://static.igem.org/mediawiki/parts/4/45/Achievmentss.jpg"></center><br />
<br><br />
<p class="title2">It is better to have "well-made rather than a well-filled head"... (Michel de Montaigne)</p><br />
<p class="texte">We developed a deep <a href="https://2014.igem.org/Team:Toulouse/ethics">ethical questioning</a>, trying to understand the relationship between Science and the protection of the Beauty. We strongly believe that neglecting the ethical aspect in a scientific project is a mistake. All the team was involved in this part and the reflexions made were very profitable to us.</p><br />
<br />
<p class="texte">We filled the <a href="https://igem.org/2014_Judging_Form?id=1364">2014 Judging Form</a> thanks to the following accomplishments:</p><br />
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<td style="border-bottom:4px solid #e5e6e6; border-top-left-radius:9px;"><center><br>Bronze Medal <img src="https://static.igem.org/mediawiki/2013/8/86/Medal_bronze.png" class="imgcontenttable"/></center><br></td><br />
<td style="border-bottom:4px solid #e5e6e6;"><center><br>Silver Medal <img src="https://static.igem.org/mediawiki/2013/d/dd/Medal_silver.png" class="imgcontenttable"/></center><br></td><br />
<td style="border-bottom:4px solid #e5e6e6; border-top-right-radius:9px;"><center><br>Gold Medal<img src="https://static.igem.org/mediawiki/2013/b/bd/Medal_gold_3.png" class="imgcontenttable"/></center><br></td><br />
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<td style="border-right:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/>Register the team, have a great summer, and plan to have fun at the Jamboree</td><br />
<td style="border-right:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/>Experimentally validate that at least three new BioBrick Part or Device of your own design and construction work as expected.</td><br />
<td><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Improve the function of an existing BioBrick Part or Device and improve the <i>Bacillus subtilis</i> genetic tools, enter this information in the Registry : <a href="http://parts.igem.org/Part:BBa_K1364019"target="_blank">BBa_K1364019</a>, <a href="http://parts.igem.org/Part:BBa_K1364016"target="_blank">BBa_K1364016</a>, <a href="http://parts.igem.org/Part:BBa_K1364017"target="_blank">BBa_K1364017</a>, <a href="http://parts.igem.org/Part:BBa_K1364021"target="_blank">BBa_K1364021</a>, </td><br />
</tr><br />
<br />
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<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Successfully complete and submit this iGEM Judging form </td><br />
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/>Document the characterization of these three parts in the 'Main Page' of that Part's/Device's Registry entry<br />
Submit this new part to the iGEM Parts Registry:<br> <a href="http://parts.igem.org/Part:BBa_K1364005"target="_blank">BBa_K1364005</a>, <a href="http://parts.igem.org/Part:BBa_K1364009"target="_blank">BBa_K1364009</a>, <a href="http://parts.igem.org/Part:BBa_K1364013"target="_blank">BBa_K1364013.</a></td><br />
<td style="border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Your project may have implications for the environment, security, safety and ethics and/or ownership and sharing. Describe a novel approach that your team has used to help you and others consider these aspects of the design and outcomes of synthetic biology efforts. → <a href="https://2014.igem.org/Team:Toulouse/ethics" target="_blank">Ethic Part</a> </td><br />
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<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Create and share a Description of the team’s project using the iGEM wiki and the team’s parts using the Registry Biological Parts </td><br />
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Submit these new part to the iGEM Parts Registry (submissions must adhere to the iGEM Registry guidelines)</td><br />
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<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Plan to present a Poster and Talk at the iGEM Jamboree </td><br />
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Your project may have implications for the environment, security, safety and ethics and/or ownership and sharing. Describe one or more ways in which these or other broader implications have been taken into consideration in the design and execution of your project. → <a href="https://2014.igem.org/Team:Toulouse/ethics" target="_blank"> Ethical Page</a></td><br />
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<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Document at least one new standard BioBrick Part or Device used in your project/central to your project and submit this part to the iGEM Registry : <a href="http://parts.igem.org/Part:BBa_K1364000" target="_blank">BBa_K1364000</a>, <a href="http://parts.igem.org/Part:BBa_K1364002"target="_blank">BBa_K1364002</a>, <a href="http://parts.igem.org/Part:BBa_K1364003"target="_blank">BBa_K1364003</a>, <a href="http://parts.igem.org/Part:BBa_K1364005"target="_blank">BBa_K1364005</a>. </td><br />
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{{:Team:Toulouse/template/footer}}</div>Jourdanhttp://2014.igem.org/Team:Toulouse/Result/achievementTeam:Toulouse/Result/achievement2014-10-18T00:48:49Z<p>Jourdan: </p>
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<h2>Achievements</h2><br />
<p>Bronze, Silver or Gold Medal? That is the question!</p><br />
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<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Results&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Achievements</p> <br />
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<p class="title1" style="text-align:left;">Let's sum up what we did during this summer!</p><br />
<p class="title2">First step...</p><br />
<p class="texte"> <br />
After a summer of hard work, our team is proud of the achieved results. The first step of our project was to have a <b>proof of concept</b> of the three functions requiered to fight against canker. The verifications of our genetic constructions were successful (PCR, Migration on an agarose gel and Sequencing). We could then proceed to specific test of the modules: capillary test, chitin beads test or fungicide tests.</p><br />
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<p class="title2">Second step...</p><br />
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Once these results obtained, we decided to move on to the next step: in order to get closer to our final objective, the three genetic modules have to be assembled and to be tested in <b>model plants</b>. In the short time we had, these tests were only performed with the antifungal module, but the results were very encouraging (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">3. In planta tests with SubtiTree</a> in the Fungicides module). <br />
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<p class="title2">Third step...</p><br />
<p class="texte"> <br />
So encouraging that we moved on to the third step of our project and think about the <b>real injection of SubtiTree in diseased trees</b>. We then considered the <a href="https://2014.igem.org/Team:Toulouse/Project/Spreading"><b>spreading</b></a> problems and proposed different strategies to avoid this major issue. <a href="https://2014.igem.org/Team:Toulouse/Modelling"><b>Modeling</b></a> enables us to predict the growth and the lifespan of SubtiTree in Plane Trees. <br />
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<p class="title2">And what comes next?</p><br />
<p class="texte"> With the support of Governmental institutions, scientific organizations and the popular enthusiasm raised by our project, we are still looking for the best way to continue and achieve our goal: save the plane trees and preserve the beauty of the Canal du Midi.<br><br />
However, SubtiTree's spreading must be controlled, fungicide production regulated, specifity improved, tests of the whole system performed. In a nutshell, we still have a lot of things to do even if we did our best during the whole summer!</p><br />
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<p class="title2">iGEM Community</p><br />
<p class="texte"><br />
Well aware that we are part of the <b>iGEM Community</b>, our team also worked for this community by improving the iGEMer's Bible: <a href="http://parts.igem.org/Catalog">the Registry of Standard Biological Parts</a>. <br><br />
We improved the EcAMP BioBrick by adding a promotor, a RBS, but mainly by the addition of a STOP codon (<a href="http://parts.igem.org/Part:BBa_K1364019">BBa_K1364019</a>).<br><br />
As <i>Bacillus subtilis</i> appears to be for us an ideal chassis, we also developed new genetic tools to facilitate its utilization. Thanks to numerous interactions with other iGEM teams, we didn't see the time flying by during this <b>amazing summer</b> !</p><br />
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<p class="title2">It is better to have "well-made rather than a well-filled head"... (Michel de Montaigne)</p><br />
<p class="texte">We developed a deep <a href="https://2014.igem.org/Team:Toulouse/ethics">ethical questioning</a>, trying to understand the relationship between Science and the protection of the Beauty. We strongly believe that neglecting the ethical aspect in a scientific project is a mistake. All the team was involved in this part and the reflexions made were very profitable to us.</p><br />
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<p class="texte">We filled the <a href="https://igem.org/2014_Judging_Form?id=1364">2014 Judging Form</a> thanks to the following accomplishments:</p><br />
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<td style="border-bottom:4px solid #e5e6e6;"><center><br>Silver Medal <img src="https://static.igem.org/mediawiki/2013/d/dd/Medal_silver.png" class="imgcontenttable"/></center><br></td><br />
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<td style="border-right:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/>Register the team, have a great summer, and plan to have fun at the Jamboree</td><br />
<td style="border-right:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/>Experimentally validate that at least three new BioBrick Part or Device of your own design and construction work as expected.</td><br />
<td><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Improve the function of an existing BioBrick Part or Device and improve the <i>Bacillus subtilis</i> genetic tools, enter this information in the Registry : <a href="http://parts.igem.org/Part:BBa_K1364019"target="_blank">BBa_K1364019</a>, <a href="http://parts.igem.org/Part:BBa_K1364016"target="_blank">BBa_K1364016</a>, <a href="http://parts.igem.org/Part:BBa_K1364017"target="_blank">BBa_K1364017</a>, <a href="http://parts.igem.org/Part:BBa_K1364021"target="_blank">BBa_K1364021</a>, </td><br />
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<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Successfully complete and submit this iGEM Judging form </td><br />
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/>Document the characterization of these three parts in the 'Main Page' of that Part's/Device's Registry entry<br />
Submit this new part to the iGEM Parts Registry:<br> <a href="http://parts.igem.org/Part:BBa_K1364005"target="_blank">BBa_K1364005</a>, <a href="http://parts.igem.org/Part:BBa_K1364009"target="_blank">BBa_K1364009</a>, <a href="http://parts.igem.org/Part:BBa_K1364013"target="_blank">BBa_K1364013.</a></td><br />
<td style="border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Your project may have implications for the environment, security, safety and ethics and/or ownership and sharing. Describe a novel approach that your team has used to help you and others consider these aspects of the design and outcomes of synthetic biology efforts. → <a href="https://2014.igem.org/Team:Toulouse/ethics" target="_blank">Ethic Part</a> </td><br />
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<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Create and share a Description of the team’s project using the iGEM wiki and the team’s parts using the Registry Biological Parts </td><br />
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Submit these new part to the iGEM Parts Registry (submissions must adhere to the iGEM Registry guidelines)</td><br />
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<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Plan to present a Poster and Talk at the iGEM Jamboree </td><br />
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Your project may have implications for the environment, security, safety and ethics and/or ownership and sharing. Describe one or more ways in which these or other broader implications have been taken into consideration in the design and execution of your project. → <a href="https://2014.igem.org/Team:Toulouse/ethics" target="_blank"> Ethical Page</a></td><br />
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<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Document at least one new standard BioBrick Part or Device used in your project/central to your project and submit this part to the iGEM Registry : <a href="http://parts.igem.org/Part:BBa_K1364000" target="_blank">BBa_K1364000</a>, <a href="http://parts.igem.org/Part:BBa_K1364002"target="_blank">BBa_K1364002</a>, <a href="http://parts.igem.org/Part:BBa_K1364003"target="_blank">BBa_K1364003</a>, <a href="http://parts.igem.org/Part:BBa_K1364005"target="_blank">BBa_K1364005</a>. </td><br />
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{{:Team:Toulouse/template/footer}}</div>Jourdanhttp://2014.igem.org/Team:Toulouse/Result/achievementTeam:Toulouse/Result/achievement2014-10-18T00:47:21Z<p>Jourdan: </p>
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<h2>Achievements</h2><br />
<p>Bronze, Silver or Gold Medal? That is the question!</p><br />
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<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Results&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Achievements</p> <br />
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<p class="title1" style="text-align:left;">Let's sum up what we did during this summer!</p><br />
<p class="title2">First step...</p><br />
<p class="texte"> <br />
After a summer of hard work, our team is proud of the achieved results. The first step of our project was to have a <b>proof of concept</b> of the three functions requiered to fight against canker. The verifications of our genetic constructions were successful (PCR, Migration on an agarose gel and Sequencing). We could then proceed to specific test of the modules: capillary test, chitin beads test or fungicide tests.</p><br />
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<p class="title2">Second step...</p><br />
<p class="texte"> <br />
Once these results obtained, we decided to move on to the next step: in order to get closer to our final objective, the three genetic modules have to be assembled and to be tested in <b>model plants</b>. In the short time we had, these tests were only performed with the antifungal module, but the results were very encouraging (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">3. In planta tests with SubtiTree</a> in the Fungicides module). <br />
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<p class="title2">Third step...</p><br />
<p class="texte"> <br />
So encouraging that we moved on to the third step of our project and think about the <b>real injection of SubtiTree in diseased trees</b>. We then considered the <a href="https://2014.igem.org/Team:Toulouse/Project/Spreading"><b>spreading</b></a> problems and proposed different strategies to avoid this major issue. <a href="https://2014.igem.org/Team:Toulouse/Modelling"><b>Modeling</b></a> enables us to predict the growth and the lifespan of SubtiTree in Plane Trees. <br />
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<p class="title2">And what comes next?</p><br />
<p class="texte"> With the support of Governmental institutions, scientific organizations and the popular enthusiasm raised by our project, we are still looking for the best way to continue and achieve our goal: save the plane trees and preserve the beauty of the Canal du Midi.<br><br />
However, SubtiTree's spreading must be controlled, fungicide production regulated, specifity improved, tests of the whole system performed. In a nutshell, we still have a lot of things to do even if we did our best during the whole summer!</p><br />
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<p class="title2">iGEM Community</p><br />
<p class="texte"><br />
Well aware that we are part of the <b>iGEM Community</b>, our team also worked for this community by improving the iGEMer's Bible: <a href="http://parts.igem.org/Catalog">the Registry of Standard Biological Parts</a>. <br><br />
We improved the EcAMP BioBrick by adding a promotor, a RBS, but mainly by the addition of a STOP codon (<a href="http://parts.igem.org/Part:BBa_K1364019">BBa_K1364019</a>).<br><br />
As <i>Bacillus subtilis</i> appears to be for us an ideal chassis, we also developed new genetic tools to facilitate its utilization. Thanks to numerous interactions with other iGEM teams, we didn't see the time flying by during this <b>amazing summer</b> !</p>https://static.igem.org/mediawiki/parts/e/e2/Ach.jpg<br />
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<center><img style="width:1000px; " src="https://static.igem.org/mediawiki/parts/4/45/Achievmentss.jpg"></center><br />
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<p class="title2">It is better to have "well-made rather than a well-filled head"... (Michel de Montaigne)</p><br />
<p class="texte">We developed a deep <a href="https://2014.igem.org/Team:Toulouse/ethics">ethical questioning</a>, trying to understand the relationship between Science and the protection of the Beauty. We strongly believe that neglecting the ethical aspect in a scientific project is a mistake. All the team was involved in this part and the reflexions made were very profitable to us.</p><br />
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<p class="texte">We filled the <a href="https://igem.org/2014_Judging_Form?id=1364">2014 Judging Form</a> thanks to the following accomplishments:</p><br />
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<td style="border-bottom:4px solid #e5e6e6; border-top-left-radius:9px;"><center><br>Bronze Medal <img src="https://static.igem.org/mediawiki/2013/8/86/Medal_bronze.png" class="imgcontenttable"/></center><br></td><br />
<td style="border-bottom:4px solid #e5e6e6;"><center><br>Silver Medal <img src="https://static.igem.org/mediawiki/2013/d/dd/Medal_silver.png" class="imgcontenttable"/></center><br></td><br />
<td style="border-bottom:4px solid #e5e6e6; border-top-right-radius:9px;"><center><br>Gold Medal<img src="https://static.igem.org/mediawiki/2013/b/bd/Medal_gold_3.png" class="imgcontenttable"/></center><br></td><br />
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<td style="border-right:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/>Register the team, have a great summer, and plan to have fun at the Jamboree</td><br />
<td style="border-right:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/>Experimentally validate that at least three new BioBrick Part or Device of your own design and construction work as expected.</td><br />
<td><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Improve the function of an existing BioBrick Part or Device and improve the <i>Bacillus subtilis</i> genetic tools, enter this information in the Registry : <a href="http://parts.igem.org/Part:BBa_K1364019"target="_blank">BBa_K1364019</a>, <a href="http://parts.igem.org/Part:BBa_K1364016"target="_blank">BBa_K1364016</a>, <a href="http://parts.igem.org/Part:BBa_K1364017"target="_blank">BBa_K1364017</a>, <a href="http://parts.igem.org/Part:BBa_K1364021"target="_blank">BBa_K1364021</a>, </td><br />
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<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Successfully complete and submit this iGEM Judging form </td><br />
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/>Document the characterization of these three parts in the 'Main Page' of that Part's/Device's Registry entry<br />
Submit this new part to the iGEM Parts Registry:<br> <a href="http://parts.igem.org/Part:BBa_K1364005"target="_blank">BBa_K1364005</a>, <a href="http://parts.igem.org/Part:BBa_K1364009"target="_blank">BBa_K1364009</a>, <a href="http://parts.igem.org/Part:BBa_K1364013"target="_blank">BBa_K1364013.</a></td><br />
<td style="border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Your project may have implications for the environment, security, safety and ethics and/or ownership and sharing. Describe a novel approach that your team has used to help you and others consider these aspects of the design and outcomes of synthetic biology efforts. → <a href="https://2014.igem.org/Team:Toulouse/ethics" target="_blank">Ethic Part</a> </td><br />
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<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Create and share a Description of the team’s project using the iGEM wiki and the team’s parts using the Registry Biological Parts </td><br />
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Submit these new part to the iGEM Parts Registry (submissions must adhere to the iGEM Registry guidelines)</td><br />
<td style="border-top:1px solid #e5e6e6;"> </td><br />
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<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Plan to present a Poster and Talk at the iGEM Jamboree </td><br />
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Your project may have implications for the environment, security, safety and ethics and/or ownership and sharing. Describe one or more ways in which these or other broader implications have been taken into consideration in the design and execution of your project. → <a href="https://2014.igem.org/Team:Toulouse/ethics" target="_blank"> Ethical Page</a></td><br />
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<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Document at least one new standard BioBrick Part or Device used in your project/central to your project and submit this part to the iGEM Registry : <a href="http://parts.igem.org/Part:BBa_K1364000" target="_blank">BBa_K1364000</a>, <a href="http://parts.igem.org/Part:BBa_K1364002"target="_blank">BBa_K1364002</a>, <a href="http://parts.igem.org/Part:BBa_K1364003"target="_blank">BBa_K1364003</a>, <a href="http://parts.igem.org/Part:BBa_K1364005"target="_blank">BBa_K1364005</a>. </td><br />
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{{:Team:Toulouse/template/footer}}</div>Jourdanhttp://2014.igem.org/Team:ToulouseTeam:Toulouse2014-10-18T00:39:07Z<p>Jourdan: </p>
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SubtiTree project<br />
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<p class="texte"><br></br><br />
Fungal diseases lead to major economic losses all over the world. Some pathogens trigger tracheomycosis by disturbing the vascular system of the plant. Canker is one of these infections especially affecting plane trees (<i>Platanus sp.</i>). Plane trees are widely present in Southern France and in particular along the famous Canal du Midi, participating to the site gorgeousness. Today, the only treatment consists in a costly preventive tree-cutting and implies significant ecological troubles. Facing this emergency, our team offers an innovative solution originating from synthetic biology. <br />
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<center><a class="button-home" href="https://2014.igem.org/Team:Toulouse/Project/project-context" style="border: 1px solid #282828;-webkit-border-radius: 5px;-moz-border-radius: 5px;border-radius: 5px; padding: 13px 25px 12px; color: #282828; text-decoration: none; font-size: 17px; background: none; display: block; width: 89px;">Learn more</a><br />
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<div style="position:absolute; top:90px; left:34px; color:white; font-family:'Open Sans'; font-size:16px; width:200px; line-height:24px;">Let's save our trees with SubtiTree!</div><br />
<div style="border-bottom: 1px solid #fff; color:white; font-family:'Open Sans'; position:absolute; bottom:25px; right:22px; font-size:16px;">Read more</div><br />
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<a href="https://2014.igem.org/Team:Toulouse/Project/Chemotaxis" class="part-large-projet" style="background: url('https://static.igem.org/mediawiki/2014/a/aa/Chimio.jpg') no-repeat center;-webkit-background-size: cover;-moz-background-size: cover;-o-background-size: cover;background-size: cover;"><br />
<div class="title-part-projet" style="color:white; ">Chemotaxis</div><br />
<div style="position:absolute; top:90px; left:34px; color:white; font-family:'Open Sans'; font-size:16px; width:200px; line-height:24px;">To target the pathogenic fungus</div><br />
<div style="border-bottom: 1px solid #white; color:white; font-family:'Open Sans'; position:absolute; bottom:25px; left:34px; font-size:16px;"><u>Read more</u></div><br />
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<a href="https://2014.igem.org/Team:Toulouse/Project/binding" class="part-large-projet" style="background: url('https://static.igem.org/mediawiki/2014/0/0b/Billes3D.png') no-repeat center;-webkit-background-size: cover;-moz-background-size: cover;-o-background-size: cover;background-size: cover;"><br />
<div class="title-part-projet style="color:white; ">Binding</div><br />
<div style="position:absolute; top:90px; left:34px; color:white; font-family:'Open Sans'; font-size:16px; width:200px; line-height:24px">To be attached to the fungal pathogen wall</div><br />
<div style="border-bottom: 1px solid #515553; color:white; font-family:'Open Sans'; position:absolute; bottom:28px; right:34px; font-size:16px;">Read more</div><br />
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<a href="https://2014.igem.org/Team:Toulouse/Modelling" class="part-little-projet"><br />
<div class="title-part-projet">Modeling</div><br />
<div style="position:absolute; top:90px; left:34px; color:white; font-family:'Open Sans'; font-size:16px; width:200px; line-height:24px">To develop a predictive model</div><br />
<div style="border-bottom: 1px solid #fff; color:white; font-family:'Open Sans'; position:absolute; bottom:28px; left:34px; font-size:16px;">Read more</div><br />
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<a href="https://2014.igem.org/Team:Toulouse/Project/Spreading" class="part-little-projet"><br />
<div class="title-part-projet">Spreading</div><br />
<div style="position:absolute; top:90px; left:34px; color:white; font-family:'Open Sans'; font-size:16px; width:200px; line-height:24px">How do we keep control on Subtitree?</div><br />
<div style="border-bottom: 1px solid #fff; color:white; font-family:'Open Sans'; position:absolute; bottom:25px; right:34px; font-size:16px;"">Read more</div><br />
</a><br />
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{{:Team:Toulouse/template/footer}}</div>Jourdanhttp://2014.igem.org/Team:Toulouse/Result/experimental-resultsTeam:Toulouse/Result/experimental-results2014-10-18T00:34:16Z<p>Jourdan: </p>
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<h2>Experimental results</h2><br />
<p> Are our modules functionnal? </p><br />
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<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Results&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Experimental results</p> <br />
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<br />
<p class="texte">How did we validate the three modules and improve our new protocols? Click below to find out…</p><br />
<br />
<p style="font-size:1.3em; margin: 0 0 30px 0;"><a href="#" onClick="ddaccordion.expandall('technology'); return false">Expand all</a> | <a href="#" onClick="ddaccordion.collapseall('technology'); return false">Collapse all</a></p><br />
<br />
<div class="technology">Chemotaxis module</div><br />
<div class="thelanguage"><br />
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<br />
<p class="texte">We performed several assays to demonstrate the chemotaxis ability of our engineered <i>Bacillus subtilis</i> strain to move towards N-acetylglucosamine (NAG), the base unit of chitin. The ability of the <i>wild-type</i> strain to move towards glucose was the positive control. <br />
</p><br />
<br />
<p class="title2">1. Petri dishes Test</p><br />
<p class="texte"><br />
The first chemotaxis assay was done in Petri dishes filled with a growth medium containing 0,3% agar. This semi-solid medium was supposed to favor bacterial motility. A paper disk soaked with the attractive compound was placed in the middle of the dish, then cells were loaded into the medium (see Figure 1). This protocol was from the <a href="https://2011.igem.org/Team:Imperial_College_London/Protocols_Chemotaxis">the Imperial College 2011 iGEM team</a>.<br />
</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/0/05/Schema_1.png" alt="schema Figure 1" style="width:500px"></center><br />
<p class="legend">Figure 1: Petri dishes chemotaxis assay. (A) pipetman was used to inject cells into the semi-solid medium. (B) Bacteria would move toward the attractive compound diffusing from a paper disk.<br />
</p><br />
<br />
<p class="texte">With this assay, we however failed to see any chemotaxis of the <i>wild-type</i> control toward glucose (Figure 2-A).As <i>B. subtilis</i> is attracted by many other glucides and amino-acids that can be found in rich medium such as in LB medium. Therefore, with the hope of improving the experimental conditions, we have challenged the cells with paper discs soaked in LB medium containing glucose (Figure 2-B).<br />
</p><br />
<br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/f/ff/Fig2_AetB.png" alt="Figure 2" style="width:750px"></center><br />
<p class="legend">Figure 2: Chemotaxis test with paper discs soaked in either a Glucose solution (A) or in Glucose containing LB medium (B) as attractive compound. Paper discs soaked with water were used as negative controls.<br />
</p><br />
<br />
<p class="texte">No difference was observed between the Petri dishes with or without glucose. With glucose containing LB medium, a large halo around the paper disk was observed. This halo might be due to cells attracted by the solution, as it was not observed when cells were not inoculated in the Petri dish (data not shown). However this result was barely reproducible. Moreover with the addition of LB medium, it was hard to make the distinction between attractive effects and cell growth resulting from random diffusion. We have therefore given up this protocol and tested alternative protocols.<br />
</p><br />
<br />
<p class="title2">2. Plug in Pond system<br />
</p><br />
<br />
<p class="texte"><br />
This protocol was from a PhD work (see references [1]). <i>B.subtilis</i> was grown overnight to a density of 8.10<sup>8</sup> cells/mL. 10mL of the culture was mixed with 15mL of LB medium containing 1.5% agar kept at 45°C. The final agar concentration was 0.9%. Tetracycline (25 µg/ml) was added to inhibit growth in order to only observe the chemotaxis phenomenon. Plates were cooled down and dried, before digging wells with either a punch or 1mL tips. The wells were then filled with the attractive compound (Figure 3). After one hour at room temperature, pictures of the plates were taken and the results analyzed.<br />
</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/c/cd/Fig3.png" alt="Figure 3" style="width:400px"></center><br />
<p class="legend">Figure 3: Plug-in-pond test design.</p><br />
<br />
<p class="texte"><br />
On our first try with <i>B. subtilis</i>, we made three wells per plate (Figure 4).The wells were filled with glucose at different concentrations and tetracycline was not added in one of the plates.<br />
</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/c/ce/Fig4.png" alt="Figure 4" style="width:400px"></center><br />
<p class="legend">Figure 4: Plates after 12h at room temperature.</p><br />
<br />
<p class="texte"><br />
After one hour, no tangible results were obtained. After 12h we observed halos around the 1M glucose containing wells in the plates without tetracycline but not in the plates with tetracycline. Again, because cells could use glucose for growth, we could not distinguish between growth and chemotaxis. Making the hypothesis that the concentration of tetracycline could have been too high and inhibited any bacterial activity, we thereafter lowered the tetracycline concentration to 15µg/mL. We repeated this protocol with this new experimental condition. We made two wells per plate (Figure 5), one with either Glucose or n-acetyl-glucosamine and one with LB medium. As previously, no results were achieved after 1h, but after 12h we could notice halos.<br />
</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/c/c3/Bsubtilis_result.png" alt="Figure 5" style="width:750px"></center><br />
<p class="legend">Figure 5: Chemotaxis test with <i>B. subtilis</i> WT. The upper well contains attractive compound and the lower well contains medium without any attractive compound.<br />
</p><br />
<br />
<p class="texte"><br />
Results were not as clear as in the previous assay (Figure 4), but halos around the wells with glucose at 250mM with and without tetracycline were observed. With N-acetyl-glucosamine (NAG) as the attractive compound, halos were observed for a concentration of 25mM with tetracycline and for a concentration of 250mM without tetracycline, thus suggesting that our <i>B. subtilis</i> 168 strain is attracted toward NAG and uses it to grow.<br />
</p><br />
<br />
<br />
<p class="texte"><br />
<b>References:</b></br><br />
[1]: Claudine Baraquet. <b>Etude de la réponse adaptative à l'oxyde de triméthylamine et de son mécanisme de détection chez <i>Escherichia coli</i> et <i>Shewanella oneidensis</i></b>. 2008, Université de la Méditerranée Aix-Marseille II.<br />
</p><br />
<br />
<br />
<br />
<br />
<p class="title2">4. Capillary test between two tubes also called the tubes test</p><br />
<p class="texte">After the experiment of the plug-in-pond, we decided to construct a system by welding two Eppendorf tubes with a capillary thanks to an electric burner.</p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/f/fb/Chemotaxis_-_eppendorf.png"></center><br />
<p class="legend">Figure 6: Photography of the first tubes system</p><br />
<br />
<p class="texte">We tested this system with a fuchsin dye and water and we were able to observe the diffusion of fuchsin dye towards water. However this construction had a leakage next to the weld seam that we could not stop. <br />
Thus, the Toulouse 2014 iGEM team asked the help from the INSA glass blower, Patrick Chekroun. He designed two systems composed of two tubes linked by a capillary.</p><br />
<br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/2/2b/Chemotaxis_-_tubes.png"><center><br />
<p class="legend">Figure 7: Scheme of the tubes system</p><br />
<br />
<p class="texte"><br />
This new system was tested with the fuchsin dye and the assay was made with WT <i>B. subtilis</i> and N-Acetylglucosamine as the attractive compound.<br />
<br><br><br />
<i>NB: We could not see any diffusion of the fushin dye from one tube to the other. We made the hypothesis that it was not visible by sight because of the small diameter of the capillary. <br />
</i><br><br />
<br><br />
The following strategy was used to avoid disturbance due to pressure and liquid movement through the capillary:<br><br />
- The first step was the addition of Wash Buffer until the capillary was full to prevent the presence of any air bubbles which could have impeded diffusion.<br><br />
- Then, the tube 2 was plugged and maintained with the thumb while another iGEM mate was adding the bacterial suspention of WT <i>B. subtilis</i> into the tube 1.<br><br />
- The tube 1 was also plugged and only then the thumb could be removed from the tube 2. <br><br />
- In the same way, the N-Acetylglucosamine was added in the tube 2.<br><br />
- The same process was made with xylose as a positive control.<br><br />
<br><br />
<i>NB: According to the paper "Chemotaxis towards sugars by </i>Bacillus subtilis", (Ordal et al., 1979),<i> glucose and xylose have the same attractant power. We have privileged a positive control instead of a negative one as we were not sure that this system was efficient.</i><br><br />
<br><br />
- The system was kept straight for 2 hours. Every 40 minutes, samples from each were removed and streaked on solid medium (dilution 1/1,000) in order to estimate the bacterial concentration.</p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/1/1b/Chemotaxis_-_tubes_photo.png"></center><br />
<p class="legend">Figure 8: Photography of the tubes system</p><br />
<br />
<br />
<p class="texte">Unfortunately, the dilution was too high to detect any chemotaxis movement. As we did not find any information in the litterature and did not have enough time to optimize this protocol, we dicided to test again the first protocol from the Imperial college 2011 iGEM team : the tips capillary test.<br />
</p><br />
<br />
<p class="title2"> 5. Tips capillary system</p><br />
<p class="title3">First tips capillary system</p><br />
<p class="texte">This protocol was adapted from The Imperial College 2011 iGEM team (See <a href="https://2014.igem.org/Team:Toulouse/Notebook/Protocols#select8">chemotaxis protocol</a>).<br />
<br><br />
In the first tips capillary system, we used parafilm to avoid any kind of air disturbance in the tips. The different steps are described below:<br><br />
- 15µL of each chemo-attractant was pipetted.<br><br />
- The bottom of tip with the pipette was then put on a piece of parafilm and the pipette was removed from the top of the tips.<br><br />
- The tip was then sealed with a piece of parafilm in order to keep the liquid sterile and inside the tip.<br><br />
- To finish, the level of the solution in the tip was marked.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/9/94/Chemotaxis_-_tip.png"></center><br />
<p class="legend">Figure 9: Sealing of a tip with parafilm</p><br />
<br />
<p class="texte">- When all the chemo-attractants were added, the were fixed on a green support. The whole process can be seen on Figure 10.<br><br />
- Each tip was then immersed into 300 µL of a bacterial suspension in the wells of an Elisa plate.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/0/05/Chemotaxis_-_tip_and_support.png"></center><br />
<p class="legend">Figure 10: First tips capillary system</p><br />
<br />
<p class="texte"><i>NB: the yellow carton was used to stabilize the system and kept it straight.</i><br><br />
<br><br />
- After one hour, the tips were removed from the bacteria suspensions and the bacteria content of the tips was monitored with a Thoma cell under the microscope.<br><br />
<br><br />
We experienced several problems with this system:<br><br />
- The liquid level decreasing so much during the course of the experiment that we did not have enough liquid to fill the Thoma cell for counting.<br><br />
- The bacteria were moving, therefore preventing us from accurately counting them. Taking into account this probelem, we decided to estimate the bacterial concentration by streaking the tips content on agar plate instead of using Thoma cell and microscopy.<br />
<br />
<p class="title3">Second tips capillary system<br />
</p><br />
<p class="texte">And then the revolution came! We found a multichannel pipette :D The same protocol was performed except that the parafilm was used to avoid the air entrance between the tips and the pipette and therefore the loss of liquid.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/e/e4/Chemotaxis_-_pipette.png"></center><br />
<p class="legend">Figure 11: Second tips capillary system</p><br />
<br />
<p class="title3">Improvement of the second tips capillary system</p><br />
<p class="texte">However this system was not optimal it is why we decided to use blu tack instead of parafilm: <br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/4/42/Chemotaxis_-_pipette_and_blu_tack.png"></center><br />
<p class="legend">Figure 12: Improvement of the second tips capillary system</p><br />
<br />
<p class="texte"><b>At that point, the protocol was approved and the final test could finally start! :-)</b><br><br />
<br><br />
There was just one tiny problem… we did not have our optimized bacterium transformed with the chemotaxis module!!! That is why we concentrated our efforts on WT <i>B. subtilis</i> strain.<br><br />
<br><br />
The main goal was to find an optimized control and to analyze the eventual chemotaxis of the WT strain. To avoid osmolality bias, we wanted to find a molecule which was non-attractant and with a similar molecular weight than that of the N-Acetylglucosamine (221.21 g/mol). Our first idea was to use fuchsin (Molecular weight: 337.85 g/mol).<br><br />
<br><br />
At the beginning, the experiment was conducted with only one negative control, the fuchsin and different NAG concentrations: 25mM, 250mM and 500mM. The tested strain was <i>Bacillus subtilis</i> 168:<br><br />
<br></p><br />
<center><br />
<table align="center"><br />
<tr><td align=center><img src="https://static.igem.org/mediawiki/2014/8/8c/Chemotaxis_-_results_fuch.png"></td><br />
<td align=center><img src="https://static.igem.org/mediawiki/2014/f/fd/Chemotaxis_-_results_fuchsin.png"></td></tr><br />
<tr><td align=center><p class="legend">Figure 13: Fuchsin - negative control (dilution 1/50)</p></td><br />
<td align=center><p class="legend">Figure 14: NAG (25mM) (dilution 1/50)</p></td></tr><br />
</table></center><br><br />
<p class="texte">The average number of colonies with the negative control is 121. In contrast, a cell layer was observed for the NAG plates with every concentration, indicating that a large number of cells have been inoculated in the plates.<br><br />
<br><br />
Thus, we assumed that WT <i>B. subtilis</i> was more attracted by NAG than by fuchsin. In addition we could neglect the bacterial growth because the test only lasted one hour. We also neglected diffusion and osmolality phenomena for the reasons explained above. <br><br />
<br><br />
Unfortunately for us we forgot one major effect… Can you believe that fuchsin solution contains about 15% of ethanol?!!! This concentration can lead to the death of some cells and thus make the fuschin dye an appropriate negative control.<br><br />
<br><br />
<b><p class="texte">This incredible and dramatic discovery destroyed all of our hopes about the God of chemotaxis! :-(</b><br><br />
<br><br />
However, our team did not give up on synthetic biology! :-) Indeed, after days of disappointment and no time left for lab work, we raised from ashes and tried to find another negative control.<br><br />
<br><br />
Hopefully, we managed to find a negative control: galactose (25mM). The article "Chemotaxis towards sugars by <i>B. subtilis</i>" (<i>Ordal et al., 1979</i>) proved that it was a poor attractant.<br><br />
<br><br />
We made our tests again with this new molecule and glucose (25mM) as positive control.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/8/86/Chemotaxis_-_final_results.png"></center><br />
<p class="legend">Figure 15: Final results (dilution : 1/10,000)</p><br />
<p class="texte"> The miracle arrived! We managed to prove that our WT <i>Bacillus subtilis</i> was indeed naturally attracted to NAG.</p><br />
<p class="texte"><i>NB: It was our last experiment. Unfortunately we were running out of time and we could not do much more test. We would like to do the experiment with a lower dilution and repeat it several times.</i><br><br />
<br><br />
<b><p class="texte">Our results are not statistically significant however this result has been proved in literature.</p></b><br></p><br />
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<div class="technology">Binding module</div><br />
<div class="thelanguage"><br />
<br />
<br />
<p class="title2">1. Preliminary experiments</p><br />
<p class="texte">For the first experiment we wanted to check if the buffer of the binding assay was compatible with <i>B. subtilis</i> survival. To do that, we tested four bacterial concentrations (from OD 0.1 to OD 0.01). These <i>B. subtilis</i> suspensions were incubated 1 hour at 4°C with 500µL of either Chitin Binding Buffer (CBB) or water. 100 µL cell suspension were plated on LB medium in order to count surviving cells.</br><br />
Cells do not seem affected by the presence of CBB or water with estimated ODs of 0.05 or 0.025.<br />
</br>We observed similar survival rates between cells treated with CBB or water (these data are not shown on figure 16).<br />
Thus, the experimental conditions of the chitin binding assay are compatible with the bacterial life.</p><br />
</br><br />
<center><img src="https://static.igem.org/mediawiki/parts/e/e0/Data_not.jpg" width="40%"></center><br />
</br><br />
<br />
<p class="legend">Figure 16: We don't want to bother you with useless data</p><br />
<br />
<p class="title2">2. Binding test using engineered <i>B. subtilis</i></p><br />
<br />
<p class="title3">Purpose</p><br />
<p class="texte"><i>B. subtilis</i> transformed with the binding module should produce a chimeric protein allowing the bacterium to bind chitin. It is composed of the Cell Wall Binding Domain of a <i>B. subtilis</i> protein LycT, and of the GbpA domain 4 of <i>Vibrio cholerae</i> able to bind chitin. The capacity to bind chitin was assessed by bringing together either the WT strain or the engineered bacterium with chitin beads. After several washes, bacteria still bound to the beads were counted.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">We measured the quantity of bacteria before the binding assay (Direct), in the eluted fraction of the first (Wash A) and the second (Wash B) washing steps, and associated with the beads.</br> <br />
<br />
Both bacterial solutions of WT and engineered bacterium used for the binding assay had the initial same concentration before the assay (Direct on Figure 17).<br />
There is also no significant difference between both strains after the first wash (Wash A on Figure 17). However, the concentration of cells quantified in the eluted fraction after the second wash was significantly higher for the wild-type strain. This suggests that the engineered strain is more retained on chitin beads. This was confirmed by a 40 times higher number of cells associated with the chitin beads for the engineered over the wild-type strain(Beads on Figure 17). <br />
<br/>Thus, we successfully engineered <i>B. subtilis</i> to promote its fixation on chitin.</p><br />
<br />
</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/e/ea/Graphe_binding_2.png" width="60%"></center><br />
</br><br />
<br />
<p class="legend">Figure 17: Attachment of WT <i>B. subtilis</i> and engineered bacterium to chitin. The <span style="color:#0000FF">WT bacteria</span> or <span style="color:#FF0000">the bacteria with the binding system</span> concentrations have been determined during the different steps of the binding test. The stars represent a significant difference observed with a Student test with p<0.05.<br />
</p><br />
<br />
<p class="title2">3. Microscopic observations</p><br />
<br />
<p class="title3">Purpose</p><br />
<p class="texte">We wanted to observe SubtiTree bound on the chitin coated beads. In order to perform a 3D reconstruction showing this interaction, we used confocal laser scanning microscope. Through the use of a green fluorochrome (Syto9), we highlighted the presence of bacteria on the surface of the beads (individualized by phase-contrast). A first calibration step determined the minimum threshold to remove the background noise and the natural fluorescence.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">We can notice the engineered bacterium is well attached to the surface of beads coated with chitin.</p><br />
</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/archive/5/53/20141013073044!Photo_billes_microscopie.png" width="45%"></center><br />
</br><br />
<p class="legend">Figure 18: Microscopic view of engineered strain associated with beads surfaces coated with chitin</p><br />
<br />
<p class="texte">Using ImageJ software, we are able to create 3D pictures and movies of those comments.</br></p><br />
<center><img src="https://static.igem.org/mediawiki/2014/5/53/Photo_billes_microscopie.png" width="45%" style="float:left;"><video width="45%" poster="https://static.igem.org/mediawiki/2014/4/4c/Video_poster.png" controls><br />
<source src="https://static.igem.org/mediawiki/2014/6/61/Beads_3D_movie.ogg" type='video/ogg; codecs="theora, vorbis"'/><br />
</video></center><br />
</br><br />
<p class="legend">Figure 19: A short movie of 3D bead surfaces coated with chitin and the engineered strain (emotional sequence for Subtitree: first movie apparition, before Cannes…)</p><br />
<br />
<p class="texte">We then performed a wash step on the chitin beads. We measured the release of bacteria on the washing solution. When our bacterium has the binding module (Right on Figure 20), there is less release than without the module (Right on Figure 20). Therefore, the engineered bacterium is retained by the beads.</p><br />
<center><img src="https://static.igem.org/mediawiki/2014/9/97/Photo_lavage_microscopie.png" width="45%"></center><br />
<p class="legend">Figure 20: Microscopic view of elution fraction of WT <i>B. subtilis</i> (Left) and engineered bacterium (Right). <br />
</p><br />
<br />
<p class="texte">To conclude, all the results are consistent with the successfull integration of a functional chitin binding system in <i>B. subtilis</i>. We thus validated the second module.</p><br />
<br />
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<br />
<div class="technology">Fungicides module</div><br />
<div class="thelanguage"><br />
<br />
<br />
<p class="title2"> 1. Preliminary experiments</p><br />
<p class="title3">Tests with commercial peptides and controls</p><br />
<p class="texte">The first tests were accomplished with commercial GAFP-1 and D4E1 peptides at different concentrations (12.5µM, 25µM, 100µM). As <i>Ceratocystis Platani</i> is pathogenic, we could not perform tests directly with this fungus. These tests were therefore performed with different non-pathogenic fungal strains from the same phylum as <i>Ceratocystis Platani</i>.<br />
</br><br />
After 1 to 6 days at 30°C depending on the fungal strain, the PDA (Potato Dextrose Agar) plates covered with fungus and containing a paper disk soaked with a commercial peptides solution were analyzed.</p><br />
<p class="texte">An inhibition halo was noticeable with commercial D4E1 peptide at 100µM on <i>Aspergillus brasiliensis</i> (Figure 21). Less bright halos were also present with lower concentrations. Concerning commercial GAFP-1, we did not notice any effect in the tested conditions. Copper Sulfate, a well-known chemical fungicide was used as a positive control.Inhibition of the fungal growth was complete with 20 mg/ml copper Sulfate, and at 10 mg/ml a darker halo appeared around the pad as can be seen on figure 21. This corresponds to a sporulating halo in response to the stress generated by the fungicide.<br />
</p><br />
<br />
<br />
<center><img style="width:450px; " src="https://static.igem.org/mediawiki/parts/a/a8/Prelim_tests_fung.jpg"><br />
<img style="width:450px; " src="https://static.igem.org/mediawiki/parts/f/f8/Controls_fung.jpg"></center><br />
<p class="legend"> Figure 21: Results of the preliminary tests of antifungal compounds</p><br />
<br />
<br />
</br><br />
<br />
<p class="texte">Regarding these results, we concluded that very high fungicide concentrations are required to inhibit the fungal growth in the tested conditions. Following these tests, new conditions were adopted in order to avoid too much fungal growth over bacterial growth. The culture medium was adjusted to fit our objective and to approximate the conditions found in the trees, and a 'sap-like' medium was elaborated (See <a href="https://2014.igem.org/Team:Toulouse/Notebook/Protocols">protocols</a> for more informations). Incubations were performed at room temperature. These new conditions were used as standard for the the next experiment.<br><br />
Camille also concluded that turning blue the Canal du Midi using high copper sulfate concentrations is not such a good idea... Thereby strengthening our faith in SubtiTree :-) ! <br />
</p><br />
<br />
<p class="title2">2. Test with antifungal bacteria</p><br />
<br />
<p class="texte">In order to test our <i>Bacillus subtilis</i> engineered strains, it was essential to find the right balance between the fungal growth and the bacterial one which was achieved with the previous modifications. Furthermore, in our genetic constructions, the antifungal peptides were designed to be exported in the extracellular medium.</br><br />
</br><br />
The transformed <i>Bacillus subtilis</i> strains were grown at 37°C during 72h before testings. Inhibitory effect of supernatant and cell pellets were tested separately. After centrifugation, the supernatant and the resuspended pellet were placed on pads and disposed on plates previously seeded with a defined number of conidia (see protocols to have more details). After several days at room temperature, an inhibition halo of <i>Trichoderma reesei</i> growth was clearly observable for the strain expressing the D4E1 peptide. The inhibition was even more noticeable with the strain carrying the GAFP-1 + D4E1 operon (Figure 22).</br><br />
However, no effect was detected for the strain expressing the GAFP-1 gene, we thus suppose a putative synergistic effect between these two peptides.</br><br />
Regarding EcAMP-1, no effect has been detected on the fungal growth. Several factors can explain these results: a number of post-transcriptional modifications are required to have a functional EcAMP-1 and in addition, the sequencing results for these constructs showed several discrepancies with the original designed sequence.<br />
<p class="texte">Inhibition halos are not visible with supernatants, probably because of either low concentrations or of instability in the extracellular medium. <br />
Another effect was noted with the same strains expressing D4E1 and GAFP-1 + D4E1 on another fungus, <i>Aspergillus brasiliensis</i> (figure 22). This effect is comparable to the one previously noted with a low concentration of copper sulfate (figure 21).</br><br />
</p><br />
<br />
</br><br />
<img style="width:400px; " src="https://static.igem.org/mediawiki/parts/c/c2/Resultfong.jpg"><br />
<img style="width:400px; " src="https://static.igem.org/mediawiki/parts/9/92/Results_fong_2.jpg"> <p class="legend">Figure 22: Results with transformed bacteria.</p><br />
<br />
<p class="texte"><br />
After this set of experiments, the strains expressing D4E1 or GAFP-1 + D4E1 have been shown to be the best candidates to play a major role in the fight against fungal diseases such as Canker stain. Keeping in mind our objective, <b>we decided to tests these strains in model plants</b>: <i>Nicotiana benthamiana</i> and <i>Arabidopsis thaliana</i>.</br><br />
These tests were performed in association with Sylvain Raffaële and Marielle Barascud in the National Institute for the Agronomic Research.</p><br />
<br />
<br />
<p class="title2">3. <i>In planta</i> tests with SubtiTree</p><br />
<br />
<br />
<center><img style="width:215px;" img src="https://static.igem.org/mediawiki/parts/a/af/In_planta.jpg" style="margin-top:5px"/></center><br />
<p class="legend"> Figure 23: Injection of antifungal <i>B. subtilis</i> in a model plant</p><br />
<br />
<p class="texte"><br />
The goal of the project is to introduce the transformed bacteria in a diseased tree. So it is necessary to perform preliminary<i> in planta </i> tests to judge the fungus-killing abilities of the two strains selected after the previous sets of experiments. </br><br />
The strain expressing fungicides was first inoculated in two model plants (<i>Arabidopsis thaliana</i> and <i>Nicotiana benthamiana</i>). After this step, a phytopathogenic fungus (<i>Sclerotinia sclerotiorum</i>) was placed on the leaves. </br><br />
</p><br />
<br />
<p class="texte">Twenty-four hours after SubtiTree inoculation, no phenotypic modification of the leaves could be detected. We can conclude that our bacterium, its introduction and the fungicides production in plants do not have deleterious effects.</br><br />
Without proper treatment, the drop of the phytopathogenic fungus on <i>Nicotiana benthamiana</i> leaves caused a necrosis halo which could be measured after 40h. The number of necrotic sites and the lesion size appeared as reduced by <i>B. subtilis</i> expressing DE41 or GAFP1-D4E1, unlike the WT bacterium. Two independant replicate of this experiments were performed successfully</br><br></br><br />
We did not observe any significant results for <i>Arabidopsis thaliana</i> because of the use of two plants batches with different ages.</br><br />
<br />
We can therefore conclude that when SubtiTree is in plant's physiological conditions, <b> it is harmless to the plant, and that the production of fungicides is effective, reducing the leaves necrosis. </b><br />
</p><br />
<br />
<center><img style="width:860px;" img src="https://static.igem.org/mediawiki/parts/f/f1/Results_d4%2B_gafp1.jpg"></center> <p class="legend">Figure 24: Results of <i>in planta</i> test</p><br />
<br />
<br />
<p class="texte">We could expect that bringing altogether the three modules (chemotaxis, binding and antifungal) should even improved the performance of SubtiTree. Thus, these results open the way towards the use of SubtiTree in plane tree</br><br />
More than ever, let's save our trees with SubtiTree!<br />
</p><br />
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<h2>Experimental results</h2><br />
<p> Are our modules functionnal? </p><br />
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<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Results&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Experimental results</p> <br />
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<br />
<p class="texte">How did we validate the three modules and improve our new protocols? Click below to find out…</p><br />
<br />
<p style="font-size:1.3em; margin: 0 0 30px 0;"><a href="#" onClick="ddaccordion.expandall('technology'); return false">Expand all</a> | <a href="#" onClick="ddaccordion.collapseall('technology'); return false">Collapse all</a></p><br />
<br />
<div class="technology">Chemotaxis Module</div><br />
<div class="thelanguage"><br />
<br />
<br />
<p class="texte">We performed several assays to demonstrate the chemotaxis ability of our engineered <i>Bacillus subtilis</i> strain to move towards N-acetylglucosamine (NAG), the base unit of chitin. The ability of the <i>wild-type</i> strain to move towards glucose was the positive control. <br />
</p><br />
<br />
<p class="title2">1. Petri dishes Test</p><br />
<p class="texte"><br />
The first chemotaxis assay was done in Petri dishes filled with a growth medium containing 0,3% agar. This semi-solid medium was supposed to favor bacterial motility. A paper disk soaked with the attractive compound was placed in the middle of the dish, then cells were loaded into the medium (see Figure 1). This protocol was from the <a href="https://2011.igem.org/Team:Imperial_College_London/Protocols_Chemotaxis">the Imperial College 2011 iGEM team</a>.<br />
</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/0/05/Schema_1.png" alt="schema Figure 1" style="width:500px"></center><br />
<p class="legend">Figure 1: Petri dishes chemotaxis assay. (A) pipetman was used to inject cells into the semi-solid medium. (B) Bacteria would move toward the attractive compound diffusing from a paper disk.<br />
</p><br />
<br />
<p class="texte">With this assay, we however failed to see any chemotaxis of the <i>wild-type</i> control toward glucose (Figure 2-A).As <i>B. subtilis</i> is attracted by many other glucides and amino-acids that can be found in rich medium such as in LB medium. Therefore, with the hope of improving the experimental conditions, we have challenged the cells with paper discs soaked in LB medium containing glucose (Figure 2-B).<br />
</p><br />
<br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/f/ff/Fig2_AetB.png" alt="Figure 2" style="width:750px"></center><br />
<p class="legend">Figure 2: Chemotaxis test with paper discs soaked in either a Glucose solution (A) or in Glucose containing LB medium (B) as attractive compound. Paper discs soaked with water were used as negative controls.<br />
</p><br />
<br />
<p class="texte">No difference was observed between the Petri dishes with or without glucose. With glucose containing LB medium, a large halo around the paper disk was observed. This halo might be due to cells attracted by the solution, as it was not observed when cells were not inoculated in the Petri dish (data not shown). However this result was barely reproducible. Moreover with the addition of LB medium, it was hard to make the distinction between attractive effects and cell growth resulting from random diffusion. We have therefore given up this protocol and tested alternative protocols.<br />
</p><br />
<br />
<p class="title2">2. Plug in Pond system<br />
</p><br />
<br />
<p class="texte"><br />
This protocol was from a PhD work (see references [1]). <i>B.subtilis</i> was grown overnight to a density of 8.10<sup>8</sup> cells/mL. 10mL of the culture was mixed with 15mL of LB medium containing 1.5% agar kept at 45°C. The final agar concentration was 0.9%. Tetracycline (25 µg/ml) was added to inhibit growth in order to only observe the chemotaxis phenomenon. Plates were cooled down and dried, before digging wells with either a punch or 1mL tips. The wells were then filled with the attractive compound (Figure 3). After one hour at room temperature, pictures of the plates were taken and the results analyzed.<br />
</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/c/cd/Fig3.png" alt="Figure 3" style="width:400px"></center><br />
<p class="legend">Figure 3: Plug-in-pond test design.</p><br />
<br />
<p class="texte"><br />
On our first try with <i>B. subtilis</i>, we made three wells per plate (Figure 4).The wells were filled with glucose at different concentrations and tetracycline was not added in one of the plates.<br />
</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/c/ce/Fig4.png" alt="Figure 4" style="width:400px"></center><br />
<p class="legend">Figure 4: Plates after 12h at room temperature.</p><br />
<br />
<p class="texte"><br />
After one hour, no tangible results were obtained. After 12h we observed halos around the 1M glucose containing wells in the plates without tetracycline but not in the plates with tetracycline. Again, because cells could use glucose for growth, we could not distinguish between growth and chemotaxis. Making the hypothesis that the concentration of tetracycline could have been too high and inhibited any bacterial activity, we thereafter lowered the tetracycline concentration to 15µg/mL. We repeated this protocol with this new experimental condition. We made two wells per plate (Figure 5), one with either Glucose or n-acetyl-glucosamine and one with LB medium. As previously, no results were achieved after 1h, but after 12h we could notice halos.<br />
</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/c/c3/Bsubtilis_result.png" alt="Figure 5" style="width:750px"></center><br />
<p class="legend">Figure 5: Chemotaxis test with <i>B. subtilis</i> WT. The upper well contains attractive compound and the lower well contains medium without any attractive compound.<br />
</p><br />
<br />
<p class="texte"><br />
Results were not as clear as in the previous assay (Figure 4), but halos around the wells with glucose at 250mM with and without tetracycline were observed. With N-acetyl-glucosamine (NAG) as the attractive compound, halos were observed for a concentration of 25mM with tetracycline and for a concentration of 250mM without tetracycline, thus suggesting that our <i>B. subtilis</i> 168 strain is attracted toward NAG and uses it to grow.<br />
</p><br />
<br />
<br />
<p class="texte"><br />
<b>References:</b></br><br />
[1]: Claudine Baraquet. <b>Etude de la réponse adaptative à l'oxyde de triméthylamine et de son mécanisme de détection chez <i>Escherichia coli</i> et <i>Shewanella oneidensis</i></b>. 2008, Université de la Méditerranée Aix-Marseille II.<br />
</p><br />
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<br />
<br />
<br />
<p class="title2">4. Capillary test between two tubes also called the tubes test</p><br />
<p class="texte">After the experiment of the plug-in-pond, we decided to construct a system by welding two Eppendorf tubes with a capillary thanks to an electric burner.</p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/f/fb/Chemotaxis_-_eppendorf.png"></center><br />
<p class="legend">Figure 6: Photography of the first tubes system</p><br />
<br />
<p class="texte">We tested this system with a fuchsin dye and water and we were able to observe the diffusion of fuchsin dye towards water. However this construction had a leakage next to the weld seam that we could not stop. <br />
Thus, the Toulouse 2014 iGEM team asked the help from the INSA glass blower, Patrick Chekroun. He designed two systems composed of two tubes linked by a capillary.</p><br />
<br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/2/2b/Chemotaxis_-_tubes.png"><center><br />
<p class="legend">Figure 7: Scheme of the tubes system</p><br />
<br />
<p class="texte"><br />
This new system was tested with the fuchsin dye and the assay was made with WT <i>B. subtilis</i> and N-Acetylglucosamine as the attractive compound.<br />
<br><br><br />
<i>NB: We could not see any diffusion of the fushin dye from one tube to the other. We made the hypothesis that it was not visible by sight because of the small diameter of the capillary. <br />
</i><br><br />
<br><br />
The following strategy was used to avoid disturbance due to pressure and liquid movement through the capillary:<br><br />
- The first step was the addition of Wash Buffer until the capillary was full to prevent the presence of any air bubbles which could have impeded diffusion.<br><br />
- Then, the tube 2 was plugged and maintained with the thumb while another iGEM mate was adding the bacterial suspention of WT <i>B. subtilis</i> into the tube 1.<br><br />
- The tube 1 was also plugged and only then the thumb could be removed from the tube 2. <br><br />
- In the same way, the N-Acetylglucosamine was added in the tube 2.<br><br />
- The same process was made with xylose as a positive control.<br><br />
<br><br />
<i>NB: According to the paper "Chemotaxis towards sugars by </i>Bacillus subtilis", (Ordal et al., 1979),<i> glucose and xylose have the same attractant power. We have privileged a positive control instead of a negative one as we were not sure that this system was efficient.</i><br><br />
<br><br />
- The system was kept straight for 2 hours. Every 40 minutes, samples from each were removed and streaked on solid medium (dilution 1/1,000) in order to estimate the bacterial concentration.</p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/1/1b/Chemotaxis_-_tubes_photo.png"></center><br />
<p class="legend">Figure 8: Photography of the tubes system</p><br />
<br />
<br />
<p class="texte">Unfortunately, the dilution was too high to detect any chemotaxis movement. As we did not find any information in the litterature and did not have enough time to optimize this protocol, we dicided to test again the first protocol from the Imperial college 2011 iGEM team : the tips capillary test.<br />
</p><br />
<br />
<p class="title2"> 5. Tips capillary system</p><br />
<p class="title3">First tips capillary system</p><br />
<p class="texte">This protocol was adapted from The Imperial College 2011 iGEM team (See <a href="https://2014.igem.org/Team:Toulouse/Notebook/Protocols#select8">chemotaxis protocol</a>).<br />
<br><br />
In the first tips capillary system, we used parafilm to avoid any kind of air disturbance in the tips. The different steps are described below:<br><br />
- 15µL of each chemo-attractant was pipetted.<br><br />
- The bottom of tip with the pipette was then put on a piece of parafilm and the pipette was removed from the top of the tips.<br><br />
- The tip was then sealed with a piece of parafilm in order to keep the liquid sterile and inside the tip.<br><br />
- To finish, the level of the solution in the tip was marked.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/9/94/Chemotaxis_-_tip.png"></center><br />
<p class="legend">Figure 9: Sealing of a tip with parafilm</p><br />
<br />
<p class="texte">- When all the chemo-attractants were added, the were fixed on a green support. The whole process can be seen on Figure 10.<br><br />
- Each tip was then immersed into 300 µL of a bacterial suspension in the wells of an Elisa plate.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/0/05/Chemotaxis_-_tip_and_support.png"></center><br />
<p class="legend">Figure 10: First tips capillary system</p><br />
<br />
<p class="texte"><i>NB: the yellow carton was used to stabilize the system and kept it straight.</i><br><br />
<br><br />
- After one hour, the tips were removed from the bacteria suspensions and the bacteria content of the tips was monitored with a Thoma cell under the microscope.<br><br />
<br><br />
We experienced several problems with this system:<br><br />
- The liquid level decreasing so much during the course of the experiment that we did not have enough liquid to fill the Thoma cell for counting.<br><br />
- The bacteria were moving, therefore preventing us from accurately counting them. Taking into account this probelem, we decided to estimate the bacterial concentration by streaking the tips content on agar plate instead of using Thoma cell and microscopy.<br />
<br />
<p class="title3">Second tips capillary system<br />
</p><br />
<p class="texte">And then the revolution came! We found a multichannel pipette :D The same protocol was performed except that the parafilm was used to avoid the air entrance between the tips and the pipette and therefore the loss of liquid.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/e/e4/Chemotaxis_-_pipette.png"></center><br />
<p class="legend">Figure 11: Second tips capillary system</p><br />
<br />
<p class="title3">Improvement of the second tips capillary system</p><br />
<p class="texte">However this system was not optimal it is why we decided to use blu tack instead of parafilm: <br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/4/42/Chemotaxis_-_pipette_and_blu_tack.png"></center><br />
<p class="legend">Figure 12: Improvement of the second tips capillary system</p><br />
<br />
<p class="texte"><b>At that point, the protocol was approved and the final test could finally start! :-)</b><br><br />
<br><br />
There was just one tiny problem… we did not have our optimized bacterium transformed with the chemotaxis module!!! That is why we concentrated our efforts on WT <i>B. subtilis</i> strain.<br><br />
<br><br />
The main goal was to find an optimized control and to analyze the eventual chemotaxis of the WT strain. To avoid osmolality bias, we wanted to find a molecule which was non-attractant and with a similar molecular weight than that of the N-Acetylglucosamine (221.21 g/mol). Our first idea was to use fuchsin (Molecular weight: 337.85 g/mol).<br><br />
<br><br />
At the beginning, the experiment was conducted with only one negative control, the fuchsin and different NAG concentrations: 25mM, 250mM and 500mM. The tested strain was <i>Bacillus subtilis</i> 168:<br><br />
<br></p><br />
<center><br />
<table align="center"><br />
<tr><td align=center><img src="https://static.igem.org/mediawiki/2014/8/8c/Chemotaxis_-_results_fuch.png"></td><br />
<td align=center><img src="https://static.igem.org/mediawiki/2014/f/fd/Chemotaxis_-_results_fuchsin.png"></td></tr><br />
<tr><td align=center><p class="legend">Figure 13: Fuchsin - negative control (dilution 1/50)</p></td><br />
<td align=center><p class="legend">Figure 14: NAG (25mM) (dilution 1/50)</p></td></tr><br />
</table></center><br><br />
<p class="texte">The average number of colonies with the negative control is 121. In contrast, a cell layer was observed for the NAG plates with every concentration, indicating that a large number of cells have been inoculated in the plates.<br><br />
<br><br />
Thus, we assumed that WT <i>B. subtilis</i> was more attracted by NAG than by fuchsin. In addition we could neglect the bacterial growth because the test only lasted one hour. We also neglected diffusion and osmolality phenomena for the reasons explained above. <br><br />
<br><br />
Unfortunately for us we forgot one major effect… Can you believe that fuchsin solution contains about 15% of ethanol?!!! This concentration can lead to the death of some cells and thus make the fuschin dye an appropriate negative control.<br><br />
<br><br />
<b><p class="texte">This incredible and dramatic discovery destroyed all of our hopes about the God of chemotaxis! :-(</b><br><br />
<br><br />
However, our team did not give up on synthetic biology! :-) Indeed, after days of disappointment and no time left for lab work, we raised from ashes and tried to find another negative control.<br><br />
<br><br />
Hopefully, we managed to find a negative control: galactose (25mM). The article "Chemotaxis towards sugars by <i>B. subtilis</i>" (<i>Ordal et al., 1979</i>) proved that it was a poor attractant.<br><br />
<br><br />
We made our tests again with this new molecule and glucose (25mM) as positive control.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/8/86/Chemotaxis_-_final_results.png"></center><br />
<p class="legend">Figure 15: Final results (dilution : 1/10,000)</p><br />
<p class="texte"> The miracle arrived! We managed to prove that our WT <i>Bacillus subtilis</i> was indeed naturally attracted to NAG.</p><br />
<p class="texte"><i>NB: It was our last experiment. Unfortunately we were running out of time and we could not do much more test. We would like to do the experiment with a lower dilution and repeat it several times.</i><br><br />
<br><br />
<b><p class="texte">Our results are not statistically significant however this result has been proved in literature.</p></b><br></p><br />
<br />
</br><br />
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<br />
<div class="technology">Binding module</div><br />
<div class="thelanguage"><br />
<br />
<br />
<p class="title2">1. Preliminary experiments</p><br />
<p class="texte">For the first experiment we wanted to check if the buffer of the binding assay was compatible with <i>B. subtilis</i> survival. To do that, we tested four bacterial concentrations (from OD 0.1 to OD 0.01). These <i>B. subtilis</i> suspensions were incubated 1 hour at 4°C with 500µL of either Chitin Binding Buffer (CBB) or water. 100 µL cell suspension were plated on LB medium in order to count surviving cells.</br><br />
Cells do not seem affected by the presence of CBB or water with estimated ODs of 0.05 or 0.025.<br />
</br>We observed similar survival rates between cells treated with CBB or water (these data are not shown on figure 16).<br />
Thus, the experimental conditions of the chitin binding assay are compatible with the bacterial life.</p><br />
</br><br />
<center><img src="https://static.igem.org/mediawiki/parts/e/e0/Data_not.jpg" width="40%"></center><br />
</br><br />
<br />
<p class="legend">Figure 16: We don't want to bother you with useless data</p><br />
<br />
<p class="title2">2. Binding test using engineered <i>B. subtilis</i></p><br />
<br />
<p class="title3">Purpose</p><br />
<p class="texte"><i>B. subtilis</i> transformed with the binding module should produce a chimeric protein allowing the bacterium to bind chitin. It is composed of the Cell Wall Binding Domain of a <i>B. subtilis</i> protein LycT, and of the GbpA domain 4 of <i>Vibrio cholerae</i> able to bind chitin. The capacity to bind chitin was assessed by bringing together either the WT strain or the engineered bacterium with chitin beads. After several washes, bacteria still bound to the beads were counted.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">We measured the quantity of bacteria before the binding assay (Direct), in the eluted fraction of the first (Wash A) and the second (Wash B) washing steps, and associated with the beads.</br> <br />
<br />
Both bacterial solutions of WT and engineered bacterium used for the binding assay had the initial same concentration before the assay (Direct on Figure 17).<br />
There is also no significant difference between both strains after the first wash (Wash A on Figure 17). However, the concentration of cells quantified in the eluted fraction after the second wash was significantly higher for the wild-type strain. This suggests that the engineered strain is more retained on chitin beads. This was confirmed by a 40 times higher number of cells associated with the chitin beads for the engineered over the wild-type strain(Beads on Figure 17). <br />
<br/>Thus, we successfully engineered <i>B. subtilis</i> to promote its fixation on chitin.</p><br />
<br />
</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/e/ea/Graphe_binding_2.png" width="60%"></center><br />
</br><br />
<br />
<p class="legend">Figure 17: Attachment of WT <i>B. subtilis</i> and engineered bacterium to chitin. The <span style="color:#0000FF">WT bacteria</span> or <span style="color:#FF0000">the bacteria with the binding system</span> concentrations have been determined during the different steps of the binding test. The stars represent a significant difference observed with a Student test with p<0.05.<br />
</p><br />
<br />
<p class="title2">3. Microscopic observations</p><br />
<br />
<p class="title3">Purpose</p><br />
<p class="texte">We wanted to observe SubtiTree bound on the chitin coated beads. In order to perform a 3D reconstruction showing this interaction, we used confocal laser scanning microscope. Through the use of a green fluorochrome (Syto9), we highlighted the presence of bacteria on the surface of the beads (individualized by phase-contrast). A first calibration step determined the minimum threshold to remove the background noise and the natural fluorescence.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">We can notice the engineered bacterium is well attached to the surface of beads coated with chitin.</p><br />
</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/archive/5/53/20141013073044!Photo_billes_microscopie.png" width="45%"></center><br />
</br><br />
<p class="legend">Figure 18: Microscopic view of engineered strain associated with beads surfaces coated with chitin</p><br />
<br />
<p class="texte">Using ImageJ software, we are able to create 3D pictures and movies of those comments.</br></p><br />
<center><img src="https://static.igem.org/mediawiki/2014/5/53/Photo_billes_microscopie.png" width="45%" style="float:left;"><video width="45%" poster="https://static.igem.org/mediawiki/2014/4/4c/Video_poster.png" controls><br />
<source src="https://static.igem.org/mediawiki/2014/6/61/Beads_3D_movie.ogg" type='video/ogg; codecs="theora, vorbis"'/><br />
</video></center><br />
</br><br />
<p class="legend">Figure 19: A short movie of 3D bead surfaces coated with chitin and the engineered strain (emotional sequence for Subtitree: first movie apparition, before Cannes…)</p><br />
<br />
<p class="texte">We then performed a wash step on the chitin beads. We measured the release of bacteria on the washing solution. When our bacterium has the binding module (Right on Figure 20), there is less release than without the module (Right on Figure 20). Therefore, the engineered bacterium is retained by the beads.</p><br />
<center><img src="https://static.igem.org/mediawiki/2014/9/97/Photo_lavage_microscopie.png" width="45%"></center><br />
<p class="legend">Figure 20: Microscopic view of elution fraction of WT <i>B. subtilis</i> (Left) and engineered bacterium (Right). <br />
</p><br />
<br />
<p class="texte">To conclude, all the results are consistent with the successfull integration of a functional chitin binding system in <i>B. subtilis</i>. We thus validated the second module.</p><br />
<br />
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<br />
<div class="technology">Fungicides module</div><br />
<div class="thelanguage"><br />
<br />
<br />
<p class="title2"> 1. Preliminary experiments</p><br />
<p class="title3">Tests with commercial peptides and controls</p><br />
<p class="texte">The first tests were accomplished with commercial GAFP-1 and D4E1 peptides at different concentrations (12.5µM, 25µM, 100µM). As <i>Ceratocystis Platani</i> is pathogenic, we could not perform tests directly with this fungus. These tests were therefore performed with different non-pathogenic fungal strains from the same phylum as <i>Ceratocystis Platani</i>.<br />
</br><br />
After 1 to 6 days at 30°C depending on the fungal strain, the PDA (Potato Dextrose Agar) plates covered with fungus and containing a paper disk soaked with a commercial peptides solution were analyzed.</p><br />
<p class="texte">An inhibition halo was noticeable with commercial D4E1 peptide at 100µM on <i>Aspergillus brasiliensis</i> (Figure 21). Less bright halos were also present with lower concentrations. Concerning commercial GAFP-1, we did not notice any effect in the tested conditions. Copper Sulfate, a well-known chemical fungicide was used as a positive control.Inhibition of the fungal growth was complete with 20 mg/ml copper Sulfate, and at 10 mg/ml a darker halo appeared around the pad as can be seen on figure 21. This corresponds to a sporulating halo in response to the stress generated by the fungicide.<br />
</p><br />
<br />
<br />
<center><img style="width:450px; " src="https://static.igem.org/mediawiki/parts/a/a8/Prelim_tests_fung.jpg"><br />
<img style="width:450px; " src="https://static.igem.org/mediawiki/parts/f/f8/Controls_fung.jpg"></center><br />
<p class="legend"> Figure 21: Results of the preliminary tests of antifungal compounds</p><br />
<br />
<br />
</br><br />
<br />
<p class="texte">Regarding these results, we concluded that very high fungicide concentrations are required to inhibit the fungal growth in the tested conditions. Following these tests, new conditions were adopted in order to avoid too much fungal growth over bacterial growth. The culture medium was adjusted to fit our objective and to approximate the conditions found in the trees, and a 'sap-like' medium was elaborated (See <a href="https://2014.igem.org/Team:Toulouse/Notebook/Protocols">protocols</a> for more informations). Incubations were performed at room temperature. These new conditions were used as standard for the the next experiment.<br><br />
Camille also concluded that turning blue the Canal du Midi using high copper sulfate concentrations is not such a good idea... Thereby strengthening our faith in SubtiTree :-) ! <br />
</p><br />
<br />
<p class="title2">2. Test with antifungal bacteria</p><br />
<br />
<p class="texte">In order to test our <i>Bacillus subtilis</i> engineered strains, it was essential to find the right balance between the fungal growth and the bacterial one which was achieved with the previous modifications. Furthermore, in our genetic constructions, the antifungal peptides were designed to be exported in the extracellular medium.</br><br />
</br><br />
The transformed <i>Bacillus subtilis</i> strains were grown at 37°C during 72h before testings. Inhibitory effect of supernatant and cell pellets were tested separately. After centrifugation, the supernatant and the resuspended pellet were placed on pads and disposed on plates previously seeded with a defined number of conidia (see protocols to have more details). After several days at room temperature, an inhibition halo of <i>Trichoderma reesei</i> growth was clearly observable for the strain expressing the D4E1 peptide. The inhibition was even more noticeable with the strain carrying the GAFP-1 + D4E1 operon (Figure 22).</br><br />
However, no effect was detected for the strain expressing the GAFP-1 gene, we thus suppose a putative synergistic effect between these two peptides.</br><br />
Regarding EcAMP-1, no effect has been detected on the fungal growth. Several factors can explain these results: a number of post-transcriptional modifications are required to have a functional EcAMP-1 and in addition, the sequencing results for these constructs showed several discrepancies with the original designed sequence.<br />
<p class="texte">Inhibition halos are not visible with supernatants, probably because of either low concentrations or of instability in the extracellular medium. <br />
Another effect was noted with the same strains expressing D4E1 and GAFP-1 + D4E1 on another fungus, <i>Aspergillus brasiliensis</i> (figure 22). This effect is comparable to the one previously noted with a low concentration of copper sulfate (figure 21).</br><br />
</p><br />
<br />
</br><br />
<img style="width:400px; " src="https://static.igem.org/mediawiki/parts/c/c2/Resultfong.jpg"><br />
<img style="width:400px; " src="https://static.igem.org/mediawiki/parts/9/92/Results_fong_2.jpg"> <p class="legend">Figure 22: Results with transformed bacteria.</p><br />
<br />
<p class="texte"><br />
After this set of experiments, the strains expressing D4E1 or GAFP-1 + D4E1 have been shown to be the best candidates to play a major role in the fight against fungal diseases such as Canker stain. Keeping in mind our objective, <b>we decided to tests these strains in model plants</b>: <i>Nicotiana benthamiana</i> and <i>Arabidopsis thaliana</i>.</br><br />
These tests were performed in association with Sylvain Raffaële and Marielle Barascud in the National Institute for the Agronomic Research.</p><br />
<br />
<br />
<p class="title2">3. <i>In planta</i> tests with SubtiTree</p><br />
<br />
<br />
<center><img style="width:215px;" img src="https://static.igem.org/mediawiki/parts/a/af/In_planta.jpg" style="margin-top:5px"/></center><br />
<p class="legend"> Figure 23: Injection of antifungal <i>B. subtilis</i> in a model plant</p><br />
<br />
<p class="texte"><br />
The goal of the project is to introduce the transformed bacteria in a diseased tree. So it is necessary to perform preliminary<i> in planta </i> tests to judge the fungus-killing abilities of the two strains selected after the previous sets of experiments. </br><br />
The strain expressing fungicides was first inoculated in two model plants (<i>Arabidopsis thaliana</i> and <i>Nicotiana benthamiana</i>). After this step, a phytopathogenic fungus (<i>Sclerotinia sclerotiorum</i>) was placed on the leaves. </br><br />
</p><br />
<br />
<p class="texte">Twenty-four hours after SubtiTree inoculation, no phenotypic modification of the leaves could be detected. We can conclude that our bacterium, its introduction and the fungicides production in plants do not have deleterious effects.</br><br />
Without proper treatment, the drop of the phytopathogenic fungus on <i>Nicotiana benthamiana</i> leaves caused a necrosis halo which could be measured after 40h. The number of necrotic sites and the lesion size appeared as reduced by <i>B. subtilis</i> expressing DE41 or GAFP1-D4E1, unlike the WT bacterium. Two independant replicate of this experiments were performed successfully</br><br></br><br />
We did not observe any significant results for <i>Arabidopsis thaliana</i> because of the use of two plants batches with different ages.</br><br />
<br />
We can therefore conclude that when SubtiTree is in plant's physiological conditions, <b> it is harmless to the plant, and that the production of fungicides is effective, reducing the leaves necrosis. </b><br />
</p><br />
<br />
<center><img style="width:860px;" img src="https://static.igem.org/mediawiki/parts/f/f1/Results_d4%2B_gafp1.jpg"></center> <p class="legend">Figure 24: Results of <i>in planta</i> test</p><br />
<br />
<br />
<p class="texte">We could expect that bringing altogether the three modules (chemotaxis, binding and antifungal) should even improved the performance of SubtiTree. Thus, these results open the way towards the use of SubtiTree in plane tree</br><br />
More than ever, let's save our trees with SubtiTree!<br />
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<h2>Experimental results</h2><br />
<p> Are our modules functionnal? </p><br />
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<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Results&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Experimental results</p> <br />
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<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 20px; padding-bottom:40px;"><br />
<br />
<p class="texte">How did we validate the three modules and improve our new protocols? Click below to find out…</p><br />
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<p style="font-size:1.3em; margin: 0 0 30px 0;"><a href="#" onClick="ddaccordion.expandall('technology'); return false">Expand all</a> | <a href="#" onClick="ddaccordion.collapseall('technology'); return false">Collapse all</a></p><br />
<br />
<div class="technology">Chemotaxis</div><br />
<div class="thelanguage"><br />
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<p class="texte">We performed several assays to demonstrate the chemotaxis ability of our engineered <i>Bacillus subtilis</i> strain to move towards N-acetylglucosamine (NAG), the base unit of chitin. The ability of the <i>wild-type</i> strain to move towards glucose was the positive control. <br />
</p><br />
<br />
<p class="title2">1. Petri dishes Test</p><br />
<p class="texte"><br />
The first chemotaxis assay was done in Petri dishes filled with a growth medium containing 0,3% agar. This semi-solid medium was supposed to favor bacterial motility. A paper disk soaked with the attractive compound was placed in the middle of the dish, then cells were loaded into the medium (see Figure 1). This protocol was from the <a href="https://2011.igem.org/Team:Imperial_College_London/Protocols_Chemotaxis">the Imperial College 2011 iGEM team</a>.<br />
</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/0/05/Schema_1.png" alt="schema Figure 1" style="width:500px"></center><br />
<p class="legend">Figure 1: Petri dishes chemotaxis assay. (A) pipetman was used to inject cells into the semi-solid medium. (B) Bacteria would move toward the attractive compound diffusing from a paper disk.<br />
</p><br />
<br />
<p class="texte">With this assay, we however failed to see any chemotaxis of the <i>wild-type</i> control toward glucose (Figure 2-A).As <i>B. subtilis</i> is attracted by many other glucides and amino-acids that can be found in rich medium such as in LB medium. Therefore, with the hope of improving the experimental conditions, we have challenged the cells with paper discs soaked in LB medium containing glucose (Figure 2-B).<br />
</p><br />
<br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/f/ff/Fig2_AetB.png" alt="Figure 2" style="width:750px"></center><br />
<p class="legend">Figure 2: Chemotaxis test with paper discs soaked in either a Glucose solution (A) or in Glucose containing LB medium (B) as attractive compound. Paper discs soaked with water were used as negative controls.<br />
</p><br />
<br />
<p class="texte">No difference was observed between the Petri dishes with or without glucose. With glucose containing LB medium, a large halo around the paper disk was observed. This halo might be due to cells attracted by the solution, as it was not observed when cells were not inoculated in the Petri dish (data not shown). However this result was barely reproducible. Moreover with the addition of LB medium, it was hard to make the distinction between attractive effects and cell growth resulting from random diffusion. We have therefore given up this protocol and tested alternative protocols.<br />
</p><br />
<br />
<p class="title2">2. Plug in Pond system<br />
</p><br />
<br />
<p class="texte"><br />
This protocol was from a PhD work (see references [1]). <i>B.subtilis</i> was grown overnight to a density of 8.10<sup>8</sup> cells/mL. 10mL of the culture was mixed with 15mL of LB medium containing 1.5% agar kept at 45°C. The final agar concentration was 0.9%. Tetracycline (25 µg/ml) was added to inhibit growth in order to only observe the chemotaxis phenomenon. Plates were cooled down and dried, before digging wells with either a punch or 1mL tips. The wells were then filled with the attractive compound (Figure 3). After one hour at room temperature, pictures of the plates were taken and the results analyzed.<br />
</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/c/cd/Fig3.png" alt="Figure 3" style="width:400px"></center><br />
<p class="legend">Figure 3: Plug-in-pond test design.</p><br />
<br />
<p class="texte"><br />
On our first try with <i>B. subtilis</i>, we made three wells per plate (Figure 4).The wells were filled with glucose at different concentrations and tetracycline was not added in one of the plates.<br />
</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/c/ce/Fig4.png" alt="Figure 4" style="width:400px"></center><br />
<p class="legend">Figure 4: Plates after 12h at room temperature.</p><br />
<br />
<p class="texte"><br />
After one hour, no tangible results were obtained. After 12h we observed halos around the 1M glucose containing wells in the plates without tetracycline but not in the plates with tetracycline. Again, because cells could use glucose for growth, we could not distinguish between growth and chemotaxis. Making the hypothesis that the concentration of tetracycline could have been too high and inhibited any bacterial activity, we thereafter lowered the tetracycline concentration to 15µg/mL. We repeated this protocol with this new experimental condition. We made two wells per plate (Figure 5), one with either Glucose or n-acetyl-glucosamine and one with LB medium. As previously, no results were achieved after 1h, but after 12h we could notice halos.<br />
</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/c/c3/Bsubtilis_result.png" alt="Figure 5" style="width:750px"></center><br />
<p class="legend">Figure 5: Chemotaxis test with <i>B. subtilis</i> WT. The upper well contains attractive compound and the lower well contains medium without any attractive compound.<br />
</p><br />
<br />
<p class="texte"><br />
Results were not as clear as in the previous assay (Figure 4), but halos around the wells with glucose at 250mM with and without tetracycline were observed. With N-acetyl-glucosamine (NAG) as the attractive compound, halos were observed for a concentration of 25mM with tetracycline and for a concentration of 250mM without tetracycline, thus suggesting that our <i>B. subtilis</i> 168 strain is attracted toward NAG and uses it to grow.<br />
</p><br />
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<br />
<p class="texte"><br />
<b>References:</b></br><br />
[1]: Claudine Baraquet. <b>Etude de la réponse adaptative à l'oxyde de triméthylamine et de son mécanisme de détection chez <i>Escherichia coli</i> et <i>Shewanella oneidensis</i></b>. 2008, Université de la Méditerranée Aix-Marseille II.<br />
</p><br />
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<p class="title2">4. Capillary test between two tubes also called the tubes test</p><br />
<p class="texte">After the experiment of the plug-in-pond, we decided to construct a system by welding two Eppendorf tubes with a capillary thanks to an electric burner.</p><br />
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<center><img src="https://static.igem.org/mediawiki/2014/f/fb/Chemotaxis_-_eppendorf.png"></center><br />
<p class="legend">Figure 6: Photography of the first tubes system</p><br />
<br />
<p class="texte">We tested this system with a fuchsin dye and water and we were able to observe the diffusion of fuchsin dye towards water. However this construction had a leakage next to the weld seam that we could not stop. <br />
Thus, the Toulouse 2014 iGEM team asked the help from the INSA glass blower, Patrick Chekroun. He designed two systems composed of two tubes linked by a capillary.</p><br />
<br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/2/2b/Chemotaxis_-_tubes.png"><center><br />
<p class="legend">Figure 7: Scheme of the tubes system</p><br />
<br />
<p class="texte"><br />
This new system was tested with the fuchsin dye and the assay was made with WT <i>B. subtilis</i> and N-Acetylglucosamine as the attractive compound.<br />
<br><br><br />
<i>NB: We could not see any diffusion of the fushin dye from one tube to the other. We made the hypothesis that it was not visible by sight because of the small diameter of the capillary. <br />
</i><br><br />
<br><br />
The following strategy was used to avoid disturbance due to pressure and liquid movement through the capillary:<br><br />
- The first step was the addition of Wash Buffer until the capillary was full to prevent the presence of any air bubbles which could have impeded diffusion.<br><br />
- Then, the tube 2 was plugged and maintained with the thumb while another iGEM mate was adding the bacterial suspention of WT <i>B. subtilis</i> into the tube 1.<br><br />
- The tube 1 was also plugged and only then the thumb could be removed from the tube 2. <br><br />
- In the same way, the N-Acetylglucosamine was added in the tube 2.<br><br />
- The same process was made with xylose as a positive control.<br><br />
<br><br />
<i>NB: According to the paper "Chemotaxis towards sugars by </i>Bacillus subtilis", (Ordal et al., 1979),<i> glucose and xylose have the same attractant power. We have privileged a positive control instead of a negative one as we were not sure that this system was efficient.</i><br><br />
<br><br />
- The system was kept straight for 2 hours. Every 40 minutes, samples from each were removed and streaked on solid medium (dilution 1/1,000) in order to estimate the bacterial concentration.</p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/1/1b/Chemotaxis_-_tubes_photo.png"></center><br />
<p class="legend">Figure 8: Photography of the tubes system</p><br />
<br />
<br />
<p class="texte">Unfortunately, the dilution was too high to detect any chemotaxis movement. As we did not find any information in the litterature and did not have enough time to optimize this protocol, we dicided to test again the first protocol from the Imperial college 2011 iGEM team : the tips capillary test.<br />
</p><br />
<br />
<p class="title2"> 5. Tips capillary system</p><br />
<p class="title3">First tips capillary system</p><br />
<p class="texte">This protocol was adapted from The Imperial College 2011 iGEM team (See <a href="https://2014.igem.org/Team:Toulouse/Notebook/Protocols#select8">chemotaxis protocol</a>).<br />
<br><br />
In the first tips capillary system, we used parafilm to avoid any kind of air disturbance in the tips. The different steps are described below:<br><br />
- 15µL of each chemo-attractant was pipetted.<br><br />
- The bottom of tip with the pipette was then put on a piece of parafilm and the pipette was removed from the top of the tips.<br><br />
- The tip was then sealed with a piece of parafilm in order to keep the liquid sterile and inside the tip.<br><br />
- To finish, the level of the solution in the tip was marked.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/9/94/Chemotaxis_-_tip.png"></center><br />
<p class="legend">Figure 9: Sealing of a tip with parafilm</p><br />
<br />
<p class="texte">- When all the chemo-attractants were added, the were fixed on a green support. The whole process can be seen on Figure 10.<br><br />
- Each tip was then immersed into 300 µL of a bacterial suspension in the wells of an Elisa plate.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/0/05/Chemotaxis_-_tip_and_support.png"></center><br />
<p class="legend">Figure 10: First tips capillary system</p><br />
<br />
<p class="texte"><i>NB: the yellow carton was used to stabilize the system and kept it straight.</i><br><br />
<br><br />
- After one hour, the tips were removed from the bacteria suspensions and the bacteria content of the tips was monitored with a Thoma cell under the microscope.<br><br />
<br><br />
We experienced several problems with this system:<br><br />
- The liquid level decreasing so much during the course of the experiment that we did not have enough liquid to fill the Thoma cell for counting.<br><br />
- The bacteria were moving, therefore preventing us from accurately counting them. Taking into account this probelem, we decided to estimate the bacterial concentration by streaking the tips content on agar plate instead of using Thoma cell and microscopy.<br />
<br />
<p class="title3">Second tips capillary system<br />
</p><br />
<p class="texte">And then the revolution came! We found a multichannel pipette :D The same protocol was performed except that the parafilm was used to avoid the air entrance between the tips and the pipette and therefore the loss of liquid.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/e/e4/Chemotaxis_-_pipette.png"></center><br />
<p class="legend">Figure 11: Second tips capillary system</p><br />
<br />
<p class="title3">Improvement of the second tips capillary system</p><br />
<p class="texte">However this system was not optimal it is why we decided to use blu tack instead of parafilm: <br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/4/42/Chemotaxis_-_pipette_and_blu_tack.png"></center><br />
<p class="legend">Figure 12: Improvement of the second tips capillary system</p><br />
<br />
<p class="texte"><b>At that point, the protocol was approved and the final test could finally start! :-)</b><br><br />
<br><br />
There was just one tiny problem… we did not have our optimized bacterium transformed with the chemotaxis module!!! That is why we concentrated our efforts on WT <i>B. subtilis</i> strain.<br><br />
<br><br />
The main goal was to find an optimized control and to analyze the eventual chemotaxis of the WT strain. To avoid osmolality bias, we wanted to find a molecule which was non-attractant and with a similar molecular weight than that of the N-Acetylglucosamine (221.21 g/mol). Our first idea was to use fuchsin (Molecular weight: 337.85 g/mol).<br><br />
<br><br />
At the beginning, the experiment was conducted with only one negative control, the fuchsin and different NAG concentrations: 25mM, 250mM and 500mM. The tested strain was <i>Bacillus subtilis</i> 168:<br><br />
<br></p><br />
<center><br />
<table align="center"><br />
<tr><td align=center><img src="https://static.igem.org/mediawiki/2014/8/8c/Chemotaxis_-_results_fuch.png"></td><br />
<td align=center><img src="https://static.igem.org/mediawiki/2014/f/fd/Chemotaxis_-_results_fuchsin.png"></td></tr><br />
<tr><td align=center><p class="legend">Figure 13: Fuchsin - negative control (dilution 1/50)</p></td><br />
<td align=center><p class="legend">Figure 14: NAG (25mM) (dilution 1/50)</p></td></tr><br />
</table></center><br><br />
<p class="texte">The average number of colonies with the negative control is 121. In contrast, a cell layer was observed for the NAG plates with every concentration, indicating that a large number of cells have been inoculated in the plates.<br><br />
<br><br />
Thus, we assumed that WT <i>B. subtilis</i> was more attracted by NAG than by fuchsin. In addition we could neglect the bacterial growth because the test only lasted one hour. We also neglected diffusion and osmolality phenomena for the reasons explained above. <br><br />
<br><br />
Unfortunately for us we forgot one major effect… Can you believe that fuchsin solution contains about 15% of ethanol?!!! This concentration can lead to the death of some cells and thus make the fuschin dye an appropriate negative control.<br><br />
<br><br />
<b><p class="texte">This incredible and dramatic discovery destroyed all of our hopes about the God of chemotaxis! :-(</b><br><br />
<br><br />
However, our team did not give up on synthetic biology! :-) Indeed, after days of disappointment and no time left for lab work, we raised from ashes and tried to find another negative control.<br><br />
<br><br />
Hopefully, we managed to find a negative control: galactose (25mM). The article "Chemotaxis towards sugars by <i>B. subtilis</i>" (<i>Ordal et al., 1979</i>) proved that it was a poor attractant.<br><br />
<br><br />
We made our tests again with this new molecule and glucose (25mM) as positive control.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/8/86/Chemotaxis_-_final_results.png"></center><br />
<p class="legend">Figure 15: Final results (dilution : 1/10,000)</p><br />
<p class="texte"> The miracle arrived! We managed to prove that our WT <i>Bacillus subtilis</i> was indeed naturally attracted to NAG.</p><br />
<p class="texte"><i>NB: It was our last experiment. Unfortunately we were running out of time and we could not do much more test. We would like to do the experiment with a lower dilution and repeat it several times.</i><br><br />
<br><br />
<b><p class="texte">Our results are not statistically significant however this result has been proved in literature.</p></b><br></p><br />
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<div class="technology">Binding module</div><br />
<div class="thelanguage"><br />
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<p class="title2">1. Preliminary experiments</p><br />
<p class="texte">For the first experiment we wanted to check if the buffer of the binding assay was compatible with <i>B. subtilis</i> survival. To do that, we tested four bacterial concentrations (from OD 0.1 to OD 0.01). These <i>B. subtilis</i> suspensions were incubated 1 hour at 4°C with 500µL of either Chitin Binding Buffer (CBB) or water. 100 µL cell suspension were plated on LB medium in order to count surviving cells.</br><br />
Cells do not seem affected by the presence of CBB or water with estimated ODs of 0.05 or 0.025.<br />
</br>We observed similar survival rates between cells treated with CBB or water (these data are not shown on figure 16).<br />
Thus, the experimental conditions of the chitin binding assay are compatible with the bacterial life.</p><br />
</br><br />
<center><img src="https://static.igem.org/mediawiki/parts/e/e0/Data_not.jpg" width="40%"></center><br />
</br><br />
<br />
<p class="legend">Figure 16: We don't want to bother you with useless data</p><br />
<br />
<p class="title2">2. Binding test using engineered <i>B. subtilis</i></p><br />
<br />
<p class="title3">Purpose</p><br />
<p class="texte"><i>B. subtilis</i> transformed with the binding module should produce a chimeric protein allowing the bacterium to bind chitin. It is composed of the Cell Wall Binding Domain of a <i>B. subtilis</i> protein LycT, and of the GbpA domain 4 of <i>Vibrio cholerae</i> able to bind chitin. The capacity to bind chitin was assessed by bringing together either the WT strain or the engineered bacterium with chitin beads. After several washes, bacteria still bound to the beads were counted.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">We measured the quantity of bacteria before the binding assay (Direct), in the eluted fraction of the first (Wash A) and the second (Wash B) washing steps, and associated with the beads.</br> <br />
<br />
Both bacterial solutions of WT and engineered bacterium used for the binding assay had the initial same concentration before the assay (Direct on Figure 17).<br />
There is also no significant difference between both strains after the first wash (Wash A on Figure 17). However, the concentration of cells quantified in the eluted fraction after the second wash was significantly higher for the wild-type strain. This suggests that the engineered strain is more retained on chitin beads. This was confirmed by a 40 times higher number of cells associated with the chitin beads for the engineered over the wild-type strain(Beads on Figure 17). <br />
<br/>Thus, we successfully engineered <i>B. subtilis</i> to promote its fixation on chitin.</p><br />
<br />
</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/e/ea/Graphe_binding_2.png" width="60%"></center><br />
</br><br />
<br />
<p class="legend">Figure 17: Attachment of WT <i>B. subtilis</i> and engineered bacterium to chitin. The <span style="color:#0000FF">WT bacteria</span> or <span style="color:#FF0000">the bacteria with the binding system</span> concentrations have been determined during the different steps of the binding test. The stars represent a significant difference observed with a Student test with p<0.05.<br />
</p><br />
<br />
<p class="title2">3. Microscopic observations</p><br />
<br />
<p class="title3">Purpose</p><br />
<p class="texte">We wanted to observe SubtiTree bound on the chitin coated beads. In order to perform a 3D reconstruction showing this interaction, we used confocal laser scanning microscope. Through the use of a green fluorochrome (Syto9), we highlighted the presence of bacteria on the surface of the beads (individualized by phase-contrast). A first calibration step determined the minimum threshold to remove the background noise and the natural fluorescence.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">We can notice the engineered bacterium is well attached to the surface of beads coated with chitin.</p><br />
</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/archive/5/53/20141013073044!Photo_billes_microscopie.png" width="45%"></center><br />
</br><br />
<p class="legend">Figure 18: Microscopic view of engineered strain associated with beads surfaces coated with chitin</p><br />
<br />
<p class="texte">Using ImageJ software, we are able to create 3D pictures and movies of those comments.</br></p><br />
<center><img src="https://static.igem.org/mediawiki/2014/5/53/Photo_billes_microscopie.png" width="45%" style="float:left;"><video width="45%" poster="https://static.igem.org/mediawiki/2014/4/4c/Video_poster.png" controls><br />
<source src="https://static.igem.org/mediawiki/2014/6/61/Beads_3D_movie.ogg" type='video/ogg; codecs="theora, vorbis"'/><br />
</video></center><br />
</br><br />
<p class="legend">Figure 19: A short movie of 3D bead surfaces coated with chitin and the engineered strain (emotional sequence for Subtitree: first movie apparition, before Cannes…)</p><br />
<br />
<p class="texte">We then performed a wash step on the chitin beads. We measured the release of bacteria on the washing solution. When our bacterium has the binding module (Right on Figure 20), there is less release than without the module (Right on Figure 20). Therefore, the engineered bacterium is retained by the beads.</p><br />
<center><img src="https://static.igem.org/mediawiki/2014/9/97/Photo_lavage_microscopie.png" width="45%"></center><br />
<p class="legend">Figure 20: Microscopic view of elution fraction of WT <i>B. subtilis</i> (Left) and engineered bacterium (Right). <br />
</p><br />
<br />
<p class="texte">To conclude, all the results are consistent with the successfull integration of a functional chitin binding system in <i>B. subtilis</i>. We thus validated the second module.</p><br />
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<div class="technology">Fungicides module</div><br />
<div class="thelanguage"><br />
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<br />
<p class="title2"> 1. Preliminary experiments</p><br />
<p class="title3">Tests with commercial peptides and controls</p><br />
<p class="texte">The first tests were accomplished with commercial GAFP-1 and D4E1 peptides at different concentrations (12.5µM, 25µM, 100µM). As <i>Ceratocystis Platani</i> is pathogenic, we could not perform tests directly with this fungus. These tests were therefore performed with different non-pathogenic fungal strains from the same phylum as <i>Ceratocystis Platani</i>.<br />
</br><br />
After 1 to 6 days at 30°C depending on the fungal strain, the PDA (Potato Dextrose Agar) plates covered with fungus and containing a paper disk soaked with a commercial peptides solution were analyzed.</p><br />
<p class="texte">An inhibition halo was noticeable with commercial D4E1 peptide at 100µM on <i>Aspergillus brasiliensis</i> (Figure 21). Less bright halos were also present with lower concentrations. Concerning commercial GAFP-1, we did not notice any effect in the tested conditions. Copper Sulfate, a well-known chemical fungicide was used as a positive control.Inhibition of the fungal growth was complete with 20 mg/ml copper Sulfate, and at 10 mg/ml a darker halo appeared around the pad as can be seen on figure 21. This corresponds to a sporulating halo in response to the stress generated by the fungicide.<br />
</p><br />
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<center><img style="width:450px; " src="https://static.igem.org/mediawiki/parts/a/a8/Prelim_tests_fung.jpg"><br />
<img style="width:450px; " src="https://static.igem.org/mediawiki/parts/f/f8/Controls_fung.jpg"></center><br />
<p class="legend"> Figure 21: Results of the preliminary tests of antifungal compounds</p><br />
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<p class="texte">Regarding these results, we concluded that very high fungicide concentrations are required to inhibit the fungal growth in the tested conditions. Following these tests, new conditions were adopted in order to avoid too much fungal growth over bacterial growth. The culture medium was adjusted to fit our objective and to approximate the conditions found in the trees, and a 'sap-like' medium was elaborated (See <a href="https://2014.igem.org/Team:Toulouse/Notebook/Protocols">protocols</a> for more informations). Incubations were performed at room temperature. These new conditions were used as standard for the the next experiment.<br><br />
Camille also concluded that turning blue the Canal du Midi using high copper sulfate concentrations is not such a good idea... Thereby strengthening our faith in SubtiTree :-) ! <br />
</p><br />
<br />
<p class="title2">2. Test with antifungal bacteria</p><br />
<br />
<p class="texte">In order to test our <i>Bacillus subtilis</i> engineered strains, it was essential to find the right balance between the fungal growth and the bacterial one which was achieved with the previous modifications. Furthermore, in our genetic constructions, the antifungal peptides were designed to be exported in the extracellular medium.</br><br />
</br><br />
The transformed <i>Bacillus subtilis</i> strains were grown at 37°C during 72h before testings. Inhibitory effect of supernatant and cell pellets were tested separately. After centrifugation, the supernatant and the resuspended pellet were placed on pads and disposed on plates previously seeded with a defined number of conidia (see protocols to have more details). After several days at room temperature, an inhibition halo of <i>Trichoderma reesei</i> growth was clearly observable for the strain expressing the D4E1 peptide. The inhibition was even more noticeable with the strain carrying the GAFP-1 + D4E1 operon (Figure 22).</br><br />
However, no effect was detected for the strain expressing the GAFP-1 gene, we thus suppose a putative synergistic effect between these two peptides.</br><br />
Regarding EcAMP-1, no effect has been detected on the fungal growth. Several factors can explain these results: a number of post-transcriptional modifications are required to have a functional EcAMP-1 and in addition, the sequencing results for these constructs showed several discrepancies with the original designed sequence.<br />
<p class="texte">Inhibition halos are not visible with supernatants, probably because of either low concentrations or of instability in the extracellular medium. <br />
Another effect was noted with the same strains expressing D4E1 and GAFP-1 + D4E1 on another fungus, <i>Aspergillus brasiliensis</i> (figure 22). This effect is comparable to the one previously noted with a low concentration of copper sulfate (figure 21).</br><br />
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</br><br />
<img style="width:400px; " src="https://static.igem.org/mediawiki/parts/c/c2/Resultfong.jpg"><br />
<img style="width:400px; " src="https://static.igem.org/mediawiki/parts/9/92/Results_fong_2.jpg"> <p class="legend">Figure 22: Results with transformed bacteria.</p><br />
<br />
<p class="texte"><br />
After this set of experiments, the strains expressing D4E1 or GAFP-1 + D4E1 have been shown to be the best candidates to play a major role in the fight against fungal diseases such as Canker stain. Keeping in mind our objective, <b>we decided to tests these strains in model plants</b>: <i>Nicotiana benthamiana</i> and <i>Arabidopsis thaliana</i>.</br><br />
These tests were performed in association with Sylvain Raffaële and Marielle Barascud in the National Institute for the Agronomic Research.</p><br />
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<p class="title2">3. <i>In planta</i> tests with SubtiTree</p><br />
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<center><img style="width:215px;" img src="https://static.igem.org/mediawiki/parts/a/af/In_planta.jpg" style="margin-top:5px"/></center><br />
<p class="legend"> Figure 23: Injection of antifungal <i>B. subtilis</i> in a model plant</p><br />
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<p class="texte"><br />
The goal of the project is to introduce the transformed bacteria in a diseased tree. So it is necessary to perform preliminary<i> in planta </i> tests to judge the fungus-killing abilities of the two strains selected after the previous sets of experiments. </br><br />
The strain expressing fungicides was first inoculated in two model plants (<i>Arabidopsis thaliana</i> and <i>Nicotiana benthamiana</i>). After this step, a phytopathogenic fungus (<i>Sclerotinia sclerotiorum</i>) was placed on the leaves. </br><br />
</p><br />
<br />
<p class="texte">Twenty-four hours after SubtiTree inoculation, no phenotypic modification of the leaves could be detected. We can conclude that our bacterium, its introduction and the fungicides production in plants do not have deleterious effects.</br><br />
Without proper treatment, the drop of the phytopathogenic fungus on <i>Nicotiana benthamiana</i> leaves caused a necrosis halo which could be measured after 40h. The number of necrotic sites and the lesion size appeared as reduced by <i>B. subtilis</i> expressing DE41 or GAFP1-D4E1, unlike the WT bacterium. Two independant replicate of this experiments were performed successfully</br><br></br><br />
We did not observe any significant results for <i>Arabidopsis thaliana</i> because of the use of two plants batches with different ages.</br><br />
<br />
We can therefore conclude that when SubtiTree is in plant's physiological conditions, <b> it is harmless to the plant, and that the production of fungicides is effective, reducing the leaves necrosis. </b><br />
</p><br />
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<center><img style="width:860px;" img src="https://static.igem.org/mediawiki/parts/f/f1/Results_d4%2B_gafp1.jpg"></center> <p class="legend">Figure 24: Results of <i>in planta</i> test</p><br />
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<p class="texte">We could expect that bringing altogether the three modules (chemotaxis, binding and antifungal) should even improved the performance of SubtiTree. Thus, these results open the way towards the use of SubtiTree in plane tree</br><br />
More than ever, let's save our trees with SubtiTree!<br />
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{{:Team:Toulouse/template/footer}}</div>Jourdanhttp://2014.igem.org/Team:Toulouse/Project/FungicidesTeam:Toulouse/Project/Fungicides2014-10-18T00:20:11Z<p>Jourdan: </p>
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<h2>Fungicides</h2><br />
<p>To eradicate fungal diseases</p><br />
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<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Project&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Fungicides</p> <br />
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<center><img style="width:700px; " src="https://static.igem.org/mediawiki/2014/0/0c/Recap_fungicides.jpg"><br />
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<p class="legend">Figure 1: Schema of the fungicide module</p></center><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.<br />
In order to achieve this goal, we built a genetic module to produce three different peptides with antifungal activities. This triple therapy provides <br />
the advantage to minimize the resistance phenomenon.</p> <br><br />
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<p class="textesimple">Originally from plants, these peptides have different targets thus increasing the lethality on <i>C. platani</i>.</p><br />
<br></br> <br />
<ul><br />
<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<br />
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><br />
<br />
<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<br />
originating from the Asiatic orchid <i>Gastrodia elata</i>, a traditional Chinese medicinal herb cultured for thousands of years. <br />
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 <br />
ascomycete and basidiomycete fungal plant pathogens.</p></li><br />
<br />
<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<br />
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.<br />
EcAMP-1 exhibits high activity against fungi of the genus <i>Fusarium</i>.</p></li><br />
</ul><br />
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<p class="title1" style="margin-top:30px;">More information about this module </p><br />
<p class="texte"><br />
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<br />
antifungal peptides are placed under the control of the constitutive promoter P<sub>veg</sub> in <i>Bacillus subtilis</i>.</p><br />
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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"><br />
<p class="legend">Figure 2: Fungicide operon</p></center><br />
<br />
<p class="title2">Added parts</p><br />
<p class="title3">EcAMP-1</p><br />
<p class="texte">EcAMP-1 was already present in the Registry, added by the Utah State 2013 iGEM team <br />
(<a href="http://parts.igem.org/Part:BBa_K1162001"_blank">BBa_K1162001</a>). This part has been modified and improved by our team <br />
(<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><br />
<p class="title3">D4E1 and GAFP-1</p><br />
<p class="texte">We added D4E1 and GAFP-1 to the Registry of Standard Biological Parts <br />
(See <a href="https://2014.igem.org/Team:Toulouse/Result/parts/Submitted_parts"_blank">Submitted parts</a>).<br />
<br> These new BioBricks were designed in order to be expressed and secreted with <i>Bacillus subtilis</i>. </p><br />
<br><br />
<p class="title2">Secretion</p><br />
<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<br />
a signal peptide (amyE signal peptide) followed by a pro peptide, cleaved during the secretion process.</p><br><br />
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<img style="width:500px; " src="https://static.igem.org/mediawiki/2014/d/d7/Fongpep.jpg"><br />
<br><p class="legend">Figure 3: Design of GAFP-1 and D4E1</p></center><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><br />
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<p class="title1">References</p><br />
<br />
<ul><br />
<li class="tree"><p class="texte">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. </p></li><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><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><br />
<li class="tree"><p class="texte">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</p></li><br />
<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><br />
<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 (Echinochloa crus-galli)</b>. The journal of Biological Chemistry. 2011, Vol. 286, 25145–25153</p></li><br />
</ul><br />
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{{:Team:Toulouse/template/footer}}</div>Jourdanhttp://2014.igem.org/Team:Toulouse/Project/bindingTeam:Toulouse/Project/binding2014-10-18T00:19:46Z<p>Jourdan: </p>
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<p>To be attached to the fungal cell wall</p><br />
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<p class="legend">Figure 1: Schema of the binding module</p><br />
<br />
<p class="texte"> In order to be highly efficient in the fight against the pythopathogen <i>Ceratocystis platani</i>, our optimized bacterium has to be anchored to the fungus. <br />
Thus, we designed a chimeric protein (<a href="http://parts.igem.org/Part:BBa_K1364005"target="_blank">BBa_K1364005</a>) capable of building <br />
a <B>bridge between the bacterial peptidoglycan and the fungal chitin</B>, the main component of the pathogen’s cell wall. <br />
According to the work of <a href="https://2010.igem.org/Team:Imperial_College_London"target="_blank">the Imperial College 2010</a> iGEM team, <br />
we used the Cell Wall Binding (CWB) domain of the LytC protein (coding for a N-acetylmuramoyl-L-alanine amidase) to attach our chimeric protein <br />
to the <I>B. subtilis</I> cell wall. On the other side of our protein, we added the domain 4 of GbpA from <I>Vibrio cholerae</I>, which is known to <br />
recognize chitin.<br />
</p><br />
<br />
<br><br />
<p class="title1"> More information about this module </p><br />
<p class="texte"> <br />
The open reading frame of the Binding Module is composed of 3 sections:</p><br />
<br />
<ul><br />
<li class="tree"><p class="texte"><B>Anchor section</B>: the CWB (Cell Wall Binding) domain is extracted from LytC gene and composes the 5' side of our binding module. As previously described by the Imperial College of London 2010 iGEM team, we kept the first 318 bp. We can note the presence of the signal peptide at the beginning of the sequence, from 1 to 24 bp.</p></li><br />
<li class="tree"><p class="texte"><B>Chitin Binding Domain (CBD) section</b>: the domain 4 of GbpA from <I>Vibrio cholerae </I> is able to bind to N-Acetyl Glucosamine oligosacchararides. Also, the 3' side of our gene is composed by a part of the GbpA sequence (from 423 to 484 bp).</p></li><br />
<li class="tree"><p class="texte"><B>Helical Linker</B>: According to the work of the 2010 Imperial College of London iGEM team, we used the same six amino acids sequence (SRGSRA) to make a bridge between the Anchor section and the Chitin Binding section.<br />
</p></li></ul><br />
<center style="margin:-30px;"><img style="width:500px; " src="https://static.igem.org/mediawiki/2014/a/a0/Binding.png"></center><br />
<p class="legend">Figure 2: Binding gene composition</p><br />
<br></br><br />
<p class="texte"> <br />
The sequence has been designed <i>in silico</i> and codon optimized for the transcription in <i>B. subtilis</i>. <br />
</p><br />
<br />
<B class="title1">Final construction</B> <br />
<br />
<B class="title2">(More details about the intermediate parts <a href="https://2014.igem.org/Team:Toulouse/Result/parts#select2"target="_blank">Here</a>)</B> <br />
<br />
<p class="texte"><br />
<br>The binding module has been placed under the control of Pveg (<a href="http://parts.igem.org/Part:BBa_K143012"target="_blank">BBa_K143012</a>),<br />
a strong constitutive promoter and we used a consensus RBS (<a href="http://parts.igem.org/Part:BBa_K090505"target="_blank">BBa_K090505</a>) as well as a<br />
double terminator (<a href="http://parts.igem.org/Part:BBa_B0015"target="_blank">B0015</a>). <br />
</p> <br />
<br />
<center style="margin:20px;"><img style="width:500px; " src="https://static.igem.org/mediawiki/2014/f/f5/BBa_K1364005.png"></center><br />
<p class="legend">Figure 3: Binding gene construction</p><br />
<br />
<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><br />
<br />
<br />
<p class="title1">References</p><br />
<br />
<ul><br />
<li class="tree"><p class="texte">M. Desvaux, E. Dumas, I. Chafsey and M. Hébraud.<b> Protein cell surface display in Gram-positive bacteria: from single protein to macromolecular protein structure </b>. FEMS Microbiol. Lett. 256, 1–15 (2006). </p></li><br />
<li class="tree"><p class="texte">E. Wong, G. Vaaje-Kolstad, A. Ghosh, R. Hurtado-Guerrero, PV. Konarev, AF. Ibrahim, DI. Svergun, VG. Eijsink, NS. Chatterjee and DM. van Aalten.<b>The Vibrio cholerae colonization factor GbpA possesses a modular structure that governs binding to different host surfaces</b>. PLoS Pathog. 8, e1002373 (2012).</p></li><br />
<br />
<li class="tree"><p class="texte">H. Yamamoto, S. Kurosawa and J. Sekiguchi. <b>Localization of the vegetative cell wall hydrolases LytC, LytE, and LytF on the Bacillus subtilis cell surface and stability of these enzymes to cell wall-bound or extracellular proteases</b>. J. Bacteriol. 185, 6666–6677 (2003).</p></li><br />
</ul><br />
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{{:Team:Toulouse/template/footer}}</div>Jourdanhttp://2014.igem.org/Team:Toulouse/Project/ChemotaxisTeam:Toulouse/Project/Chemotaxis2014-10-18T00:18:58Z<p>Jourdan: </p>
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<h2>Chemotaxis</h2><br />
<p>To target the pathogenic fungus</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Project&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Chemotaxis</p> <br />
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<br />
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<br />
<br />
<br />
<center><img style="width:420px; " src="https://static.igem.org/mediawiki/parts/e/e9/Recap_chemotax.jpg"></center><br />
<p class="legend">Figure 1: Schema of the chemotaxis module</p><br />
<br />
<p class="title1">What is chemotaxis?</p><br />
<br />
<p class="texte"><br />
Chemotaxis is a bacterial function which induces a movement toward a gradient of concentration of a molecule of interest. With this system the bacteria are able to swim to a location containing higher concentrations of molecules such as sugar, amino acid, vitamins... <br />
Chemotactic-signal transducers respond to changes in the concentration of attractants and repellents in the environment, transduce the signal from the outside to the inside of the cell, and facilitate sensory adaptation through the variation of the level of methylation. <br />
<br />
<p class="title1">More information on this module</p><br />
<p class="texte"><br />
Chemotaxis is used as a way to detect and come close to the location of fungi infection. During its growth, fungi release N-acetylglucosamine (NAG), the basic unit of chitin which composed its cell wall. Thus, there should exist a gradient of the concentration of NAG around the fungi.</p><br />
<p class="texte"><br />
It is known that <i>B. subtilis</i> is able to detect and to swim towards glucose using the Methyl-accepting chemotaxis protein, henceforth called <b>McpA</b> (<a href="http://www.uniprot.org/uniprot/P39214"_blanck">MCPA_BACSU</a>).<br><br />
Some bacteria are attracted by NAG, like <i>Vibrio cholerae</i> which has a NAG regulated methyl-accepting chemotaxis protein: <b>VCD</b> (<a href="http://www.uniprot.org/uniprot/C3NYT2"_blank">VCD_000306</a>).</p><br />
<br />
<center><img width="500px" SRC="https://static.igem.org/mediawiki/2014/4/47/Chimio1.png" alt="schema" style="margin-bottom:60px;"></center><br />
<p class="legend">Figure 2: Chimeric protein of chemotaxis</p><br />
<br />
<p class="texte"><br />
Therefore, our idea is to switch the natural glucose specificity of <i>B. subtilis'</i>, mediated by McpA, to a NAG specificity. To achieve this, we need to change the extracellular domain of McpA, responsible for the specificity, by the extracellular domain of VCD.<br />
The whole sequence has been designed <i>in silico</i> and codon optimized for the transcription in <i>B. subtilis</i> before its synthesis.</p><br />
<br />
<center><img width="600px" SRC="https://static.igem.org/mediawiki/2014/e/e4/Chimio2.png" alt="gene construct" style="margin-bottom:40px;"></center><br />
<p class="legend">Figure 3: Construction of the chemotaxis gene</p><br />
<br />
<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><br />
<br />
<br />
<p class="title1">References</p><br />
<ul><br />
<br />
<li class="tree"><p class="texte">K. Meibom,L. Xibing, A. Nielsen, CY. Wu, S. Roseman and G. Schoolnik.<b> The Vibrio cholerae chitin utilization program </b>. The National Academy of Sciences of the USA (2004).</p></li><br />
<li class="tree"><p class="texte">C. Kristich and GW. Ordal. <b><i>Bacillus subtilis</i> CheD is a chemoreceptor modification enzyme required for chemotaxis</b>. J Biol Chem. 2002 Jul 12;277(28):25356-62. Epub 2002 May 13.<br></p></li><br />
</ul><br />
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{{:Team:Toulouse/template/footer}}</div>Jourdanhttp://2014.igem.org/Team:Toulouse/Project/ChemotaxisTeam:Toulouse/Project/Chemotaxis2014-10-18T00:17:03Z<p>Jourdan: </p>
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<h2>Chemotaxis</h2><br />
<p>To target the pathogenic fungus</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Project&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Chemotaxis</p> <br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 65px; padding-bottom:40px;"><br />
<br />
<!--Short description : à changer!!!--><br />
<br />
<br />
<br />
<center><img style="width:420px; " src="https://static.igem.org/mediawiki/parts/e/e9/Recap_chemotax.jpg"></center><br />
<p class="legend">Figure 1: Schema of the chemotaxis module</p><br />
<br />
<p class="title1">What is chemotaxis?</p><br />
<br />
<p class="texte"><br />
Chemotaxis is a bacterial function which induces a movement toward a gradient of concentration of a molecule of interest. With this system the bacteria are able to swim to a location containing higher concentrations of molecules such as sugar, amino acid, vitamins... <br />
Chemotactic-signal transducers respond to changes in the concentration of attractants and repellents in the environment, transduce the signal from the outside to the inside of the cell, and facilitate sensory adaptation through the variation of the level of methylation. <br />
<br />
<p class="title1">More information on this module</p><br />
<p class="texte"><br />
Chemotaxis is used as a way to detect and come close to the location of fungi infection. During its growth, fungi release N-acetylglucosamine (NAG), the basic unit of chitin which composed its cell wall. Thus, there should exist a gradient of the concentration of NAG around the fungi.</p><br />
<p class="texte"><br />
It is known that <i>B. subtilis</i> is able to detect and to swim towards glucose using the Methyl-accepting chemotaxis protein, henceforth called <b>McpA</b> (<a href="http://www.uniprot.org/uniprot/P39214"_blanck">MCPA_BACSU</a>).<br><br />
Some bacteria are attracted by NAG, like <i>Vibrio cholerae</i> which has a NAG regulated methyl-accepting chemotaxis protein: <b>VCD</b> (<a href="http://www.uniprot.org/uniprot/C3NYT2"_blank">VCD_000306</a>).</p><br />
<br />
<center><img width="500px" SRC="https://static.igem.org/mediawiki/2014/4/47/Chimio1.png" alt="schema" style="margin-bottom:60px;"></center><br />
<p class="legend">Figure 2: Chimeric protein of chemotaxis</p><br />
<br />
<p class="texte"><br />
Therefore, our idea is to switch the natural glucose specificity of <i>B. subtilis'</i>, mediated by McpA, to a NAG specificity. To achieve this, we need to change the extracellular domain of McpA, responsible for the specificity, by the extracellular domain of VCD.<br />
The whole sequence has been designed <i>in silico</i> and codon optimized for the transcription in <i>B. subtilis</i> before its synthesis.</p><br />
<br />
<center><img width="600px" SRC="https://static.igem.org/mediawiki/2014/e/e4/Chimio2.png" alt="gene construct" style="margin-bottom:40px;"></center><br />
<p class="legend">Figure 3: Construction of the chemotaxis gene</p><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results"> <img src="http://parts.igem.org/File:Jump.jpg"> </a><br />
<p class="title1">References</p><br />
<ul><br />
<br />
<li class="tree"><p class="texte">K. Meibom,L. Xibing, A. Nielsen, CY. Wu, S. Roseman and G. Schoolnik.<b> The Vibrio cholerae chitin utilization program </b>. The National Academy of Sciences of the USA (2004).</p></li><br />
<li class="tree"><p class="texte">C. Kristich and GW. Ordal. <b><i>Bacillus subtilis</i> CheD is a chemoreceptor modification enzyme required for chemotaxis</b>. J Biol Chem. 2002 Jul 12;277(28):25356-62. Epub 2002 May 13.<br></p></li><br />
</ul><br />
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{{:Team:Toulouse/template/footer}}</div>Jourdanhttp://2014.igem.org/Team:Toulouse/Notebook/ProtocolsTeam:Toulouse/Notebook/Protocols2014-10-17T23:58:06Z<p>Jourdan: </p>
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<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Notebook&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Protocols</p> <br />
<ul class="topnav" id="topnav" style="top:15px;"><br />
<br />
</ul><br />
</div><br />
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<h3 class="title2" style="margin-top:10px; color:#333;">Summary :</h3><br />
<ul class="menuleft"><br />
<li style="margin-top:25px;"><a href="#select1"><i>E. coli</i> competent cells</a></li><br />
<li><a href="#select2"><i>E. coli</i> transformation protocol</a></li><br />
<li><a href="#select3">Miniprep and alcaline lysis</a></li><br />
<li><a href="#select4">Cloning</a></li><br />
<li><a href="#select5">Checking of the genetic constructions</a></li><br />
<li><a href="#select6"><i>B. subtilis</i> transformation</a></li><br />
<li><a href="#select7">Test of the pSB<sub>BS</sub>4S plasmid integration in <i>Bacillus subtilis</i> genome on the threonine site</a></li><br />
<li><a href="#select8">Final Tests</a></li><br />
</ul><br />
</div><br />
<div class="column-right" style="width:75%; float:right;"><br />
<br />
<p class="texte"> All the following protocols were inspired by one or several protocols, used, improved and optimized (which took more or less time...). Finally they gave us some <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">results</a> :-).</p><br />
<br />
<p class="title1" id="select1"><I>E. coli</I> competent cells</p><br />
<p class="texte"><I> <CENTER> MANIPULATION IN ICE </CENTER> </I></p><br />
<p class="texte"><B> Day 0 </B><br />
<br>- Make an <i>Escherichia coli</i> cell culture in LB medium overnight<br />
<p><br />
<p class="texte"><B> Day 1 </B><br />
<br/> <br />
- Freeze 0.1M CaCl<sub>2</sub> and 4 Falcon tubes of 50mL at 4°C<br />
<br/> <br />
- Streak 2% culture in LB to get an OD of 0.3 to 0.4 (it takes approximately 1h30)<br />
<br/> <br />
- Centrifuge 10 minutes at 4500 RPM<br />
<br/><br />
- Remove the supernatant<br />
<br/><br />
- Resuspend the pellet in 7.5 mL of 0.1 M frozen CaCl<sub>2</sub> <br />
<br/><br />
- Centrifuge 10 minutes at 4500 RPM<br />
<br/><br />
- Resuspend the pellet in 500 µL of 0.1 M CaCl<sub>2</sub><br />
<br/><br />
- Add glycerol to a final concentration of 15%<br />
<br/><br />
- Keep the tubes at -80°C<br />
</p><br />
<br />
<p class="title1" id="select2"> <I>E. coli</I> transformation protocol </p><br />
<p class="texte"><br />
- Let the LB agar medium plates dry in a sterile area<br />
<br/><br />
- Thaw out the competent cell aliquotes for about 10 to 20 minutes<br />
<br/><br />
- Add 20 to 100 ng of plasmid or 3 µL of kit plate DNA <br />
<br/><br />
<i>NB: for kit plate, resuspend the well in 10 µL of sterile water</i><br />
- Put the tubes 20 minutes in the ice </i><br />
<br/><br />
- Put the tubes 2 minutes at 42°C in the water bath<br />
<br/><br />
- Put the tubes back in ice immediately to create the thermic shock<br />
<br/><br />
- Add 1mL of LB medium<br />
<br/><br />
- Put the tube 2 hours in the 37°C water bath (1 hour if it concerns an ampicillin resistant strain) to allow the phenotypic expression<br />
<br/><br />
- Centrifuge for 1 minute at 13000 RPM<br />
<br/><br />
- Remove the supernatant <br />
<br/><br />
- Resuspend in 250 µL of LB medium<br />
<br/><br />
- Streak the final mix on LB agar selective medium: 200 µL on one plate, 50 µL on the second plate<br />
</p><br />
<br />
<p class="title1" id="select3"> Miniprep and alcaline lysis </p><br />
<p class="texte"><B> Day 0 </B><br />
<br/>- Resuspend 4 to 12 colonies from the plate and name each colony taken on the tubes and on the plate (A, B, C, …)<br />
<br/><br />
- Resuspend one colony per culture tube in 5 mL of LB medium with antibiotic<br />
<br/><br />
- Let the culture grow overnight at 37°C in a shaking incubator</p> <br />
<br />
<p class="texte"><B> Day 1 </B><br />
<br>- Use the QIAprep spin Miniprep Kit for each culture tube. The last step consisting in the elution of the DNA is made with elution buffer or water at 55°C.<br />
<br/><br />
- Keep the tubes at -20°C <br />
<br><br />
<br />
<br/><i>NB: It is possible to purify the plasmid with an alcaline lysis without any purification column. For 2 mL of culture, 200 µL of buffer 1 is added to resuspend the pellet, 400 µL of buffer 2 to allow the lysis of the cells and the denaturation of the protein and 300 µL of buffer 3 to precipitate the DNA and the proteins. The solution is then centrifuged 10 minutes at 13 000 RPM.<br />
600 µL of isopropanol is added to the supernatant and the solution is centrifuged again. The pellet is then resuspended in 100 µL of pH 7.4 TE buffer. A part of the contamination by the RNA can avoid by the addition of pH 7.4 TE buffer + 0.2 µL of RNAse. </i><br />
<br><br />
<br> <b>Buffer 1:</b> Tris 10 mM pH 8 + EDTA 1mM<br />
<br> <b>Buffer 2:</b> NaOH 2 mM + SDS 1%<br />
<br> <b>Buffer 3:</b> Potassium acetate 3 M + 15% glacial acetic acid<br />
</I></p><br />
<br />
<p class="title1" id="select4">Cloning </p><br />
<p class="texte">Cloning is the step after taking the competent cells, transforming the BioBricks and miniprep them.<br />
<br><br />
<p class="title2">First step</p><br />
<p class="title3">Both parts have the same antibiotic resistance</p><br />
<p class="texte"><b>1) Digestion mix</b><br />
<br> For the vector :<br />
<br>- 5 µL of miniprep plasmid <br />
<br><br />
- 2 µL of each restriction enzymes<br />
<br><br />
- 2 µL of Green Buffer<br />
<br><br />
- 9 µL of Milli-Q water <br />
<br><br />
<br> For the insert :<br />
<br>- 10 µL of miniprep plasmid <br />
<br><br />
- 2 µL of each restriction enzymes<br />
<br><br />
- 2 µL of Green Buffer<br />
<br><br />
- 4 µL of Milli-Q water <br />
<br><br />
- Incubate 15 minutes at 37°C <br />
<br />
<br><br><b>2) Gel extraction</b><br />
<br><br />
- Prepare a 1% or 2% electrophoresis agarose gel with 0.5x TAE buffer<br />
<br><br />
- Put 20 µL of sample + 6 µL of marker (1 kb for 1% gel and 100 pb for 2%) into the well<br />
<br><br />
- Migration for 30 min at 100 V or 1 hour at 50V<br />
<br><br />
- The revelation is made in BET (10 minutes). Then wash in water for 5 minutes<br />
<br><br />
- The gel extraction is realized thanks to the THERMO SCIENTIFIC GeneJET Gel Extraction and DNA Clean Up Microkit<br />
<br />
<br><br>3) Inactivation of the enzymes for the vector<br />
<br>There are two ways to inactivate the enzymes:<br />
<br>- Use of DNA Clean up kit for the DNA fragment above 200 pb<br />
<br>- Heat inactivation at 95°C for 10 minutes.</p><br />
<br />
<p class="title3">The two parts have a different antibiotic resistance</p><br />
<p class="texte"><b>1) Digestion mix</b> <br />
<br>For each part, add: <br />
<br>- 5 µL of miniprep plasmid <br />
<br>- 1 µL of each restriction enzymes<br />
<br>- 2 µL of Green Buffer<br />
<br>- 9 µL of Milli-Q water <br />
<br>- Incubate 15 minutes at 37°C <br />
<br />
<p class="texte"><br />
<b>2) Inactivation of the enzymes for the vector</b><br />
<br>There are two ways to inactivate the enzymes:<br />
<br>- Use of DNA Clean up kit for the DNA fragment above 200 pb<br />
<br>- Heat inactivation at 95°C for 10 minutes.</p><br />
<br />
<p class="title2">Second step</p><br />
<p class="title3">Ligation</p><br />
<p class="texte">- Mix 10 µL of insert + 4 µL of vector + 2 µL of 10x T4 buffer + 0.5 µL of T4 ligase + 3.5 µL of Milli-Q water<br />
<br/><br />
A control without insert must be made<br />
<br/><br />
- Incubate the ligation mix 15 minutes at room temperature (22°C) and keep the tubes in ice or at -20°C to prepare the transformation</p><br />
<br />
<p class="title3">Transformation</p><br />
<p class="texte"><br />
- Take 5µL of the ligation mix for 50 µL of competent cells and use the <a href="https://2014.igem.org/Team:Toulouse/Notebook/Protocols#select2">Toulouse iGEM Team 2014 transformation protocol</a>.<br />
<br/><br />
- Plate the solution on selective medium overnight at 37°C.</p><br />
<br />
<p class="title1 " id="select5">Checking of the genetic constructions </p><br />
<p class="title2">1) Colony PCR</p><br />
<p class="texte"><br />
- Add 0.5 µL of plasmid + 25 µL of DreamTAQ MasterMix + 2 µL of each 10 µM primer (VR and VF2) + H<sub>2</sub>0 qsp 25 µL and take a colony.<br />
<br/><br />
- Look for the number of necessary cycles and the proper temperature thanks to AmplifX or Serial Cloner 2.1 softwares.<br />
<br/>The following cycles have been used : <br />
<br/>- 94°C - 5 min<br />
<br/>- (94°C 45 sec ; 55°C 45 sec ; 72°C 1min/kb ) * 25 cycles <br />
<br/>- 72°C - 5min<br />
<br/>- Then 4°C</p><br />
<br />
<p class="title2">2) Analytic digestion</p><br />
<p class="texte"><br />
- Put a colony in 5 mL of LB selective medium and wait for 6 hours<br />
<br/><br />
- Make a purification thanks to the Miniprep kit<br />
<br/><br />
- Mix 2 µL of plasmid + 2 µL of Fast Digest Green Buffer + 1µL of each enzyme + Milli-Q water qsp 20µL<br />
<br/><br />
- Wait 15 minutes at 37°C and put the mix on a 1% or 2% gel for 30 minutes at 100 V.</p><br />
<br />
<p class="title2">3) Sequencing</p><br />
<p class="texte"><br/>The sequencing of the genetic constructions was performed by Eurofins Genomics Company by mixing 15µL of pure plasmid solution with 2µL of one primer.</p><br />
<br />
<p class="title1" id="select6"> <I>B. subtilis</I> transformation</p><br />
<br />
<p class="texte"><br />
<B> Day 0 </B><br />
<br/>- Streak out the <i>Bacillus</i> strain and plate this on an LB agar plate overnight at 37°C</p><br />
<br />
<p class="texte"><B> Day 1 </B><br />
<br />
<br/>- Pick up a nice big colony of <I>B. Subtilis </I> strain and drop it in 2ml of completed 1x MC<br />
<br/><br />
- Grow at 37°C for 5 hours<br />
<br/><br />
- Mix 400µl of culture in a fresh tube (tubes loosely closed for the aeration) and put 5µL of Miniprep DNA.<br />
<br/><br />
- Grow the cells at 37°C for an additional 2 hours<br />
<br/><br />
- Spread the complete 400 µl reaction mix on selective antibiotic plates (100µl per plate), and incubate at 37°C overnight <br />
<br/></p><br />
<br />
<p class="texte"><b> Preparation of solutions: </b><br><br />
<I> <br> 300 mM Tri-Na Citrate:</I><br />
<br>- 0.88 g Tri-Na Citrate<br />
<br>- 10mL MQ water<br />
<br><br><I>Ferric NH4 citrate:</I><br />
<br>- 0.22g Ferric NH4<br />
<br>- 10mL MQ water<br />
<br><br><I>10x Competence Medium </I><br />
<br> For 10mL:<br />
<br>- 1.40g K2HPO4<br />
<br>- 0.52g KH2PO4<br />
<br>- 2g glucose<br />
<br>- 1 mL 300 mM Tri-Na citrate <br />
<br>- 0.1 mL Ferric NH4 citrate<br />
<br>- 0.1g Casein Hydrolysate<br />
<br>- 0.2 g Potassium glutamate<br />
<br>The complete mixture should be dissolved in 10 ml. First add 5 ml milliQ water and mix. When everything is dissolved add MQ water till 10 ml. Filter sterilize the complete mixture and store at -20°C.<br />
<br><br><I>1x Competence Medium </I><br />
<br>- 1.8 mL MQ water<br />
<br>- 200 µL 10x Competence Medium solution (previously filter sterilized)<br />
<br>- 6.7 µL 1M MgSO4 (previously autoclaved)<br />
<br>- 10 µL 1% tryptophan (previously filter sterilized and stored in aluminium foil)<br />
<br>The complete mixture should be dissolved in 100 ml. First add 50 ml milliQ water and mix. When everything is dissolved add MQ water till 100 ml. Filter sterilize the complete mixture and store at -20°C.<br />
<br />
<br />
<p class="title1" id="select7">Test of the pSB<sub>BS</sub>4S plasmid integration in <i>Bacillus subtilis</i> genome on the threonine site</p><br />
<p class="texte"><br />
<br>- Plate the transformed <i>Bacillus</i> strain on a selective medium (LB + spectinomycin) overnight <br />
<br>- The obtained clones are then plated on different media: Medium Competence (Thr+), Medium Competence (Thr-) and LB + Spectinomycin. <br />
<br>When the plasmid is integrated, the clone can grow on minimum medium with threonine and on LB + Spectinomycin but can not grow on the minimum medium without thronine.<br />
Moreover, a colony PCR can be performed with the same protocol as previously presented. The used primers are VFBS and VRBS.<br />
<center><img src="https://static.igem.org/mediawiki/2014/a/a3/Thr.png" width="400px"></center><br />
<p class="legend">Threnonine test (Left: MC Thr+; Right: MC Thr -)</p><br />
<br />
<p class="title1" id="select8">Final Tests</p><br />
<p class="title2">Chemotaxis test</p><br />
<p class="texte"><br />
Many chemotaxis tests exist such as plate tests or capillary tests. We tried all of them and we decided to optimize the <a href="https://2011.igem.org/Team:Imperial_College_London/Protocols_Chemotaxis">« Capillary essay »</a> from Imperial College 2011 iGEM team.<br />
<br>- Prepare the bacteria in LB medium until they reach an OD between 0.5 and 0.6 (exponential growth phase). This step takes about 5 hours.<br />
<br>- Prepare the multichannel pipette: improve the cohesion between the tips and the pipette with Blu-Tack to avoid air and the possibility of leakage.<br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2014/b/b1/Installation_1.gif" width="650px"></center><br />
<p class="texte"><br />
<br>- Put 200 µl of the different chemoattractants in the wells of the ELISA plate and pipet 15 µL of each with the multichannel pipette: galactose which represents our negative control, glucose which represents our positive control and N-acetylglucosamine (NAG). The volume in the tips must be marked.<br><br />
<br><i>NB: The NAG is the most important test because it is the monosaccharide which composes the chitin on <i>Ceratocystis platani</i> wall.</i><br><br />
<br>- Put the tips with chemoattractants in 300 µL of the bacterial solution in exponential growth phase in the ELISA plate.<br />
<br>- Let the installation settle for 1 hour at room temperature.<br />
<br>- After an hour, put the volume of the tips on parafilm. <br />
<br>- Each solution is diluted 1/10,000 and 100 µL is spread on LA medium.<br />
<br>- The plates are then incubated overnight at 37°C.</p><br />
<br />
<p class="title2">Binding test</p><br />
<p class="texte"><i>CBB (Chitin Binding Buffer):</i><br />
<br>- 500 mM NaCl<br />
<br>- 20 mM Tris-HCl<br />
<br>- 1 mM EDTA<br />
<br>- 0,05% Triton X-100, 25°C, pH=8<br />
</p><br />
<br />
<p class="title3">Column activation:</p><br />
<p class="texte">- Vortex the beads <br />
<br>- Put 50 µL of beads in a 1.5mL centrifuge tube<br />
<br>- Wash with 500 µL of CBB<br />
<br>- Put the centrifuge tube on a magnetic rack<br />
<br>- Wait 30 seconds<br />
<br>- Remove supernatant<br />
<br>- Repeat the wash<br />
</p><br />
<br />
<br />
<p class="title3">Bacterial fixation on the chitin beads:</p><p class="texte"><br />
- Add 200 µL of bacteria solution (10<sup>5</sup> bacteria/mL) to the washed beads <br />
<br>- Shake during 1h at 4°C<br />
<br>- Add 500µL of CBB (washing A)<br />
<br>- Put the centrifuge tube on a magnetic rack<br />
<br>- Wait 30 seconds<br />
<br>- Remove supernatant<br />
<br>- Add 500µL of CBB (washing B)<br />
<br>- Put the centrifuge tube on a magnetic rack<br />
<br>- Wait 30 seconds<br />
<br>- Remove supernatant<br />
<br>- Add 500µL of CBB to recover the beads directly<br />
</p><br />
<br />
<p class="title3">Bacteria count:</p><br />
<p class="texte">- Make different dilutions : 10<sup>-1</sup>, 10<sup>-3</sup>, 10<sup>-5</sup> of the first bacterial culture and spread on LA plates<br />
<br>- Make different dilutions : 1, 10<sup>-2</sup>, 10<sup>-4</sup> of washings (A and B) and of the beads in CBB medium and spread on LA plates<br />
<br>- Place the plates at 37°C overnight<br />
<br>- Count colonies on different plates<br />
</p><br />
<br />
<p class="title2">Fungicide test: anti-fungal activities</p><br />
<p class="texte"><br />
CAUTION : all the lab equipment must be desinfected before and after the manipulations with the fungi. <br><br />
<br>Three different fungus strains were used : <i>Aspergillus brasiliensis</i>, <i>Aspergillus nidulans</i> and <i>Trichoderma reesei</i><br />
<br>- The conidia can be taken by adding one drop of Tween 80 on the fungus plate.<br />
<br>- Then the drop is mixed with 1mL of sterile water in an Eppedorf.<br />
<br>- A microscopy count can be performed thanks to Thoma cell to determine the conidia concentration.<br />
<br><i>NB : The conidia solutions are then diluted and spread on sap medium to get 10,000 conidia/plate.</i><br />
<br>- After 72 hours of liquid culture of the different clones of <i>B. subtilis</i> with the fungicides module, the culture can be centrifugated.<br />
<br>- 130µL of the supernatant is used to soak a pad placed on the conidia plate. The bacterial pellet is resuspended in 130µL of LB medium and also put on a pad. <br />
<br>- The plates containing 10,000 conidia and the soaked pads are then put at room temperature for a few days according to the growth speed of the fungi. Controls are also realized with wild type strains or copper sulfate at 10 and 20mg/mL.<br />
</p><br />
<p class= "texte"><br />
<b> Sap-like Medium (250 mL) (see references)</b>: <br><br />
<br />
2,5g de tryptone<br />
<br />
<br>1,25g de YE<br />
<br />
<br>2,5g de NaCl<br />
<br />
<br>Glucose : 1,175g<br />
<br />
<br>Fructose : 1,125g<br />
<br />
<br>Sucrose : 0,125g<br />
<br />
<br>Inositol : 0,084 g<br />
<br />
<p class="title2">Fungicide test: <i>in planta</i> assay</p><br />
<p class="title3">First step</p><br />
<p class="texte"><br />
The first step is related to the inoculation of SubtiTree in plants through stomata (opened in wet condition). <br>We diluted our bacterial samples to get two concentrations: 5.10<sup>6</sup> and 10<sup>8</sup> bacteria per mL. The WT and transformed bacteria are introduced into plants (a control test without bacteria is performed). Thanks to a 1 ml syringe (without needle), the plant was injected with bacteria by pressure. Five leaves of each plant were used, marked with a marker point. It is necessary to repeat the operation on both sides of the leaf and the excess is wipe off. The plants are placed in a growth chamber (phytotron) to control light, high humidity, temperature and bacterial non-proliferation. Bacterial growth in the plant is left for 24 h.<br />
</p><br />
<p class="title3">Second step</p><br />
<p class="texte"><br />
The next step begins with the preparation of the fungal samples. Fungal culture is crushed and mixed with PDB (Potato Dextrose Broth). Then the mix passes through a 100 µm filter (to remove large aggregates) and through a 40 µm filter. The caught hyphae are mixed with PDB for 24 to 48 hours until it reaches an OD of 2.5 at 600nm. The previously seeded leaves are taken from the plant using a scalpel and placed in boxes above wet absorbent paper (leaves are kept alive for a week). Above each leaf, 5µl of the fungal suspension is deposited (using beveled tips because it is too viscous). As control, we kept inoculated leaves without fungus and leaves with only fungus. Pictures are taken at different times. All the plants are destroyed by autoclaving.<br />
</p><br />
<br><br />
<p class="texte"><br />
<b>References</b><br><br />
Véronique Amiard, Annette Morvan-Bertrand, Jean-Bernard Cliquet, Jean-Pierre Billard,<br />
Claude Huault, Jonas P. Sandström, and Marie-Pascale Prud’homme. <b>Carbohydrate and amino<br />
acid composition in phloem sap of Lolium perenne L. before and after defoliation</b>. Can. J. Bot.<br />
Vol. 82: 1594–1601, 2004.</p><br />
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<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Notebook&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Protocols</p> <br />
<ul class="topnav" id="topnav" style="top:15px;"><br />
<br />
</ul><br />
</div><br />
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<br />
<div id="column-left"><br />
<h3 class="title2" style="margin-top:10px; color:#333;">Summary :</h3><br />
<ul class="menuleft"><br />
<li style="margin-top:25px;"><a href="#select1"><i>E. coli</i> competent cells</a></li><br />
<li><a href="#select2"><i>E. coli</i> transformation protocol</a></li><br />
<li><a href="#select3">Miniprep and alcaline lysis</a></li><br />
<li><a href="#select4">Cloning</a></li><br />
<li><a href="#select5">Checking of the genetic constructions</a></li><br />
<li><a href="#select6"><i>B. subtilis</i> transformation</a></li><br />
<li><a href="#select7">Test of the pSB<sub>BS</sub>4S plasmid integration in <i>Bacillus subtilis</i> genome on the threonine site</a></li><br />
<li><a href="#select8">Final Tests</a></li><br />
</ul><br />
</div><br />
<div class="column-right" style="width:75%; float:right;"><br />
<br />
<p class="texte"> All the following protocols were inspired by one or several protocols, used, improved and optimized (which took more or less time...). Finally they gave us some <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">results</a> :-).</p><br />
<br />
<p class="title1" id="select1"><I>E. coli</I> competent cells</p><br />
<p class="texte"><I> <CENTER> MANIPULATION IN ICE </CENTER> </I></p><br />
<p class="texte"><B> Day 0 </B><br />
<br>- Make an <i>Escherichia coli</i> cell culture in LB medium overnight<br />
<p><br />
<p class="texte"><B> Day 1 </B><br />
<br/> <br />
- Freeze 0.1M CaCl<sub>2</sub> and 4 Falcon tubes of 50mL at 4°C<br />
<br/> <br />
- Streak 2% culture in LB to get an OD of 0.3 to 0.4 (it takes approximately 1h30)<br />
<br/> <br />
- Centrifuge 10 minutes at 4500 RPM<br />
<br/><br />
- Remove the supernatant<br />
<br/><br />
- Resuspend the pellet in 7.5 mL of 0.1 M frozen CaCl<sub>2</sub> <br />
<br/><br />
- Centrifuge 10 minutes at 4500 RPM<br />
<br/><br />
- Resuspend the pellet in 500 µL of 0.1 M CaCl<sub>2</sub><br />
<br/><br />
- Add glycerol to a final concentration of 15%<br />
<br/><br />
- Keep the tubes at -80°C<br />
</p><br />
<br />
<p class="title1" id="select2"> <I>E. coli</I> transformation protocol </p><br />
<p class="texte"><br />
- Let the LB agar medium plates dry in a sterile area<br />
<br/><br />
- Thaw out the competent cell aliquotes for about 10 to 20 minutes<br />
<br/><br />
- Add 20 to 100 ng of plasmid or 3 µL of kit plate DNA <br />
<br/><br />
<i>NB: for kit plate, resuspend the well in 10 µL of sterile water</i><br />
- Put the tubes 20 minutes in the ice </i><br />
<br/><br />
- Put the tubes 2 minutes at 42°C in the water bath<br />
<br/><br />
- Put the tubes back in ice immediately to create the thermic shock<br />
<br/><br />
- Add 1mL of LB medium<br />
<br/><br />
- Put the tube 2 hours in the 37°C water bath (1 hour if it concerns an ampicillin resistant strain) to allow the phenotypic expression<br />
<br/><br />
- Centrifuge for 1 minute at 13000 RPM<br />
<br/><br />
- Remove the supernatant <br />
<br/><br />
- Resuspend in 250 µL of LB medium<br />
<br/><br />
- Streak the final mix on LB agar selective medium: 200 µL on one plate, 50 µL on the second plate<br />
</p><br />
<br />
<p class="title1" id="select3"> Miniprep and alcaline lysis </p><br />
<p class="texte"><B> Day 0 </B><br />
<br/>- Resuspend 4 to 12 colonies from the plate and name each colony taken on the tubes and on the plate (A, B, C, …)<br />
<br/><br />
- Resuspend one colony per culture tube in 5 mL of LB medium with antibiotic<br />
<br/><br />
- Let the culture grow overnight at 37°C in a shaking incubator</p> <br />
<br />
<p class="texte"><B> Day 1 </B><br />
<br>- Use the QIAprep spin Miniprep Kit for each culture tube. The last step consisting in the elution of the DNA is made with elution buffer or water at 55°C.<br />
<br/><br />
- Keep the tubes at -20°C <br />
<br><br />
<br />
<br/><i>NB: It is possible to purify the plasmid with an alcaline lysis without any purification column. For 2 mL of culture, 200 µL of buffer 1 is added to resuspend the pellet, 400 µL of buffer 2 to allow the lysis of the cells and the denaturation of the protein and 300 µL of buffer 3 to precipitate the DNA and the proteins. The solution is then centrifuged 10 minutes at 13 000 RPM.<br />
600 µL of isopropanol is added to the supernatant and the solution is centrifuged again. The pellet is then resuspended in 100 µL of pH 7.4 TE buffer. A part of the contamination by the RNA can avoid by the addition of pH 7.4 TE buffer + 0.2 µL of RNAse. </i><br />
<br><br />
<br> <b>Buffer 1:</b> Tris 10 mM pH 8 + EDTA 1mM<br />
<br> <b>Buffer 2:</b> NaOH 2 mM + SDS 1%<br />
<br> <b>Buffer 3:</b> Potassium acetate 3 M + 15% glacial acetic acid<br />
</I></p><br />
<br />
<p class="title1" id="select4">Cloning </p><br />
<p class="texte">Cloning is the step after taking the competent cells, transforming the BioBricks and miniprep them.<br />
<br><br />
<p class="title2">First step</p><br />
<p class="title3">Both parts have the same antibiotic resistance</p><br />
<p class="texte"><b>1) Digestion mix</b><br />
<br> For the vector :<br />
<br>- 5 µL of miniprep plasmid <br />
<br><br />
- 2 µL of each restriction enzymes<br />
<br><br />
- 2 µL of Green Buffer<br />
<br><br />
- 9 µL of Milli-Q water <br />
<br><br />
<br> For the insert :<br />
<br>- 10 µL of miniprep plasmid <br />
<br><br />
- 2 µL of each restriction enzymes<br />
<br><br />
- 2 µL of Green Buffer<br />
<br><br />
- 4 µL of Milli-Q water <br />
<br><br />
- Incubate 15 minutes at 37°C <br />
<br />
<br><br><b>2) Gel extraction</b><br />
<br><br />
- Prepare a 1% or 2% electrophoresis agarose gel with 0.5x TAE buffer<br />
<br><br />
- Put 20 µL of sample + 6 µL of marker (1 kb for 1% gel and 100 pb for 2%) into the well<br />
<br><br />
- Migration for 30 min at 100 V or 1 hour at 50V<br />
<br><br />
- The revelation is made in BET (10 minutes). Then wash in water for 5 minutes<br />
<br><br />
- The gel extraction is realized thanks to the THERMO SCIENTIFIC GeneJET Gel Extraction and DNA Clean Up Microkit<br />
<br />
<br><br>3) Inactivation of the enzymes for the vector<br />
<br>There are two ways to inactivate the enzymes:<br />
<br>- Use of DNA Clean up kit for the DNA fragment above 200 pb<br />
<br>- Heat inactivation at 95°C for 10 minutes.</p><br />
<br />
<p class="title3">The two parts have a different antibiotic resistance</p><br />
<p class="texte"><b>1) Digestion mix</b> <br />
<br>For each part, add: <br />
<br>- 5 µL of miniprep plasmid <br />
<br>- 1 µL of each restriction enzymes<br />
<br>- 2 µL of Green Buffer<br />
<br>- 9 µL of Milli-Q water <br />
<br>- Incubate 15 minutes at 37°C <br />
<br />
<p class="texte"><br />
<b>2) Inactivation of the enzymes for the vector</b><br />
<br>There are two ways to inactivate the enzymes:<br />
<br>- Use of DNA Clean up kit for the DNA fragment above 200 pb<br />
<br>- Heat inactivation at 95°C for 10 minutes.</p><br />
<br />
<p class="title2">Second step</p><br />
<p class="title3">Ligation</p><br />
<p class="texte">- Mix 10 µL of insert + 4 µL of vector + 2 µL of 10x T4 buffer + 0.5 µL of T4 ligase + 3.5 µL of Milli-Q water<br />
<br/><br />
A control without insert must be made<br />
<br/><br />
- Incubate the ligation mix 15 minutes at room temperature (22°C) and keep the tubes in ice or at -20°C to prepare the transformation</p><br />
<br />
<p class="title3">Transformation</p><br />
<p class="texte"><br />
- Take 5µL of the ligation mix for 50 µL of competent cells and use the <a href="https://2014.igem.org/Team:Toulouse/Notebook/Protocols#select2">Toulouse iGEM Team 2014 transformation protocol</a>.<br />
<br/><br />
- Plate the solution on selective medium overnight at 37°C.</p><br />
<br />
<p class="title1 " id="select5">Checking of the genetic constructions </p><br />
<p class="title2">1) Colony PCR</p><br />
<p class="texte"><br />
- Add 0.5 µL of plasmid + 25 µL of DreamTAQ MasterMix + 2 µL of each 10 µM primer (VR and VF2) + H<sub>2</sub>0 qsp 25 µL and take a colony.<br />
<br/><br />
- Look for the number of necessary cycles and the proper temperature thanks to AmplifX or Serial Cloner 2.1 softwares.<br />
<br/>The following cycles have been used : <br />
<br/>- 94°C - 5 min<br />
<br/>- (94°C 45 sec ; 55°C 45 sec ; 72°C 1min/kb ) * 25 cycles <br />
<br/>- 72°C - 5min<br />
<br/>- Then 4°C</p><br />
<br />
<p class="title2">2) Analytic digestion</p><br />
<p class="texte"><br />
- Put a colony in 5 mL of LB selective medium and wait for 6 hours<br />
<br/><br />
- Make a purification thanks to the Miniprep kit<br />
<br/><br />
- Mix 2 µL of plasmid + 2 µL of Fast Digest Green Buffer + 1µL of each enzyme + Milli-Q water qsp 20µL<br />
<br/><br />
- Wait 15 minutes at 37°C and put the mix on a 1% or 2% gel for 30 minutes at 100 V.</p><br />
<br />
<p class="title2">3) Sequencing</p><br />
<p class="texte"><br/>The sequencing of the genetic constructions was performed by Eurofins Genomics Company by mixing 15µL of pure plasmid solution with 2µL of one primer.</p><br />
<br />
<p class="title1" id="select6"> <I>B. subtilis</I> transformation</p><br />
<br />
<p class="texte"><br />
<B> Day 0 </B><br />
<br/>- Streak out the <i>Bacillus</i> strain and plate this on an LB agar plate overnight at 37°C</p><br />
<br />
<p class="texte"><B> Day 1 </B><br />
<br />
<br/>- Pick up a nice big colony of <I>B. Subtilis </I> strain and drop it in 2ml of completed 1x MC<br />
<br/><br />
- Grow at 37°C for 5 hours<br />
<br/><br />
- Mix 400µl of culture in a fresh tube (tubes loosely closed for the aeration) and put 5µL of Miniprep DNA.<br />
<br/><br />
- Grow the cells at 37°C for an additional 2 hours<br />
<br/><br />
- Spread the complete 400 µl reaction mix on selective antibiotic plates (100µl per plate), and incubate at 37°C overnight <br />
<br/></p><br />
<br />
<p class="texte"><b> Preparation of solutions: </b><br><br />
<I> <br> 300 mM Tri-Na Citrate:</I><br />
<br>- 0.88 g Tri-Na Citrate<br />
<br>- 10mL MQ water<br />
<br><br><I>Ferric NH4 citrate:</I><br />
<br>- 0.22g Ferric NH4<br />
<br>- 10mL MQ water<br />
<br><br><I>10x Competence Medium </I><br />
<br> For 10mL:<br />
<br>- 1.40g K2HPO4<br />
<br>- 0.52g KH2PO4<br />
<br>- 2g glucose<br />
<br>- 1 mL 300 mM Tri-Na citrate <br />
<br>- 0.1 mL Ferric NH4 citrate<br />
<br>- 0.1g Casein Hydrolysate<br />
<br>- 0.2 g Potassium glutamate<br />
<br>The complete mixture should be dissolved in 10 ml. First add 5 ml milliQ water and mix. When everything is dissolved add MQ water till 10 ml. Filter sterilize the complete mixture and store at -20°C.<br />
<br><br><I>1x Competence Medium </I><br />
<br>- 1.8 mL MQ water<br />
<br>- 200 µL 10x Competence Medium solution (previously filter sterilized)<br />
<br>- 6.7 µL 1M MgSO4 (previously autoclaved)<br />
<br>- 10 µL 1% tryptophan (previously filter sterilized and stored in aluminium foil)<br />
<br>The complete mixture should be dissolved in 100 ml. First add 50 ml milliQ water and mix. When everything is dissolved add MQ water till 100 ml. Filter sterilize the complete mixture and store at -20°C.<br />
<br />
<br />
<p class="title1" id="select7">Test of the pSB<sub>BS</sub>4S plasmid integration in <i>Bacillus subtilis</i> genome on the threonine site</p><br />
<p class="texte"><br />
<br>- Plate the transformed <i>Bacillus</i> strain on a selective medium (LB + spectinomycin) overnight <br />
<br>- The obtained clones are then plated on different media: Medium Competence (Thr+), Medium Competence (Thr-) and LB + Spectinomycin. <br />
<br>When the plasmid is integrated, the clone can grow on minimum medium with threonine and on LB + Spectinomycin but can not grow on the minimum medium without thronine.<br />
Moreover, a colony PCR can be performed with the same protocol as previously presented. The used primers are VFBS and VRBS.<br />
<center><img src="https://static.igem.org/mediawiki/2014/a/a3/Thr.png" width="400px"></center><br />
<p class="legend">Threnonine test (Left: MC Thr+; Right: MC Thr -)</p><br />
<br />
<p class="title1" id="select8">Final Tests</p><br />
<p class="title2">Chemotaxis test</p><br />
<p class="texte"><br />
Many chemotaxis tests exist such as plate tests or capillary tests. We tried all of them and we decided to optimize the <a href="https://2011.igem.org/Team:Imperial_College_London/Protocols_Chemotaxis">« Capillary essay »</a> from Imperial College 2011 iGEM team.<br />
<br>- Prepare the bacteria in LB medium until they reach an OD between 0.5 and 0.6 (exponential growth phase). This step takes about 5 hours.<br />
<br>- Prepare the multichannel pipette: improve the cohesion between the tips and the pipette with Blu-Tack to avoid air and the possibility of leakage.<br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2014/b/b1/Installation_1.gif" width="650px"></center><br />
<p class="texte"><br />
<br>- Put 200 µl of the different chemoattractants in the wells of the ELISA plate and pipet 15 µL of each with the multichannel pipette: galactose which represents our negative control, glucose which represents our positive control and N-acetylglucosamine (NAG). The volume in the tips must be marked.<br><br />
<br><i>NB: The NAG is the most important test because it is the monosaccharide which composes the chitin on <i>Ceratocystis platani</i> wall.</i><br><br />
<br>- Put the tips with chemoattractants in 300 µL of the bacterial solution in exponential growth phase in the ELISA plate.<br />
<br>- Let the installation settle for 1 hour at room temperature.<br />
<br>- After an hour, put the volume of the tips on parafilm. <br />
<br>- Each solution is diluted 1/10,000 and 100 µL is spread on LA medium.<br />
<br>- The plates are then incubated overnight at 37°C.</p><br />
<br />
<p class="title2">Binding test</p><br />
<p class="texte"><i>CBB (Chitin Binding Buffer):</i><br />
<br>- 500 mM NaCl<br />
<br>- 20 mM Tris-HCl<br />
<br>- 1 mM EDTA<br />
<br>- 0,05% Triton X-100, 25°C, pH=8<br />
</p><br />
<br />
<p class="title3">Column activation:</p><br />
<p class="texte">- Vortex the beads <br />
<br>- Put 50 µL of beads in a 1.5mL centrifuge tube<br />
<br>- Wash with 500 µL of CBB<br />
<br>- Put the centrifuge tube on a magnetic rack<br />
<br>- Wait 30 seconds<br />
<br>- Remove supernatant<br />
<br>- Repeat the wash<br />
</p><br />
<br />
<br />
<p class="title3">Bacterial fixation on the chitin beads:</p><p class="texte"><br />
- Add 200 µL of bacteria solution (10<sup>5</sup> bacteria/mL) to the washed beads <br />
<br>- Shake during 1h at 4°C<br />
<br>- Add 500µL of CBB (washing A)<br />
<br>- Put the centrifuge tube on a magnetic rack<br />
<br>- Wait 30 seconds<br />
<br>- Remove supernatant<br />
<br>- Add 500µL of CBB (washing B)<br />
<br>- Put the centrifuge tube on a magnetic rack<br />
<br>- Wait 30 seconds<br />
<br>- Remove supernatant<br />
<br>- Add 500µL of CBB to recover the beads directly<br />
</p><br />
<br />
<p class="title3">Bacteria count:</p><br />
<p class="texte">- Make different dilutions : 10<sup>-1</sup>, 10<sup>-3</sup>, 10<sup>-5</sup> of the first bacterial culture and spread on LA plates<br />
<br>- Make different dilutions : 1, 10<sup>-2</sup>, 10<sup>-4</sup> of washings (A and B) and of the beads in CBB medium and spread on LA plates<br />
<br>- Place the plates at 37°C overnight<br />
<br>- Count colonies on different plates<br />
</p><br />
<br />
<p class="title2">Fungicide test: anti-fungal activities</p><br />
<p class="texte"><br />
CAUTION : all the lab equipment must be desinfected before and after the manipulations with the fungi. <br><br />
<br>Three different fungus strains were used : <i>Aspergillus brasiliensis</i>, <i>Aspergillus nidulans</i> and <i>Trichoderma reesei</i><br />
<br>- The conidia can be taken by adding one drop of Tween 80 on the fungus plate.<br />
<br>- Then the drop is mixed with 1mL of sterile water in an Eppedorf.<br />
<br>- A microscopy count can be performed thanks to Thoma cell to determine the conidia concentration.<br />
<br><i>NB : The conidia solutions are then diluted and spread on sap medium to get 10,000 conidia/plate.</i><br />
<br>- After 72 hours of liquid culture of the different clones of <i>B. subtilis</i> with the fungicides module, the culture can be centrifugated.<br />
<br>- 130µL of the supernatant is used to soak a pad placed on the conidia plate. The bacterial pellet is resuspended in 130µL of LB medium and also put on a pad. <br />
<br>- The plates containing 10,000 conidia and the soaked pads are then put at room temperature for a few days according to the growth speed of the fungi. Controls are also realized with wild type strains or copper sulfate at 10 and 20mg/mL.<br />
</p><br />
<p class= "texte"><br />
<b> Sap-like Medium (250 mL) (see references)</b> : <br><br />
<br />
2,5g de tryptone<br><br />
<br />
<br>1,25g de YE<br />
<br />
<br>2,5g de NaCl<br />
<br />
<br>Glucose : 1,175g<br />
<br />
<br>Fructose : 1,125g<br />
<br />
<br>Sucrose : 0,125g<br />
<br />
<br>Inositol : 0,084 g<br />
<br />
<p class="title2">Fungicide test: <i>in planta</i> assay</p><br />
<p class="title3">First step</p><br />
<p class="texte"><br />
The first step is related to the inoculation of SubtiTree in plants through stomata (opened in wet condition). <br>We diluted our bacterial samples to get two concentrations: 5.10<sup>6</sup> and 10<sup>8</sup> bacteria per mL. The WT and transformed bacteria are introduced into plants (a control test without bacteria is performed). Thanks to a 1 ml syringe (without needle), the plant was injected with bacteria by pressure. Five leaves of each plant were used, marked with a marker point. It is necessary to repeat the operation on both sides of the leaf and the excess is wipe off. The plants are placed in a growth chamber (phytotron) to control light, high humidity, temperature and bacterial non-proliferation. Bacterial growth in the plant is left for 24 h.<br />
</p><br />
<p class="title3">Second step</p><br />
<p class="texte"><br />
The next step begins with the preparation of the fungal samples. Fungal culture is crushed and mixed with PDB (Potato Dextrose Broth). Then the mix passes through a 100 µm filter (to remove large aggregates) and through a 40 µm filter. The caught hyphae are mixed with PDB for 24 to 48 hours until it reaches an OD of 2.5 at 600nm. The previously seeded leaves are taken from the plant using a scalpel and placed in boxes above wet absorbent paper (leaves are kept alive for a week). Above each leaf, 5µl of the fungal suspension is deposited (using beveled tips because it is too viscous). As control, we kept inoculated leaves without fungus and leaves with only fungus. Pictures are taken at different times. All the plants are destroyed by autoclaving.<br />
</p><br />
<br><br />
<b>References</b><br><br />
Véronique Amiard, Annette Morvan-Bertrand, Jean-Bernard Cliquet, Jean-Pierre Billard,<br />
Claude Huault, Jonas P. Sandström, and Marie-Pascale Prud’homme. <b>Carbohydrate and amino<br />
acid composition in phloem sap of Lolium perenne L. before and after defoliation</b>. Can. J. Bot.<br />
Vol. 82: 1594–1601, 2004.<br />
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<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Notebook&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Protocols</p> <br />
<ul class="topnav" id="topnav" style="top:15px;"><br />
<br />
</ul><br />
</div><br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<div id="column-left"><br />
<h3 class="title2" style="margin-top:10px; color:#333;">Summary :</h3><br />
<ul class="menuleft"><br />
<li style="margin-top:25px;"><a href="#select1"><i>E. coli</i> competent cells</a></li><br />
<li><a href="#select2"><i>E. coli</i> transformation protocol</a></li><br />
<li><a href="#select3">Miniprep and alcaline lysis</a></li><br />
<li><a href="#select4">Cloning</a></li><br />
<li><a href="#select5">Checking of the genetic constructions</a></li><br />
<li><a href="#select6"><i>B. subtilis</i> transformation</a></li><br />
<li><a href="#select7">Test of the pSB<sub>BS</sub>4S plasmid integration in <i>Bacillus subtilis</i> genome on the threonine site</a></li><br />
<li><a href="#select8">Final Tests</a></li><br />
</ul><br />
</div><br />
<div class="column-right" style="width:75%; float:right;"><br />
<br />
<p class="texte"> All the following protocols were inspired by one or several protocols, used, improved and optimized (which took more or less time...). Finally they gave us some <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">results</a> :-).</p><br />
<br />
<p class="title1" id="select1"><I>E. coli</I> competent cells</p><br />
<p class="texte"><I> <CENTER> MANIPULATION IN ICE </CENTER> </I></p><br />
<p class="texte"><B> Day 0 </B><br />
<br>- Make an <i>Escherichia coli</i> cell culture in LB medium overnight<br />
<p><br />
<p class="texte"><B> Day 1 </B><br />
<br/> <br />
- Freeze 0.1M CaCl<sub>2</sub> and 4 Falcon tubes of 50mL at 4°C<br />
<br/> <br />
- Streak 2% culture in LB to get an OD of 0.3 to 0.4 (it takes approximately 1h30)<br />
<br/> <br />
- Centrifuge 10 minutes at 4500 RPM<br />
<br/><br />
- Remove the supernatant<br />
<br/><br />
- Resuspend the pellet in 7.5 mL of 0.1 M frozen CaCl<sub>2</sub> <br />
<br/><br />
- Centrifuge 10 minutes at 4500 RPM<br />
<br/><br />
- Resuspend the pellet in 500 µL of 0.1 M CaCl<sub>2</sub><br />
<br/><br />
- Add glycerol to a final concentration of 15%<br />
<br/><br />
- Keep the tubes at -80°C<br />
</p><br />
<br />
<p class="title1" id="select2"> <I>E. coli</I> transformation protocol </p><br />
<p class="texte"><br />
- Let the LB agar medium plates dry in a sterile area<br />
<br/><br />
- Thaw out the competent cell aliquotes for about 10 to 20 minutes<br />
<br/><br />
- Add 20 to 100 ng of plasmid or 3 µL of kit plate DNA <br />
<br/><br />
<i>NB: for kit plate, resuspend the well in 10 µL of sterile water</i><br />
- Put the tubes 20 minutes in the ice </i><br />
<br/><br />
- Put the tubes 2 minutes at 42°C in the water bath<br />
<br/><br />
- Put the tubes back in ice immediately to create the thermic shock<br />
<br/><br />
- Add 1mL of LB medium<br />
<br/><br />
- Put the tube 2 hours in the 37°C water bath (1 hour if it concerns an ampicillin resistant strain) to allow the phenotypic expression<br />
<br/><br />
- Centrifuge for 1 minute at 13000 RPM<br />
<br/><br />
- Remove the supernatant <br />
<br/><br />
- Resuspend in 250 µL of LB medium<br />
<br/><br />
- Streak the final mix on LB agar selective medium: 200 µL on one plate, 50 µL on the second plate<br />
</p><br />
<br />
<p class="title1" id="select3"> Miniprep and alcaline lysis </p><br />
<p class="texte"><B> Day 0 </B><br />
<br/>- Resuspend 4 to 12 colonies from the plate and name each colony taken on the tubes and on the plate (A, B, C, …)<br />
<br/><br />
- Resuspend one colony per culture tube in 5 mL of LB medium with antibiotic<br />
<br/><br />
- Let the culture grow overnight at 37°C in a shaking incubator</p> <br />
<br />
<p class="texte"><B> Day 1 </B><br />
<br>- Use the QIAprep spin Miniprep Kit for each culture tube. The last step consisting in the elution of the DNA is made with elution buffer or water at 55°C.<br />
<br/><br />
- Keep the tubes at -20°C <br />
<br><br />
<br />
<br/><I>NB: It is possible to purify the plasmid with an alcaline lysis without any purification column. For 2 mL of culture, 200 µL of buffer 1 is added to resuspend the pellet, 400 µL of buffer 2 to allow the lysis of the cells and the denaturation of the protein and 300 µL of buffer 3 to precipitate the DNA and the proteins. The solution is then centrifuged 10 minutes at 13 000 RPM.<br />
600 µL of isopropanol is added to the supernatant and the solution is centrifuged again. The pellet is then resuspended in 100 µL of pH 7.4 TE buffer. A part of the contamination by the RNA can avoid by the addition of pH 7.4 TE buffer + 0.2 µL of RNAse. </i><br />
<br><br />
<br> <b>Buffer 1:</b> Tris 10 mM pH 8 + EDTA 1mM<br />
<br> <b>Buffer 2:</b> NaOH 2 mM + SDS 1%<br />
<br> <b>Buffer 3:</b> Potassium acetate 3 M + 15% glacial acetic acid<br />
</I></p><br />
<br />
<p class="title1" id="select4"> Cloning </p><br />
<p class="texte">Cloning is the step after taking the competent cells, transforming the BioBricks and miniprep them.<br />
<br><br />
<p class="title2">First step</p><br />
<p class="title3">Both parts have the same antibiotic resistance</p><br />
<p class="texte"><b>1) Digestion mix</b><br />
<br> For the vector :<br />
<br>- 5 µL of miniprep plasmid <br />
<br><br />
- 2 µL of each restriction enzymes<br />
<br><br />
- 2 µL of Green Buffer<br />
<br><br />
- 9 µL of Milli-Q water <br />
<br><br />
<br> For the insert :<br />
<br>- 10 µL of miniprep plasmid <br />
<br><br />
- 2 µL of each restriction enzymes<br />
<br><br />
- 2 µL of Green Buffer<br />
<br><br />
- 4 µL of Milli-Q water <br />
<br><br />
- Incubate 15 minutes at 37°C <br />
<br />
<br><br><b>2) Gel extraction</b><br />
<br><br />
- Prepare a 1% or 2% electrophoresis agarose gel with 0.5x TAE buffer<br />
<br><br />
- Put 20 µL of sample + 6 µL of marker (1 kb for 1% gel and 100 pb for 2%) into the well<br />
<br><br />
- Migration for 30 min at 100 V or 1 hour at 50V<br />
<br><br />
- The revelation is made in BET (10 minutes). Then wash in water for 5 minutes<br />
<br><br />
- The gel extraction is realized thanks to the THERMO SCIENTIFIC GeneJET Gel Extraction and DNA Clean Up Microkit<br />
<br />
<br><br>3) Inactivation of the enzymes for the vector<br />
<br>There are two ways to inactivate the enzymes:<br />
<br>- Use of DNA Clean up kit for the DNA fragment above 200 pb<br />
<br>- Heat inactivation at 95°C for 10 minutes.</p><br />
<br />
<p class="title3">The two parts have a different antibiotic resistance</p><br />
<p class="texte"><b>1) Digestion mix</b> <br />
<br>For each part, add: <br />
<br>- 5 µL of miniprep plasmid <br />
<br>- 1 µL of each restriction enzymes<br />
<br>- 2 µL of Green Buffer<br />
<br>- 9 µL of Milli-Q water <br />
<br>- Incubate 15 minutes at 37°C <br />
<br />
<p class="texte"><br />
<b>2) Inactivation of the enzymes for the vector</b><br />
<br>There are two ways to inactivate the enzymes:<br />
<br>- Use of DNA Clean up kit for the DNA fragment above 200 pb<br />
<br>- Heat inactivation at 95°C for 10 minutes.</p><br />
<br />
<p class="title2">Second step</p><br />
<p class="title3">Ligation</p><br />
<p class="texte">- Mix 10 µL of insert + 4 µL of vector + 2 µL of 10x T4 buffer + 0.5 µL of T4 ligase + 3.5 µL of Milli-Q water<br />
<br/><br />
A control without insert must be made<br />
<br/><br />
- Incubate the ligation mix 15 minutes at room temperature (22°C) and keep the tubes in ice or at -20°C to prepare the transformation</p><br />
<br />
<p class="title3">Transformation</p><br />
<p class="texte"><br />
- Take 5µL of the ligation mix for 50 µL of competent cells and use the <a href="https://2014.igem.org/Team:Toulouse/Notebook/Protocols#select2">Toulouse iGEM Team 2014 transformation protocol</a>.<br />
<br/><br />
- Plate the solution on selective medium overnight at 37°C.</p><br />
<br />
<p class="title1 " id="select5">Checking of the genetic constructions </p><br />
<p class="title2">1) Colony PCR</p><br />
<p class="texte"><br />
- Add 0.5 µL of plasmid + 25 µL of DreamTAQ MasterMix + 2 µL of each 10 µM primer (VR and VF2) + H<sub>2</sub>0 qsp 25 µL and take a colony.<br />
<br/><br />
- Look for the number of necessary cycles and the proper temperature thanks to AmplifX or Serial Cloner 2.1 softwares.<br />
<br/>The following cycles have been used : <br />
<br/>- 94°C - 5 min<br />
<br/>- (94°C 45 sec ; 55°C 45 sec ; 72°C 1min/kb ) * 25 cycles <br />
<br/>- 72°C - 5min<br />
<br/>- Then 4°C</p><br />
<br />
<p class="title2">2) Analytic digestion</p><br />
<p class="texte"><br />
- Put a colony in 5 mL of LB selective medium and wait for 6 hours<br />
<br/><br />
- Make a purification thanks to the Miniprep kit<br />
<br/><br />
- Mix 2 µL of plasmid + 2 µL of Fast Digest Green Buffer + 1µL of each enzyme + Milli-Q water qsp 20µL<br />
<br/><br />
- Wait 15 minutes at 37°C and put the mix on a 1% or 2% gel for 30 minutes at 100 V.</p><br />
<br />
<p class="title2">3) Sequencing</p><br />
<p class="texte"><br/>The sequencing of the genetic constructions was performed by Eurofins Genomics Company by mixing 15µL of pure plasmid solution with 2µL of one primer.</p><br />
<br />
<p class="title1" id="select6"> <I>B. subtilis</I> transformation</p><br />
<br />
<p class="texte"><br />
<B> Day 0 </B><br />
<br/>- Streak out the <i>Bacillus</i> strain and plate this on an LB agar plate overnight at 37°C</p><br />
<br />
<p class="texte"><B> Day 1 </B><br />
<br />
<br/>- Pick up a nice big colony of <I>B. Subtilis </I> strain and drop it in 2ml of completed 1x MC<br />
<br/><br />
- Grow at 37°C for 5 hours<br />
<br/><br />
- Mix 400µl of culture in a fresh tube (tubes loosely closed for the aeration) and put 5µL of Miniprep DNA.<br />
<br/><br />
- Grow the cells at 37°C for an additional 2 hours<br />
<br/><br />
- Spread the complete 400 µl reaction mix on selective antibiotic plates (100µl per plate), and incubate at 37°C overnight <br />
<br/></p><br />
<br />
<p class="texte"><b> Preparation of solutions: </b><br><br />
<I> <br> 300 mM Tri-Na Citrate:</I><br />
<br>- 0.88 g Tri-Na Citrate<br />
<br>- 10mL MQ water<br />
<br><br><I>Ferric NH4 citrate:</I><br />
<br>- 0.22g Ferric NH4<br />
<br>- 10mL MQ water<br />
<br><br><I>10x Competence Medium </I><br />
<br> For 10mL:<br />
<br>- 1.40g K2HPO4<br />
<br>- 0.52g KH2PO4<br />
<br>- 2g glucose<br />
<br>- 1 mL 300 mM Tri-Na citrate <br />
<br>- 0.1 mL Ferric NH4 citrate<br />
<br>- 0.1g Casein Hydrolysate<br />
<br>- 0.2 g Potassium glutamate<br />
<br>The complete mixture should be dissolved in 10 ml. First add 5 ml milliQ water and mix. When everything is dissolved add MQ water till 10 ml. Filter sterilize the complete mixture and store at -20°C.<br />
<br><br><I>1x Competence Medium </I><br />
<br>- 1.8 mL MQ water<br />
<br>- 200 µL 10x Competence Medium solution (previously filter sterilized)<br />
<br>- 6.7 µL 1M MgSO4 (previously autoclaved)<br />
<br>- 10 µL 1% tryptophan (previously filter sterilized and stored in aluminium foil)<br />
<br>The complete mixture should be dissolved in 100 ml. First add 50 ml milliQ water and mix. When everything is dissolved add MQ water till 100 ml. Filter sterilize the complete mixture and store at -20°C.<br />
<br />
<br />
<p class="title1" id="select7">Test of the pSB<sub>BS</sub>4S plasmid integration in <i>Bacillus subtilis</i> genome on the threonine site</p><br />
<p class="texte"><br />
<br>- Plate the transformed <i>Bacillus</i> strain on a selective medium (LB + spectinomycin) overnight <br />
<br>- The obtained clones are then plated on different media: Medium Competence (Thr+), Medium Competence (Thr-) and LB + Spectinomycin. <br />
<br>When the plasmid is integrated, the clone can grow on minimum medium with threonine and on LB + Spectinomycin but can not grow on the minimum medium without thronine.<br />
Moreover, a colony PCR can be performed with the same protocol as previously presented. The used primers are VFBS and VRBS.<br />
<center><img src="https://static.igem.org/mediawiki/2014/a/a3/Thr.png" width="400px"></center><br />
<p class="legend">Threnonine test (Left: MC Thr+; Right: MC Thr -)</p><br />
<br />
<p class="title1" id="select8">Final Tests</p><br />
<p class="title2">Chemotaxis test</p><br />
<p class="texte"><br />
Many chemotaxis tests exist such as plate tests or capillary tests. We tried all of them and we decided to optimize the <a href="https://2011.igem.org/Team:Imperial_College_London/Protocols_Chemotaxis">« Capillary essay »</a> from Imperial College 2011 iGEM team.<br />
<br>- Prepare the bacteria in LB medium until they reach an OD between 0.5 and 0.6 (exponential growth phase). This step takes about 5 hours.<br />
<br>- Prepare the multichannel pipette: improve the cohesion between the tips and the pipette with Blu-Tack to avoid air and the possibility of leakage.<br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2014/b/b1/Installation_1.gif" width="650px"></center><br />
<p class="texte"><br />
<br>- Put 200 µl of the different chemoattractants in the wells of the ELISA plate and pipet 15 µL of each with the multichannel pipette: galactose which represents our negative control, glucose which represents our positive control and N-acetylglucosamine (NAG). The volume in the tips must be marked.<br><br />
<br><i>NB: The NAG is the most important test because it is the monosaccharide which composes the chitin on <i>Ceratocystis platani</i> wall.<br> <br />
<br>- Put the tips with chemoattractants in 300 µL of the bacterial solution in exponential growth phase in the ELISA plate.<br />
<br>- Let the installation settle for 1 hour at room temperature.<br />
<br>- After an hour, put the volume of the tips on parafilm. <br />
<br>- Each solution is diluted 1/10,000 and 100 µL is spread on LA medium.<br />
<br>- The plates are then incubated overnight at 37°C.</p><br />
<br />
<p class="title2">Binding test</p><br />
<p class="texte"><i>CBB (Chitin Binding Buffer):</i><br />
<br>- 500 mM NaCl<br />
<br>- 20 mM Tris-HCl<br />
<br>- 1 mM EDTA<br />
<br>- 0,05% Triton X-100, 25°C, pH=8<br />
</p><br />
<br />
<p class="title3">Column activation:</p><br />
<p class="texte">- Vortex the beads <br />
<br>- Put 50 µL of beads in a 1.5mL centrifuge tube<br />
<br>- Wash with 500 µL of CBB<br />
<br>- Put the centrifuge tube on a magnetic rack<br />
<br>- Wait 30 seconds<br />
<br>- Remove supernatant<br />
<br>- Repeat the wash<br />
</p><br />
<br />
<br />
<p class="title3">Bacterial fixation on the chitin beads:</p><p class="texte"><br />
- Add 200 µL of bacteria solution (10<sup>5</sup> bacteria/mL) to the washed beads <br />
<br>- Shake during 1h at 4°C<br />
<br>- Add 500µL of CBB (washing A)<br />
<br>- Put the centrifuge tube on a magnetic rack<br />
<br>- Wait 30 seconds<br />
<br>- Remove supernatant<br />
<br>- Add 500µL of CBB (washing B)<br />
<br>- Put the centrifuge tube on a magnetic rack<br />
<br>- Wait 30 seconds<br />
<br>- Remove supernatant<br />
<br>- Add 500µL of CBB to recover the beads directly<br />
</p><br />
<br />
<p class="title3">Bacteria count:</p><br />
<p class="texte">- Make different dilutions : 10<sup>-1</sup>, 10<sup>-3</sup>, 10<sup>-5</sup> of the first bacterial culture and spread on LA plates<br />
<br>- Make different dilutions : 1, 10<sup>-2</sup>, 10<sup>-4</sup> of washings (A and B) and of the beads in CBB medium and spread on LA plates<br />
<br>- Place the plates at 37°C overnight<br />
<br>- Count colonies on different plates<br />
</p><br />
<br />
<p class="title2">Fungicide test: anti-fungal activities</p><br />
<p class="texte"><br />
CAUTION : all the lab equipment must be desinfected before and after the manipulations with the fungi. <br><br />
<br>Three different fungus strains were used : <i>Aspergillus brasiliensis</i>, <i>Aspergillus nidulans</i> and <i>Trichoderma reesei</i><br />
<br>- The conidia can be taken by adding one drop of Tween 80 on the fungus plate.<br />
<br>- Then the drop is mixed with 1mL of sterile water in an Eppedorf.<br />
<br>- A microscopy count can be performed thanks to Thoma cell to determine the conidia concentration.<br />
<br><i>NB : The conidia solutions are then diluted and spread on sap medium to get 10,000 conidia/plate.</i><br />
<br>- After 72 hours of liquid culture of the different clones of <i>B. subtilis</i> with the fungicides module, the culture can be centrifugated.<br />
<br>- 130µL of the supernatant is used to soak a pad placed on the conidia plate. The bacterial pellet is resuspended in 130µL of LB medium and also put on a pad. <br />
<br>- The plates containing 10,000 conidia and the soaked pads are then put at room temperature for a few days according to the growth speed of the fungi. Controls are also realized with wild type strains or copper sulfate at 10 and 20mg/mL.<br />
</p><br />
<p class= "texte"><br />
<b> Sap-like Medium (250 mL) (see references)</b> : <br><br />
<br />
2,5g de tryptone<br><br />
<br />
<br>1,25g de YE<br />
<br />
<br>2,5g de NaCl<br />
<br />
<br>Glucose : 1,175g<br />
<br />
<br>Fructose : 1,125g<br />
<br />
<br>Sucrose : 0,125g<br />
<br />
<br>Inositol : 0,084 g<br />
<br />
<p class="title2">Fungicide test: <i>in planta</i> assay</p><br />
<p class="title3">First step</p><br />
<p class="texte"><br />
The first step is related to the inoculation of SubtiTree in plants through stomata (opened in wet condition). <br>We diluted our bacterial samples to get two concentrations: 5.10<sup>6</sup> and 10<sup>8</sup> bacteria per mL. The WT and transformed bacteria are introduced into plants (a control test without bacteria is performed). Thanks to a 1 ml syringe (without needle), the plant was injected with bacteria by pressure. Five leaves of each plant were used, marked with a marker point. It is necessary to repeat the operation on both sides of the leaf and the excess is wipe off. The plants are placed in a growth chamber (phytotron) to control light, high humidity, temperature and bacterial non-proliferation. Bacterial growth in the plant is left for 24 h.<br />
</p><br />
<p class="title3">Second step</p><br />
<p class="texte"><br />
The next step begins with the preparation of the fungal samples. Fungal culture is crushed and mixed with PDB (Potato Dextrose Broth). Then the mix passes through a 100 µm filter (to remove large aggregates) and through a 40 µm filter. The caught hyphae are mixed with PDB for 24 to 48 hours until it reaches an OD of 2.5 at 600nm. The previously seeded leaves are taken from the plant using a scalpel and placed in boxes above wet absorbent paper (leaves are kept alive for a week). Above each leaf, 5µl of the fungal suspension is deposited (using beveled tips because it is too viscous). As control, we kept inoculated leaves without fungus and leaves with only fungus. Pictures are taken at different times. All the plants are destroyed by autoclaving.<br />
</p><br />
<br><br />
<b>References</b><br><br />
Véronique Amiard, Annette Morvan-Bertrand, Jean-Bernard Cliquet, Jean-Pierre Billard,<br />
Claude Huault, Jonas P. Sandström, and Marie-Pascale Prud’homme. <b>Carbohydrate and amino<br />
acid composition in phloem sap of Lolium perenne L. before and after defoliation</b>. Can. J. Bot.<br />
Vol. 82: 1594–1601, 2004.<br />
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<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Notebook&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Protocols</p> <br />
<ul class="topnav" id="topnav" style="top:15px;"><br />
<br />
</ul><br />
</div><br />
</div><br />
<br />
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<br />
<div id="column-left"><br />
<h3 class="title2" style="margin-top:10px; color:#333;">Summary :</h3><br />
<ul class="menuleft"><br />
<li style="margin-top:25px;"><a href="#select1"><i>E. coli</i> competent cells</a></li><br />
<li><a href="#select2"><i>E. coli</i> transformation protocol</a></li><br />
<li><a href="#select3">Miniprep and alcaline lysis</a></li><br />
<li><a href="#select4">Cloning</a></li><br />
<li><a href="#select5">Checking of the genetic constructions</a></li><br />
<li><a href="#select6"><i>B. subtilis</i> transformation</a></li><br />
<li><a href="#select7">Test of the pSB<sub>BS</sub>4S plasmid integration in <i>Bacillus subtilis</i> genome on the threonine site</a></li><br />
<li><a href="#select8">Final Tests</a></li><br />
</ul><br />
</div><br />
<div class="column-right" style="width:75%; float:right;"><br />
<br />
<p class="texte"> All the following protocols were inspired by one or several protocols, used, improved and optimized (which took more or less time...). Finally they gave us some <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">results</a> :-).</p><br />
<br />
<p class="title1" id="select1"><I>E. coli</I> competent cells</p><br />
<p class="texte"><I> <CENTER> MANIPULATION IN ICE </CENTER> </I></p><br />
<p class="texte"><B> Day 0 </B><br />
<br>- Make an <i>Escherichia coli</i> cell culture in LB medium overnight<br />
<p><br />
<p class="texte"><B> Day 1 </B><br />
<br/> <br />
- Freeze 0.1M CaCl<sub>2</sub> and 4 Falcon tubes of 50mL at 4°C<br />
<br/> <br />
- Streak 2% culture in LB to get an OD of 0.3 to 0.4 (it takes approximately 1h30)<br />
<br/> <br />
- Centrifuge 10 minutes at 4500 RPM<br />
<br/><br />
- Remove the supernatant<br />
<br/><br />
- Resuspend the pellet in 7.5 mL of 0.1 M frozen CaCl<sub>2</sub> <br />
<br/><br />
- Centrifuge 10 minutes at 4500 RPM<br />
<br/><br />
- Resuspend the pellet in 500 µL of 0.1 M CaCl<sub>2</sub><br />
<br/><br />
- Add glycerol to a final concentration of 15%<br />
<br/><br />
- Keep the tubes at -80°C<br />
</p><br />
<br />
<p class="title1" id="select2"> <I>E. coli</I> transformation protocol </p><br />
<p class="texte"><br />
- Let the LB agar medium plates dry in a sterile area<br />
<br/><br />
- Thaw out the competent cell aliquotes for about 10 to 20 minutes<br />
<br/><br />
- Add 20 to 100 ng of plasmid or 3 µL of kit plate DNA <br />
<br/><br />
<i>NB: for kit plate, resuspend the well in 10 µL of sterile water</i><br />
- Put the tubes 20 minutes in the ice </i><br />
<br/><br />
- Put the tubes 2 minutes at 42°C in the water bath<br />
<br/><br />
- Put the tubes back in ice immediately to create the thermic shock<br />
<br/><br />
- Add 1mL of LB medium<br />
<br/><br />
- Put the tube 2 hours in the 37°C water bath (1 hour if it concerns an ampicillin resistant strain) to allow the phenotypic expression<br />
<br/><br />
- Centrifuge for 1 minute at 13000 RPM<br />
<br/><br />
- Remove the supernatant <br />
<br/><br />
- Resuspend in 250 µL of LB medium<br />
<br/><br />
- Streak the final mix on LB agar selective medium: 200 µL on one plate, 50 µL on the second plate<br />
</p><br />
<br />
<p class="title1" id="select3"> Miniprep and alcaline lysis </p><br />
<p class="texte"><B> Day 0 </B><br />
<br/>- Resuspend 4 to 12 colonies from the plate and name each colony taken on the tubes and on the plate (A, B, C, …)<br />
<br/><br />
- Resuspend one colony per culture tube in 5 mL of LB medium with antibiotic<br />
<br/><br />
- Let the culture grow overnight at 37°C in a shaking incubator</p> <br />
<br />
<p class="texte"><B> Day 1 </B><br />
<br>- Use the QIAprep spin Miniprep Kit for each culture tube. The last step consisting in the elution of the DNA is made with elution buffer or water at 55°C.<br />
<br/><br />
- Keep the tubes at -20°C <br />
<br><br />
<br />
<br/><I>NB: It is possible to purify the plasmid with an alcaline lysis without any purification column. For 2 mL of culture, 200 µL of buffer 1 is added to resuspend the pellet, 400 µL of buffer 2 to allow the lysis of the cells and the denaturation of the protein and 300 µL of buffer 3 to precipitate the DNA and the proteins. The solution is then centrifuged 10 minutes at 13 000 RPM.<br />
600 µL of isopropanol is added to the supernatant and the solution is centrifuged again. The pellet is then resuspended in 100 µL of pH 7.4 TE buffer. A part of the contamination by the RNA can avoid by the addition of pH 7.4 TE buffer + 0.2 µL of RNAse. <br />
<br><br />
<br> <b>Buffer 1:</b> Tris 10 mM pH 8 + EDTA 1mM<br />
<br> <b>Buffer 2:</b> NaOH 2 mM + SDS 1%<br />
<br> <b>Buffer 3:</b> Potassium acetate 3 M + 15% glacial acetic acid<br />
</I></p><br />
<br />
<p class="title1" id="select4"> Cloning </p><br />
<p class="texte">Cloning is the step after taking the competent cells, transforming the BioBricks and miniprep them.<br />
<br><br />
<p class="title2">First step</p><br />
<p class="title3">Both parts have the same antibiotic resistance</p><br />
<p class="texte"><b>1) Digestion mix</b><br />
<br> For the vector :<br />
<br>- 5 µL of miniprep plasmid <br />
<br><br />
- 2 µL of each restriction enzymes<br />
<br><br />
- 2 µL of Green Buffer<br />
<br><br />
- 9 µL of Milli-Q water <br />
<br><br />
<br> For the insert :<br />
<br>- 10 µL of miniprep plasmid <br />
<br><br />
- 2 µL of each restriction enzymes<br />
<br><br />
- 2 µL of Green Buffer<br />
<br><br />
- 4 µL of Milli-Q water <br />
<br><br />
- Incubate 15 minutes at 37°C <br />
<br />
<br><br><b>2) Gel extraction</b><br />
<br><br />
- Prepare a 1% or 2% electrophoresis agarose gel with 0.5x TAE buffer<br />
<br><br />
- Put 20 µL of sample + 6 µL of marker (1 kb for 1% gel and 100 pb for 2%) into the well<br />
<br><br />
- Migration for 30 min at 100 V or 1 hour at 50V<br />
<br><br />
- The revelation is made in BET (10 minutes). Then wash in water for 5 minutes<br />
<br><br />
- The gel extraction is realized thanks to the THERMO SCIENTIFIC GeneJET Gel Extraction and DNA Clean Up Microkit<br />
<br />
<br><br>3) Inactivation of the enzymes for the vector<br />
<br>There are two ways to inactivate the enzymes:<br />
<br>- Use of DNA Clean up kit for the DNA fragment above 200 pb<br />
<br>- Heat inactivation at 95°C for 10 minutes.</p><br />
<br />
<p class="title3">The two parts have a different antibiotic resistance</p><br />
<p class="texte"><b>1) Digestion mix</b> <br />
<br>For each part, add: <br />
<br>- 5 µL of miniprep plasmid <br />
<br>- 1 µL of each restriction enzymes<br />
<br>- 2 µL of Green Buffer<br />
<br>- 9 µL of Milli-Q water <br />
<br>- Incubate 15 minutes at 37°C <br />
<br />
<p class="texte"><br />
<b>2) Inactivation of the enzymes for the vector</b><br />
<br>There are two ways to inactivate the enzymes:<br />
<br>- Use of DNA Clean up kit for the DNA fragment above 200 pb<br />
<br>- Heat inactivation at 95°C for 10 minutes.</p><br />
<br />
<p class="title2">Second step</p><br />
<p class="title3">Ligation</p><br />
<p class="texte">- Mix 10 µL of insert + 4 µL of vector + 2 µL of 10x T4 buffer + 0.5 µL of T4 ligase + 3.5 µL of Milli-Q water<br />
<br/><br />
A control without insert must be made<br />
<br/><br />
- Incubate the ligation mix 15 minutes at room temperature (22°C) and keep the tubes in ice or at -20°C to prepare the transformation</p><br />
<br />
<p class="title3">Transformation</p><br />
<p class="texte"><br />
- Take 5µL of the ligation mix for 50 µL of competent cells and use the <a href="https://2014.igem.org/Team:Toulouse/Notebook/Protocols#select2">Toulouse iGEM Team 2014 transformation protocol</a>.<br />
<br/><br />
- Plate the solution on selective medium overnight at 37°C.</p><br />
<br />
<p class="title1 " id="select5">Checking of the genetic constructions </p><br />
<p class="title2">1) Colony PCR</p><br />
<p class="texte"><br />
- Add 0.5 µL of plasmid + 25 µL of DreamTAQ MasterMix + 2 µL of each 10 µM primer (VR and VF2) + H<sub>2</sub>0 qsp 25 µL and take a colony.<br />
<br/><br />
- Look for the number of necessary cycles and the proper temperature thanks to AmplifX or Serial Cloner 2.1 softwares.<br />
<br/>The following cycles have been used : <br />
<br/>- 94°C - 5 min<br />
<br/>- (94°C 45 sec ; 55°C 45 sec ; 72°C 1min/kb ) * 25 cycles <br />
<br/>- 72°C - 5min<br />
<br/>- Then 4°C</p><br />
<br />
<p class="title2">2) Analytic digestion</p><br />
<p class="texte"><br />
- Put a colony in 5 mL of LB selective medium and wait for 6 hours<br />
<br/><br />
- Make a purification thanks to the Miniprep kit<br />
<br/><br />
- Mix 2 µL of plasmid + 2 µL of Fast Digest Green Buffer + 1µL of each enzyme + Milli-Q water qsp 20µL<br />
<br/><br />
- Wait 15 minutes at 37°C and put the mix on a 1% or 2% gel for 30 minutes at 100 V.</p><br />
<br />
<p class="title2">3) Sequencing</p><br />
<p class="texte"><br/>The sequencing of the genetic constructions was performed by Eurofins Genomics Company by mixing 15µL of pure plasmid solution with 2µL of one primer.</p><br />
<br />
<p class="title1" id="select6"> <I>B. subtilis</I> transformation</p><br />
<br />
<p class="texte"><br />
<B> Day 0 </B><br />
<br/>- Streak out the <i>Bacillus</i> strain and plate this on an LB agar plate overnight at 37°C</p><br />
<br />
<p class="texte"><B> Day 1 </B><br />
<br />
<br/>- Pick up a nice big colony of <I>B. Subtilis </I> strain and drop it in 2ml of completed 1x MC<br />
<br/><br />
- Grow at 37°C for 5 hours<br />
<br/><br />
- Mix 400µl of culture in a fresh tube (tubes loosely closed for the aeration) and put 5µL of Miniprep DNA.<br />
<br/><br />
- Grow the cells at 37°C for an additional 2 hours<br />
<br/><br />
- Spread the complete 400 µl reaction mix on selective antibiotic plates (100µl per plate), and incubate at 37°C overnight <br />
<br/></p><br />
<br />
<p class="texte"><b> Preparation of solutions: </b><br><br />
<I> <br> 300 mM Tri-Na Citrate:</I><br />
<br>- 0.88 g Tri-Na Citrate<br />
<br>- 10mL MQ water<br />
<br><br><I>Ferric NH4 citrate:</I><br />
<br>- 0.22g Ferric NH4<br />
<br>- 10mL MQ water<br />
<br><br><I>10x Competence Medium </I><br />
<br> For 10mL:<br />
<br>- 1.40g K2HPO4<br />
<br>- 0.52g KH2PO4<br />
<br>- 2g glucose<br />
<br>- 1 mL 300 mM Tri-Na citrate <br />
<br>- 0.1 mL Ferric NH4 citrate<br />
<br>- 0.1g Casein Hydrolysate<br />
<br>- 0.2 g Potassium glutamate<br />
<br>The complete mixture should be dissolved in 10 ml. First add 5 ml milliQ water and mix. When everything is dissolved add MQ water till 10 ml. Filter sterilize the complete mixture and store at -20°C.<br />
<br><br><I>1x Competence Medium </I><br />
<br>- 1.8 mL MQ water<br />
<br>- 200 µL 10x Competence Medium solution (previously filter sterilized)<br />
<br>- 6.7 µL 1M MgSO4 (previously autoclaved)<br />
<br>- 10 µL 1% tryptophan (previously filter sterilized and stored in aluminium foil)<br />
<br>The complete mixture should be dissolved in 100 ml. First add 50 ml milliQ water and mix. When everything is dissolved add MQ water till 100 ml. Filter sterilize the complete mixture and store at -20°C.<br />
<br />
<br />
<p class="title1" id="select7">Test of the pSB<sub>BS</sub>4S plasmid integration in <i>Bacillus subtilis</i> genome on the threonine site</p><br />
<p class="texte"><br />
<br>- Plate the transformed <i>Bacillus</i> strain on a selective medium (LB + spectinomycin) overnight <br />
<br>- The obtained clones are then plated on different media: Medium Competence (Thr+), Medium Competence (Thr-) and LB + Spectinomycin. <br />
<br>When the plasmid is integrated, the clone can grow on minimum medium with threonine and on LB + Spectinomycin but can not grow on the minimum medium without thronine.<br />
Moreover, a colony PCR can be performed with the same protocol as previously presented. The used primers are VFBS and VRBS.<br />
<center><img src="https://static.igem.org/mediawiki/2014/a/a3/Thr.png" width="400px"></center><br />
<p class="legend">Threnonine test (Left: MC Thr+; Right: MC Thr -)</p><br />
<br />
<p class="title1" id="select8">Final Tests</p><br />
<p class="title2">Chemotaxis test</p><br />
<p class="texte"><br />
Many chemotaxis tests exist such as plate tests or capillary tests. We tried all of them and we decided to optimize the <a href="https://2011.igem.org/Team:Imperial_College_London/Protocols_Chemotaxis">« Capillary essay »</a> from Imperial College 2011 iGEM team.<br />
<br>- Prepare the bacteria in LB medium until they reach an OD between 0.5 and 0.6 (exponential growth phase). This step takes about 5 hours.<br />
<br>- Prepare the multichannel pipette: improve the cohesion between the tips and the pipette with Blu-Tack to avoid air and the possibility of leakage.<br />
<br><br />
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<br>- Put 200 µl of the different chemoattractants in the wells of the ELISA plate and pipet 15 µL of each with the multichannel pipette: galactose which represents our negative control, glucose which represents our positive control and N-acetylglucosamine (NAG). The volume in the tips must be marked.<br><br />
<br><i>NB: The NAG is the most important test because it is the monosaccharide which composes the chitin on <i>Ceratocystis platani</i> wall.<br> <br />
<br>- Put the tips with chemoattractants in 300 µL of the bacterial solution in exponential growth phase in the ELISA plate.<br />
<br>- Let the installation settle for 1 hour at room temperature.<br />
<br>- After an hour, put the volume of the tips on parafilm. <br />
<br>- Each solution is diluted 1/10,000 and 100 µL is spread on LA medium.<br />
<br>- The plates are then incubated overnight at 37°C.</p><br />
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<p class="title2">Binding test</p><br />
<p class="texte"><i>CBB (Chitin Binding Buffer):</i><br />
<br>- 500 mM NaCl<br />
<br>- 20 mM Tris-HCl<br />
<br>- 1 mM EDTA<br />
<br>- 0,05% Triton X-100, 25°C, pH=8<br />
</p><br />
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<p class="title3">Column activation:</p><br />
<p class="texte">- Vortex the beads <br />
<br>- Put 50 µL of beads in a 1.5mL centrifuge tube<br />
<br>- Wash with 500 µL of CBB<br />
<br>- Put the centrifuge tube on a magnetic rack<br />
<br>- Wait 30 seconds<br />
<br>- Remove supernatant<br />
<br>- Repeat the wash<br />
</p><br />
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<p class="title3">Bacterial fixation on the chitin beads:</p><p class="texte"><br />
- Add 200 µL of bacteria solution (10<sup>5</sup> bacteria/mL) to the washed beads <br />
<br>- Shake during 1h at 4°C<br />
<br>- Add 500µL of CBB (washing A)<br />
<br>- Put the centrifuge tube on a magnetic rack<br />
<br>- Wait 30 seconds<br />
<br>- Remove supernatant<br />
<br>- Add 500µL of CBB (washing B)<br />
<br>- Put the centrifuge tube on a magnetic rack<br />
<br>- Wait 30 seconds<br />
<br>- Remove supernatant<br />
<br>- Add 500µL of CBB to recover the beads directly<br />
</p><br />
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<p class="title3">Bacteria count:</p><br />
<p class="texte">- Make different dilutions : 10<sup>-1</sup>, 10<sup>-3</sup>, 10<sup>-5</sup> of the first bacterial culture and spread on LA plates<br />
<br>- Make different dilutions : 1, 10<sup>-2</sup>, 10<sup>-4</sup> of washings (A and B) and of the beads in CBB medium and spread on LA plates<br />
<br>- Place the plates at 37°C overnight<br />
<br>- Count colonies on different plates<br />
</p><br />
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<p class="title2">Fungicide test: anti-fungal activities</p><br />
<p class="texte"><br />
CAUTION : all the lab equipment must be desinfected before and after the manipulations with the fungi. <br><br />
<br>Three different fungus strains were used : <i>Aspergillus brasiliensis</i>, <i>Aspergillus nidulans</i> and <i>Trichoderma reesei</i><br />
<br>- The conidia can be taken by adding one drop of Tween 80 on the fungus plate.<br />
<br>- Then the drop is mixed with 1mL of sterile water in an Eppedorf.<br />
<br>- A microscopy count can be performed thanks to Thoma cell to determine the conidia concentration.<br />
<br><i>NB : The conidia solutions are then diluted and spread on sap medium to get 10,000 conidia/plate.</i><br />
<br>- After 72 hours of liquid culture of the different clones of <i>B. subtilis</i> with the fungicides module, the culture can be centrifugated.<br />
<br>- 130µL of the supernatant is used to soak a pad placed on the conidia plate. The bacterial pellet is resuspended in 130µL of LB medium and also put on a pad. <br />
<br>- The plates containing 10,000 conidia and the soaked pads are then put at room temperature for a few days according to the growth speed of the fungi. Controls are also realized with wild type strains or copper sulfate at 10 and 20mg/mL.<br />
</p><br />
<br />
<p class="title2">Fungicide test: <i>in planta</i> assay</p><br />
<p class="title3">First step</p><br />
<p class="texte"><br />
The first step is related to the inoculation of SubtiTree in plants through stomata (opened in wet condition). <br>We diluted our bacterial samples to get two concentrations: 5.10<sup>6</sup> and 10<sup>8</sup> bacteria per mL. The WT and transformed bacteria are introduced into plants (a control test without bacteria is performed). Thanks to a 1 ml syringe (without needle), the plant was injected with bacteria by pressure. Five leaves of each plant were used, marked with a marker point. It is necessary to repeat the operation on both sides of the leaf and the excess is wipe off. The plants are placed in a growth chamber (phytotron) to control light, high humidity, temperature and bacterial non-proliferation. Bacterial growth in the plant is left for 24 h.<br />
</p><br />
<p class="title3">Second step</p><br />
<p class="texte"><br />
The next step begins with the preparation of the fungal samples. Fungal culture is crushed and mixed with PDB (Potato Dextrose Broth). Then the mix passes through a 100 µm filter (to remove large aggregates) and through a 40 µm filter. The caught hyphae are mixed with PDB for 24 to 48 hours until it reaches an OD of 2.5 at 600nm. The previously seeded leaves are taken from the plant using a scalpel and placed in boxes above wet absorbent paper (leaves are kept alive for a week). Above each leaf, 5µl of the fungal suspension is deposited (using beveled tips because it is too viscous). As control, we kept inoculated leaves without fungus and leaves with only fungus. Pictures are taken at different times. All the plants are destroyed by autoclaving.<br />
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{{:Team:Toulouse/template/footer}}</div>Jourdanhttp://2014.igem.org/Team:Toulouse/Result/achievementTeam:Toulouse/Result/achievement2014-10-17T23:14:03Z<p>Jourdan: </p>
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<h2>Achievement</h2><br />
<p>Bronze, Silver or Gold Medal? That is the question!</p><br />
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<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Results&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Achievement</p> <br />
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<p class="title1" style="text-align:left;">Let's sum up what we did during this summer!</p><br />
<p class="title2">First step...</p><br />
<p class="texte"> <br />
After a summer of hard work, our team is proud of the achieved results. The first step of our project was to have a <b>proof of concept</b> of the three functions requiered to fight against canker. The verifications of our genetic constructions were successful (PCR, Migration on an agarose gel and Sequencing). We could then proceed to specific test of the modules: capillary test, chitin beads test or fungicide tests.</p><br />
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<p class="title2">Second step...</p><br />
<p class="texte"> <br />
Once these results obtained, we decided to move on to the next step: in order to get closer to our final objective, the three genetic modules have to be assembled and to be tested in <b>model plants</b>. In the short time we had, these tests were only performed with the antifungal module, but the results were very encouraging (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">3. In planta tests with SubtiTree</a> in the Fungicides module). <br />
<br />
<p class="title2">Third step...</p><br />
<p class="texte"> <br />
So encouraging that we moved on to the third step of our project and think about the <b>real injection of SubtiTree in diseased trees</b>. We then considered the <a href="https://2014.igem.org/Team:Toulouse/Project/Spreading"><b>spreading</b></a> problems and proposed different strategies to avoid this major issue. <a href="https://2014.igem.org/Team:Toulouse/Modelling"><b>Modeling</b></a> enables us to predict the growth and the lifespan of SubtiTree in Plane Trees. <br />
<br />
<p class="title2">And what comes next?</p><br />
<p class="texte"> With the support of Governmental institutions, scientific organizations and the popular enthusiasm raised by our project, we are still looking for the best way to continue and achieve our goal: save the plane trees and preserve the beauty of the Canal du Midi.<br><br />
However, SubtiTree's spreading must be controlled, fungicide production regulated, specifity improved, tests of the whole system performed. In a nutshell, we still have a lot of things to do even if we did our best during the whole summer!</p><br />
<br />
<p class="title2">iGEM Community</p><br />
<p class="texte"><br />
Well aware that we are part of the <b>iGEM Community</b>, our team also worked for this community by improving the iGEMer's Bible: <a href="http://parts.igem.org/Catalog">the Registry of Standard Biological Parts</a>. <br><br />
We improved the EcAMP BioBrick by adding a promotor, a RBS, but mainly by the addition of a STOP codon (<a href="http://parts.igem.org/Part:BBa_K1364019">BBa_K1364019</a>).<br><br />
As <i>Bacillus subtilis</i> appears to be for us an ideal chassis, we also developed new genetic tools to facilitate its utilization. Thanks to numerous interactions with other iGEM teams, we didn't see the time flying by during this <b>amazing summer</b> !</p><br />
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<center><img style="width:1000px; " src="https://static.igem.org/mediawiki/parts/e/e2/Ach.jpg"></center><br />
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<p class="title2">It is better to have "well-made rather than a well-filled head"... (Michel de Montaigne)</p><br />
<p class="texte">We developed a deep <a href="https://2014.igem.org/Team:Toulouse/ethics">ethical questioning</a>, trying to understand the relationship between Science and the protection of the Beauty. We strongly believe that neglecting the ethical aspect in a scientific project is a mistake. All the team was involved in this part and the reflexions made were very profitable to us.</p><br />
<br />
<p class="texte">We filled the <a href="https://igem.org/2014_Judging_Form?id=1364">2014 Judging Form</a> thanks to the following accomplishments:</p><br />
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<td style="border-bottom:4px solid #e5e6e6; border-top-left-radius:9px;"><center><br>Bronze Medal <img src="https://static.igem.org/mediawiki/2013/8/86/Medal_bronze.png" class="imgcontenttable"/></center><br></td><br />
<td style="border-bottom:4px solid #e5e6e6;"><center><br>Silver Medal <img src="https://static.igem.org/mediawiki/2013/d/dd/Medal_silver.png" class="imgcontenttable"/></center><br></td><br />
<td style="border-bottom:4px solid #e5e6e6; border-top-right-radius:9px;"><center><br>Gold Medal<img src="https://static.igem.org/mediawiki/2013/b/bd/Medal_gold_3.png" class="imgcontenttable"/></center><br></td><br />
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<td style="border-right:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/>Register the team, have a great summer, and plan to have fun at the Jamboree</td><br />
<td style="border-right:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/>Experimentally validate that at least three new BioBrick Part or Device of your own design and construction work as expected.</td><br />
<td><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Improve the function of an existing BioBrick Part or Device and improve the <i>Bacillus subtilis</i> genetic tools, enter this information in the Registry : <a href="http://parts.igem.org/Part:BBa_K1364019"target="_blank">BBa_K1364019</a>, <a href="http://parts.igem.org/Part:BBa_K1364016"target="_blank">BBa_K1364016</a>, <a href="http://parts.igem.org/Part:BBa_K1364017"target="_blank">BBa_K1364017</a>, <a href="http://parts.igem.org/Part:BBa_K1364021"target="_blank">BBa_K1364021</a>, </td><br />
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<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Successfully complete and submit this iGEM Judging form </td><br />
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/>Document the characterization of these three parts in the 'Main Page' of that Part's/Device's Registry entry<br />
Submit this new part to the iGEM Parts Registry:<br> <a href="http://parts.igem.org/Part:BBa_K1364005"target="_blank">BBa_K1364005</a>, <a href="http://parts.igem.org/Part:BBa_K1364009"target="_blank">BBa_K1364009</a>, <a href="http://parts.igem.org/Part:BBa_K1364013"target="_blank">BBa_K1364013.</a></td><br />
<td style="border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Your project may have implications for the environment, security, safety and ethics and/or ownership and sharing. Describe a novel approach that your team has used to help you and others consider these aspects of the design and outcomes of synthetic biology efforts. → <a href="https://2014.igem.org/Team:Toulouse/ethics" target="_blank">Ethic Part</a> </td><br />
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<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Create and share a Description of the team’s project using the iGEM wiki and the team’s parts using the Registry Biological Parts </td><br />
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Submit these new part to the iGEM Parts Registry (submissions must adhere to the iGEM Registry guidelines)</td><br />
<td style="border-top:1px solid #e5e6e6;"> </td><br />
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<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Plan to present a Poster and Talk at the iGEM Jamboree </td><br />
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Your project may have implications for the environment, security, safety and ethics and/or ownership and sharing. Describe one or more ways in which these or other broader implications have been taken into consideration in the design and execution of your project. → <a href="https://2014.igem.org/Team:Toulouse/ethics" target="_blank"> Ethical Page</a></td><br />
<td style="border-top:1px solid #e5e6e6;"></td><br />
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<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Document at least one new standard BioBrick Part or Device used in your project/central to your project and submit this part to the iGEM Registry : <a href="http://parts.igem.org/Part:BBa_K1364000" target="_blank">BBa_K1364000</a>, <a href="http://parts.igem.org/Part:BBa_K1364002"target="_blank">BBa_K1364002</a>, <a href="http://parts.igem.org/Part:BBa_K1364003"target="_blank">BBa_K1364003</a>, <a href="http://parts.igem.org/Part:BBa_K1364005"target="_blank">BBa_K1364005</a>. </td><br />
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{{:Team:Toulouse/template/footer}}</div>Jourdanhttp://2014.igem.org/Team:Toulouse/Result/achievementTeam:Toulouse/Result/achievement2014-10-17T23:00:08Z<p>Jourdan: </p>
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<h2>Achievement</h2><br />
<p>Bronze, Silver or Gold Medal? That is the question!</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Results&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Achievement</p> <br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<p class="title1" style="text-align:left;">Let's sum up what we did during this summer!</p><br />
<p class="title2">First step...</p><br />
<p class="texte"> <br />
After a summer of hard work, our team is proud of the achieved results. The first step of our project was to have a <b>proof of concept</b> of the three functions requiered to fight against canker. The three modules tested were Chemotaxis, Binding and Fungicides. The verifications of our genetic constructions were successful (PCR, Migration on an agarose gel and Sequencing). We could then proceed to specific test of the modules: capillary test, chitin beads test or fungicide tests.</p><br />
<br />
<p class="title2">Second step...</p><br />
<p class="texte"> <br />
Once these results obtained, we decided to move on to the next step: in order to get closer to our final objective, the three genetic modules have to be assembled and to be tested in <b>model plants</b>. In the short time left that we had, these tests were only performed with the antifungal module, but the results were very encouraging (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">3. In planta tests with SubtiTree</a> in the Fungicides module). <br />
<br />
<p class="title2">Third step...</p><br />
<p class="texte"> <br />
So encouraging that we moved on to the third step of our project and think about the <b>real injection of SubtiTree in diseased trees</b>. We then considered the <a href="https://2014.igem.org/Team:Toulouse/Project/Spreading"><b>spreading</b></a> problems and proposed different strategies to avoid this major issue. <a href="https://2014.igem.org/Team:Toulouse/Modelling"><b>Modeling</b></a> enables us to predict the growth and the lifespan of SubtiTree in Plane Trees. <br />
<br />
<p class="title2">And what comes next?</p><br />
<p class="texte"> With the support of Governmental institutions, scientific organizations and the popular enthusiasm raised by our project, we are still looking for the best way to continue and achieve our goal: save the plane trees and preserve the beauty of the Canal du Midi.<br><br />
However, SubtiTree's spreading must be controlled, fungicide production regulated, and tests of the whole system performed. In a nutshell, we still have a lot of things to do even if we did our best during the whole summer!</p><br />
<br />
<p class="title2">iGEM Community</p><br />
<p class="texte"><br />
Well aware that we are part of the <b>iGEM Community</b>, our team also worked for this community by improving the iGEMer's Bible: <a href="http://parts.igem.org/Catalog">the Registry of Standard Biological Parts</a>. <br><br />
We improved the EcAMP BioBrick by adding a promotor, a RBS, but mainly by the addition of a STOP codon (<a href="http://parts.igem.org/Part:BBa_K1364019">BBa_K1364019</a>).<br><br />
As <i>Bacillus subtilis</i> appears to be for us an ideal chassis, we also developed new genetic tools to facilitate its utilization. Thanks to numerous interactions with other iGEM teams, we didn't see the time flying by during this <b>amazing summer</b> !</p><br />
<br />
<center><img style="width:1000px; " src="https://static.igem.org/mediawiki/parts/e/e2/Ach.jpg"></center><br />
<br><br />
<p class="title2">It is better to have "well-made rather than a well-filled head"... (Michel de Montaigne)</p><br />
<p class="texte">We developed a deep <a href="https://2014.igem.org/Team:Toulouse/ethics">ethical questioning</a>, trying to understand if we had the right to act against canker. We strongly believe that neglecting the ethical aspect in a scientific project is a mistake. All the team was involved in this part and the reflexions made were very profitable to us.</p><br />
<br />
<p class="texte">We filled the <a href="https://igem.org/2014_Judging_Form?id=1364">2014 Judging Form</a> thanks to the following accomplishments:</p><br />
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<td style="border-bottom:4px solid #e5e6e6;"><center><br>Silver Medal <img src="https://static.igem.org/mediawiki/2013/d/dd/Medal_silver.png" class="imgcontenttable"/></center><br></td><br />
<td style="border-bottom:4px solid #e5e6e6; border-top-right-radius:9px;"><center><br>Gold Medal<img src="https://static.igem.org/mediawiki/2013/b/bd/Medal_gold_3.png" class="imgcontenttable"/></center><br></td><br />
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<td style="border-right:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/>Register the team, have a great summer, and plan to have fun at the Jamboree</td><br />
<td style="border-right:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/>Experimentally validate that at least three new BioBrick Part or Device of your own design and construction work as expected.</td><br />
<td><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Improve the function of an existing BioBrick Part or Device and improve the <i>Bacillus subtilis</i> genetic tools, enter this information in the Registry : <a href="http://parts.igem.org/Part:BBa_K1364019"target="_blank">BBa_K1364019</a>, <a href="http://parts.igem.org/Part:BBa_K1364016"target="_blank">BBa_K1364016</a>, <a href="http://parts.igem.org/Part:BBa_K1364017"target="_blank">BBa_K1364017</a>, <a href="http://parts.igem.org/Part:BBa_K1364021"target="_blank">BBa_K1364021</a>, </td><br />
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<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Successfully complete and submit this iGEM Judging form </td><br />
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/>Document the characterization of these three parts in the 'Main Page' of that Part's/Device's Registry entry<br />
Submit this new part to the iGEM Parts Registry:<br> <a href="http://parts.igem.org/Part:BBa_K1364005"target="_blank">BBa_K1364005</a>, <a href="http://parts.igem.org/Part:BBa_K1364009"target="_blank">BBa_K1364009</a>, <a href="http://parts.igem.org/Part:BBa_K1364013"target="_blank">BBa_K1364013.</a></td><br />
<td style="border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Your project may have implications for the environment, security, safety and ethics and/or ownership and sharing. Describe a novel approach that your team has used to help you and others consider these aspects of the design and outcomes of synthetic biology efforts. → <a href="https://2014.igem.org/Team:Toulouse/ethics" target="_blank">Ethic Part</a> </td><br />
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<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Create and share a Description of the team’s project using the iGEM wiki and the team’s parts using the Registry Biological Parts </td><br />
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Submit these new part to the iGEM Parts Registry (submissions must adhere to the iGEM Registry guidelines)</td><br />
<td style="border-top:1px solid #e5e6e6;"> </td><br />
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<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Plan to present a Poster and Talk at the iGEM Jamboree </td><br />
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Your project may have implications for the environment, security, safety and ethics and/or ownership and sharing. Describe one or more ways in which these or other broader implications have been taken into consideration in the design and execution of your project. → <a href="https://2014.igem.org/Team:Toulouse/ethics" target="_blank"> Ethical Page</a></td><br />
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<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Document at least one new standard BioBrick Part or Device used in your project/central to your project and submit this part to the iGEM Registry : <a href="http://parts.igem.org/Part:BBa_K1364000" target="_blank">BBa_K1364000</a>, <a href="http://parts.igem.org/Part:BBa_K1364002"target="_blank">BBa_K1364002</a>, <a href="http://parts.igem.org/Part:BBa_K1364003"target="_blank">BBa_K1364003</a>, <a href="http://parts.igem.org/Part:BBa_K1364005"target="_blank">BBa_K1364005</a>. </td><br />
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{{:Team:Toulouse/template/footer}}</div>Jourdanhttp://2014.igem.org/Team:Toulouse/Result/achievementTeam:Toulouse/Result/achievement2014-10-17T22:50:45Z<p>Jourdan: </p>
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<h2>Achievement</h2><br />
<p>Bronze, Silver or Gold Medal? That is the question!</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Results&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Achievement</p> <br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<p class="title1" style="text-align:left;">Let's sum up what we did during this summer!</p><br />
<p class="title2">First step...</p><br />
<p class="texte"> <br />
After a summer of hard work, our team is proud of the achieved results. The first step of our project was to have a <b>proof of concept</b> of the three functions requiered to fight against canker. The three modules tested were Chemotaxis, Binding and Fungicides. The verifications of our genetic constructions were successful (PCR, Migration on an agarose gel and Sequencing). We could then proceed to specific test of the modules: capillary test, chitin beads test or fungicide tests.</p><br />
<br />
<p class="title2">Second step...</p><br />
<p class="texte"> <br />
Once these results obtained, we decided to move on to the next step: in order to get closer to our final objective, the three genetic modules have to be assembled and to be tested in <b>model plants</b>. In the short time left that we had, these tests were only performed with the antifungal module, but the results were very encouraging (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">3. In planta tests with SubtiTree</a> in the Fungicides module). <br />
<br />
<p class="title2">Third step...</p><br />
<p class="texte"> <br />
So encouraging that we moved on to the third step of our project and think about the <b>real injection of SubtiTree in diseased trees</b>. We then considered the <a href="https://2014.igem.org/Team:Toulouse/Project/Spreading"><b>spreading</b></a> problems and proposed different strategies to avoid this major issue. <a href="https://2014.igem.org/Team:Toulouse/Modelling"><b>Modeling</b></a> enables us to predict the growth and the lifespan of SubtiTree in Plane Trees. <br />
<p class="title2">And what comes next?</p><br />
<p class="texte">This project could be carried on as a thesis because the problem of canker killing plane trees is a major concern. Moreover, the support of the Ministry and VNF could help us to continue and achieve our goal: save the plane trees and preserve the beauty of the Canal du Midi.<br><br />
However, SubtiTree dissemination must be controlled, fungicide production regulated, and tests of the whole system performed. In a nutshell, we still have a lot of things to do even if we did our best during the whole summer!</p><br />
<br />
<p class="title2">iGEM Community</p><br />
<p class="texte"><br />
Well aware that we are part of the <b>iGEM Community</b>, our team also worked for this community by improving the iGEMer's Bible: <a href="http://parts.igem.org/Catalog">the Registry of Standard Biological Parts</a>. <br><br />
We improved the EcAMP BioBrick by adding a promotor, a RBS, but mainly by the addition of a STOP codon (<a href="http://parts.igem.org/Part:BBa_K1364019">BBa_K1364019</a>).<br><br />
As <i>Bacillus subtilis</i> appears to be for us an ideal chassis, we also developed new genetic tools to facilitate its utilization. Thanks to numerous interactions with other iGEM teams, we didn't see the time flying by during this <b>amazing summer</b> !</p><br />
<br />
<center><img style="width:1000px; " src="https://static.igem.org/mediawiki/parts/e/e2/Ach.jpg"></center><br />
<br><br />
<p class="title2">It is better to have "well-made rather than a well-filled head"... (Michel de Montaigne)</p><br />
<p class="texte">We developed a deep <a href="https://2014.igem.org/Team:Toulouse/ethics">ethical questioning</a>, trying to understand if we had the right to act against canker. We strongly believe that neglecting the ethical aspect in a scientific project is a mistake. All the team was involved in this part and the reflexions made were very profitable to us.</p><br />
<br />
<p class="texte">We filled the <a href="https://igem.org/2014_Judging_Form?id=1364">2014 Judging Form</a> thanks to the following accomplishments:</p><br />
<br />
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<p class="title1" style="text-align:left;">Medals Fulfillment</p> <br />
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<td style="border-bottom:4px solid #e5e6e6; border-top-left-radius:9px;"><center><br>Bronze Medal <img src="https://static.igem.org/mediawiki/2013/8/86/Medal_bronze.png" class="imgcontenttable"/></center><br></td><br />
<td style="border-bottom:4px solid #e5e6e6;"><center><br>Silver Medal <img src="https://static.igem.org/mediawiki/2013/d/dd/Medal_silver.png" class="imgcontenttable"/></center><br></td><br />
<td style="border-bottom:4px solid #e5e6e6; border-top-right-radius:9px;"><center><br>Gold Medal<img src="https://static.igem.org/mediawiki/2013/b/bd/Medal_gold_3.png" class="imgcontenttable"/></center><br></td><br />
</tr><br />
<tr><br />
<td style="border-right:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/>Register the team, have a great summer, and plan to have fun at the Jamboree</td><br />
<td style="border-right:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/>Experimentally validate that at least three new BioBrick Part or Device of your own design and construction work as expected.</td><br />
<td><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Improve the function of an existing BioBrick Part or Device and improve the <i>Bacillus subtilis</i> genetic tools, enter this information in the Registry : <a href="http://parts.igem.org/Part:BBa_K1364019"target="_blank">BBa_K1364019</a>, <a href="http://parts.igem.org/Part:BBa_K1364016"target="_blank">BBa_K1364016</a>, <a href="http://parts.igem.org/Part:BBa_K1364017"target="_blank">BBa_K1364017</a>, <a href="http://parts.igem.org/Part:BBa_K1364021"target="_blank">BBa_K1364021</a>, </td><br />
</tr><br />
<br />
<tr style="border-top:1px solid #e5e6e6"><br />
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Successfully complete and submit this iGEM Judging form </td><br />
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/>Document the characterization of these three parts in the 'Main Page' of that Part's/Device's Registry entry<br />
Submit this new part to the iGEM Parts Registry:<br> <a href="http://parts.igem.org/Part:BBa_K1364005"target="_blank">BBa_K1364005</a>, <a href="http://parts.igem.org/Part:BBa_K1364009"target="_blank">BBa_K1364009</a>, <a href="http://parts.igem.org/Part:BBa_K1364013"target="_blank">BBa_K1364013.</a></td><br />
<td style="border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Your project may have implications for the environment, security, safety and ethics and/or ownership and sharing. Describe a novel approach that your team has used to help you and others consider these aspects of the design and outcomes of synthetic biology efforts. → <a href="https://2014.igem.org/Team:Toulouse/ethics" target="_blank">Ethic Part</a> </td><br />
<br />
</tr><br />
<tr><br />
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Create and share a Description of the team’s project using the iGEM wiki and the team’s parts using the Registry Biological Parts </td><br />
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Submit these new part to the iGEM Parts Registry (submissions must adhere to the iGEM Registry guidelines)</td><br />
<td style="border-top:1px solid #e5e6e6;"> </td><br />
</tr><br />
<tr><br />
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Plan to present a Poster and Talk at the iGEM Jamboree </td><br />
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Your project may have implications for the environment, security, safety and ethics and/or ownership and sharing. Describe one or more ways in which these or other broader implications have been taken into consideration in the design and execution of your project. → <a href="https://2014.igem.org/Team:Toulouse/ethics" target="_blank"> Ethical Page</a></td><br />
<td style="border-top:1px solid #e5e6e6;"></td><br />
</tr><br />
<tr><br />
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Document at least one new standard BioBrick Part or Device used in your project/central to your project and submit this part to the iGEM Registry : <a href="http://parts.igem.org/Part:BBa_K1364000" target="_blank">BBa_K1364000</a>, <a href="http://parts.igem.org/Part:BBa_K1364002"target="_blank">BBa_K1364002</a>, <a href="http://parts.igem.org/Part:BBa_K1364003"target="_blank">BBa_K1364003</a>, <a href="http://parts.igem.org/Part:BBa_K1364005"target="_blank">BBa_K1364005</a>. </td><br />
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"></td><br />
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{{:Team:Toulouse/template/footer}}</div>Jourdanhttp://2014.igem.org/Team:Toulouse/ethicsTeam:Toulouse/ethics2014-10-17T22:03:42Z<p>Jourdan: </p>
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<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Human practice&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Ethics</p> <br />
</div> <br />
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<div id="innercontenthome"><br />
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<h3 class="title2" style="margin-top:10px; color:#333;">Summary :</h3><br />
<ul class="menuleft"><br />
<li style="margin-top:25px;"><a href="#select1">Protection of the beauty</a></li><br />
<li><a href="#select2">Human intervention in the nature</a></li><br />
<li><a href="#select3">SubtiTree</a></li><br />
</ul><br />
</div><br />
<br />
<div class="column-right" style="width:75%; float:right;"><br />
<br />
<!--CITATION--><br />
<p class="citation"><br />
"Ethics is as important as laws."<br />
</p><br />
<br />
<p class="texte">The ethical<br />
questioning turned out to be one of the major starting points of our project.<br />
Acting on an established environment and modifying it is by no mean trivial and<br />
our combined technical and philosophical points of view. The actual purpose of our project also leads us to undertake an<br />
ethical questioning about the role of the scientist regarding “useless” things<br />
such as the trees lining along the Canal du Midi. </p><br />
<br />
<p class="title1" id="select1">Protection of the beauty</p><br />
<p class="title2">Is it the scientists’ role to protect beauty?<br />
</p><br />
<br />
<p class="texte"> Beauty is a<br />
feeling of satisfaction and is selfless. It is more a feeling than the property<br />
of a thing, this is not a notion we can clearly understand. Indeed, we can find<br />
something beautiful even when we don’t know the purpose of the object...</p><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2014/f/fa/Fontaine_Duchamp.jpg" width="400px"><br />
<p class="legend">Figure 1: Fontaine (Marcel Duchamp). Yes this is also art...</p></center><br />
<br />
<p class="texte">There is always<br />
a distinction between natural beauty and artistic beauty according to Hegel,<br />
the famous philosopher. The artistic beauty is born from our mind and our<br />
spirit: it is an element of signification of the work of art whereas the<br />
natural beauty of the object is external. In a way, the Canal du Midi combines<br />
both types of beauty: a natural one regarding the Nature, the centenary plane<br />
trees but also an artistic one since the Canal was built by the human hands.<br />
Usually, science judges beauty as a superficial feature not deserving to<br />
undertake any kind of scientific efforts to maintain it. The traditional role<br />
of science is to solve global issues and to elaborate complex strategies in<br />
order to find useful solutions for everyone’s life. Once made this observation,<br />
one may wonder why synthetic biology would be used only to protect the useless<br />
beauty of a local heritage such as the trees lining the Canal du Midi. <br />
</p><br />
<br />
<p class="texte"><br />
This crucial<br />
interrogation leads us to consider science and synthetic biology from a<br />
different point of view. <b>What if the role of scientists was also to make<br />
people rediscovering the beauty of Nature? What if the bases of new scientific<br />
challenges resulted from a more local scale? </b>Science does not have to be it has so much to gain opening itself to these<br />
challenges. First scientifically, as research is never<br />
useless and as we never know the impact and the scope of our results.<br />
class=GramE>Then socially, as we could measure the deep interest raised by our<br />
project within the population and the media. Adopting a new vision of synthetic<br />
biology, we will probably make people change their mind about this innovative<br />
discipline. <br><br />
The traditional cold objectivity of science distances itself from the society.<br />
However, scientists are also being capable of feeling the beauty, sensitive to<br />
the charm of landscapes and <b>able to understand the usefulness of<br />
"useless" trees</b>…<br><br />
The design of a strategy to protect useless beauty may seem senseless but we<br />
believe that it is also the scientist’s duty. We have to remember that thinking<br />
is what distinguish <i>Homo sapiens</i> from other species on earth and this<br />
"thinking" feature allows us to understand the world and be conscious<br />
of our human Nature (Descartes: <i>Cogito ergo sum</i>). The art is an object<br />
of philosophical thought. Consciousness raises humans above all others living<br />
creatures. Thus, it is necessary to respect and protect art. And thus, it<br />
becomes essential to preserve the beauty of the Canal du Midi. <br />
</p><br />
<br />
<br />
<p class="title1" id="select2">Human<br />
intervention on Nature</p><br />
<p class="texte">Our main<br />
question is to understand the complicated relationship between man and Nature.<br />
Does mankind have the proper right to change the Nature? Is modified Nature<br />
considered as artificial?</p><br />
<p class="title2"> Mankind & Nature</p><br />
<br />
<p class="texte"> Nature deserves<br />
to be respected and loved. Mankind has always been linked to Nature as its<br />
survival depends on what comes out of the ground, the trees, the oceans… The<br />
Nature is a source of wealth for mankind. It ensures survival and development<br />
by giving men the wood, the rocks, the soil to build shelters. Being in contact<br />
with Nature can allow men to feel strong emotion, as describe by poets like<br />
Hugo and Lamartine.</p><br />
<br />
<br />
<p class="texte">Since the birth<br />
of humanity, man himself understood the importance of studying and mastering<br />
Nature to develop the civilization. Still today the most advanced technologies<br />
often try to mimic natural phenomena. With the development of civilization, men<br />
modified their environment, changing it for their own comfort depending on<br />
their own desire. With the increase of human activity, the natural environment<br />
is modified profundly. With industrialization, the<br />
natural environment suffered from waste discharges, oil slicks, intensive<br />
fishing (and many others...) but also the introduction of devastating species<br />
such as the pathogen,<i> Ceratocystis<br />
platani</i>. However, despite these negative aspects, men<br />
are capable of favorable actions to help the environment and fix their<br />
mistakes. The current trend is to limit the impact of human interventions on<br />
Nature, and hopefully this trend is not transient and will not vanish. A new<br />
desire is born, a wish to protect Nature and wilderness. Humanity can adhere to<br />
this position: human take advantage of the<br />
environment and the environment takes advantage of the reasoned human<br />
interventions. There is an adaptation of mankind to Nature. Moreover, humans<br />
can have empathy: people are capable of understanding emotions and cognitive<br />
states of other organisms. To respond to these feelings, humans have<br />
technological tools allowing them to fight against enemies. This is the case<br />
with our project: fighting <i>Ceratocystis<br />
platani</i>. <br />
</p><br />
<br />
<p class="texte">In conclusion,<br />
by destroying and hammering the Nature, we jeopardize our lives. We need<br />
Nature, we come from Nature and we depend on Nature for survival, food,<br />
discoveries and civilisation. Respecting, loving and<br />
preserving the beauty of it is also a question of<br />
survival.</p><br />
<br />
<p class="title2">Nature and artifice<br />
</p><br />
<br />
<p class="texte">Talking about<br />
the Nature refers to the whole world with an exception: all the transformations<br />
made by mankind. Nature exists regardless of men and his interventions whereas<br />
artificial is everything that exists because to humans.<br />
</p><br />
<br />
<p class="texte">However,<br />
pretending that natural and artificial are opposite does not seem to be true.<br />
Man cannot create without the various elements provided by Nature, he then justs transform Nature. Thus we may wonder if there is a<br />
true difference between natural and artificial. The border between these two<br />
notions is not as obvious as it seems. The landscapes are shaped by the hand of<br />
man, animals are domesticated, and now bacteria are considered as cell<br />
factories. A natural reserve is artificially preserved as a result of human<br />
actions. Is there still something natural since the birth of mankind? Actually,<br />
the artifice is a slight modification of Nature and couldn’t exist by itself.<br />
The distinction between natural and artificial seems sterile and we clearly<br />
understand that these notions are inextricably linked and need each other to<br />
exist. </p><br />
<br />
<p class="texte">In conclusion,<br />
isn't it our duty to use our unique position in the history of life and our human<br />
approach to try to replace the evolutive processes?</p><br />
<br />
<br />
<p class="title2">Back to our project</p><br />
<br />
<br />
<p class="texte">These<br />
inextricable links are obviously the basis of our project. We aim to<br />
artificially preserve a natural heritage shaped by Pierre Paul Riquet hundreds years ago.Fighting a<br />
naturally occurring form of life that threatens it maybe just an imitation of<br />
the natural evolution process. What is considered today as ‘non-natural’<br />
may be one day regarded differently. To the extent that everything is done not<br />
to unbalance the ecosystem, our intervention can be judged rightful, even more<br />
than the use of chemicals.</p><br />
<br />
<br />
<p class="title1" id="select3">SubtiTree</p><br />
<br />
<p class="title2"> Potential strategies discussed<br />
<br> (See more details in the <a href="https://2014.igem.org/Team:Toulouse/Project/Spreading">Spreading</a> dedicated page)<br />
</p><br />
<br />
<p class="texte">To be sure that<br />
SubtiTree will not survive and spread in the<br />
environment, many strategies were discussed to improve our bacterium: <br />
<br />
<br>- Avoid the survival in the natural environment (outside the tree) thanks to a proline auxotrophy system <br />
<br>- Prevent the sporulation of <i>B. subtilis</i> to make it annual <br />
<br>- Avoid gene transfers between SubtiTree and a wild<br />
type bacterium thanks to a toxin-antitoxin system <br />
<br>- Use an integrative plasmid to improve the genetic stability<br />
</p><br />
<br />
<br />
<br />
<p class="title2">Public perception<br />
</p><br />
<br />
<p class="texte"><I><CENTER>Political and public adhesion</I></CENTER></p><br />
<br />
<p class="texte">Due to our<br />
strong implication in preserving this magnificent work of art, our project<br />
interested several governmental services. Indeed some municipalities and<br />
regional councils supported our local engagement. Beyond that, our project<br />
interests the highest level of the “Canal du Midi” administration: the national<br />
navigation authority (VNF) and the Ministry of agriculture. Both of them funded<br />
this project. They are now looking for the continuation of the project after<br />
the iGEM competition. This is clearly a sign that we targeted the right<br />
question. </p><br />
<br />
<p class="texte">This project<br />
also received the attention of the public through several articles in<br />
newspapers, television, radio and internet. First we had just a local coverage,<br />
but days after days there were more and more media interested in SubtiTree. This mediatic coverage<br />
allowed us to contact concerned citizens who participated to the development of<br />
this project. This interaction with the public allowed us to explain and<br />
promote public knowledge of synthetic biology. </p><br />
<br />
<br />
<p class="texte"><I><CENTER>Safety principle</I></CENTER></p><br />
<br />
<p class="texte">One single tree<br />
infected by Canker, and all the trees located in an area of a couple of hundred<br />
meters around are included in the prophylactic cut. We acted to preserve the<br />
surrounding trees. The modification of the endophytic<br />
microbial fauna generated by the introduction of the engineered bacterium has<br />
to be compared to the introduction of chemicals. They contain chlorine atom and<br />
aromatic hydrocarbon, so their remediation is complicated and they represent a<br />
source of pollution. By shortening the lifespan to one season and minimizing<br />
the risks of spreading, we plan a safe and environmental-friendly way to fight<br />
Canker. <br />
<br />
<br />
<p class="title2">Feasability<br />
</p><br />
<br />
<p class="texte">We wonder about<br />
the feasibility of tree’s treatment. As we used endophytic<br />
bacteria, we can count on the natural growth of SubtiTree<br />
inside the sap. So we can inject few bacteria to be sure to have enough<br />
bacteria to protect the tree. Some researchers (Xianling<br />
Ji<sup>1</sup> et al) already injected <i>Bacillus subtilis</i> in plants and<br />
observe an increase of bacteria concentration to a maximum of 10<sup>5</sup><br />
bacteria/mL</p><br />
<br />
<p class="texte">As we aim to<br />
inject a small quantity of bacteria, this treatment remains cheaper than the<br />
injection of several liters of chemical fungicides. In addition, this injection<br />
prevents the preventive tree cutting, which is very expensive. Cutting one tree<br />
cost around € 3000. The administration in charge of the protection of the<br />
“Canal du Midi” already plans to spend 220 million euros to cut and replant all<br />
trees along the Canal. Besides the important cost of cutting trees, it will<br />
destroy one of the symbols of south-western France. </p><br />
<br />
<p class="texte">We know that SubtiTree could be improved in many ways, but in the <br />
iGEM’s circumstances we could not have the time to go<br />
deeper. First, we can improve the fixation module. Using chitin as fixation<br />
anchor is simple but not enough specific to fix just one fungus type. That’s<br />
why we first think to fix SubtiTree to one protein<br />
included in the <i>Ceratocystis<br />
platani</i>’s<br />
membrane: CP. The bacterial prototype designed this summer can be optimized to<br />
trigger the fungicides production when the binding is completed, and to be more<br />
specific changing the peptides produced.</p><br />
<br />
<p class="title1">References</p><br />
<br />
<li class="tree"><p class="texte"> Xianling Ji, Guobing Lu, Yingping Gai, Chengchao Zheng & Zhimei Mu.<b> Biological control against bacterialwilt and colonization of<br />
mulberry byan endophyticBacillus subtilis strain </b>. FEMS Microbiol Ecol. 65 (2008) 565–573. </p></li><br />
</div><br />
<br />
<div class="clear"></div><br />
<br />
<br />
</div><br />
</div><br />
</body><br />
</html><br />
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<!-------------------------------- FOOTER ---------------------------------><br />
{{:Team:Toulouse/template/footer}}</div>Jourdanhttp://2014.igem.org/Team:Toulouse/ethicsTeam:Toulouse/ethics2014-10-17T22:01:46Z<p>Jourdan: </p>
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<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Human practice&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Ethics</p> <br />
</div> <br />
</div><br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<div id="column-left"><br />
<h3 class="title2" style="margin-top:10px; color:#333;">Summary :</h3><br />
<ul class="menuleft"><br />
<li style="margin-top:25px;"><a href="#select1">Protection of the beauty</a></li><br />
<li><a href="#select2">Human intervention in the nature</a></li><br />
<li><a href="#select3">SubtiTree</a></li><br />
</ul><br />
</div><br />
<br />
<div class="column-right" style="width:75%; float:right;"><br />
<br />
<!--CITATION--><br />
<p class="citation"><br />
"Ethics is as important as laws."<br />
</p><br />
<br />
<p class="texte">The ethical<br />
questioning turned out to be one of the major starting points of our project.<br />
Acting on an established environment and modifying it is by no mean trivial and<br />
our combined technical and philosophical points of view. The actual purpose of our project also leads us to undertake an<br />
ethical questioning about the role of the scientist regarding “useless” things<br />
such as the trees lining along the Canal du Midi. </p><br />
<br />
<p class="title1" id="select1">Protection of the beauty</p><br />
<p class="title2">Is it the scientists’ role to protect beauty?<br />
</p><br />
<br />
<p class="texte"> Beauty is a<br />
feeling of satisfaction and is selfless. It is more a feeling than the property<br />
of a thing, this is not a notion we can clearly understand. Indeed, we can find<br />
something beautiful even when we don’t know the purpose of the object...</p><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2014/f/fa/Fontaine_Duchamp.jpg" width="400px"><br />
<p class="legend">Figure 1: Fontaine (Marcel Duchamp). Yes this is also art...</p></center><br />
<br />
<p class="texte">There is always<br />
a distinction between natural beauty and artistic beauty according to Hegel,<br />
the famous philosopher. The artistic beauty is born from our mind and our<br />
spirit: it is an element of signification of the work of art whereas the<br />
natural beauty of the object is external. In a way, the Canal du Midi combines<br />
both types of beauty: a natural one regarding the Nature, the centenary plane<br />
trees but also an artistic one since the Canal was built by the human hands.<br />
Usually, science judges beauty as a superficial feature not deserving to<br />
undertake any kind of scientific efforts to maintain it. The traditional role<br />
of science is to solve global issues and to elaborate complex strategies in<br />
order to find useful solutions for everyone’s life. Once made this observation,<br />
one may wonder why synthetic biology would be used only to protect the useless<br />
beauty of a local heritage such as the trees lining the Canal du Midi. <br />
</p><br />
<br />
<p class="texte"><br />
This crucial<br />
interrogation leads us to consider science and synthetic biology from a<br />
different point of view. <b>What if the role of scientists was also to make<br />
people rediscovering the beauty of Nature? What if the bases of new scientific<br />
challenges resulted from a more local scale? </b>Science does not have to be it has so much to gain opening itself to these<br />
challenges. First scientifically, as research is never<br />
useless and as we never know the impact and the scope of our results.<br />
class=GramE>Then socially, as we could measure the deep interest raised by our<br />
project within the population and the media. Adopting a new vision of synthetic<br />
biology, we will probably make people change their mind about this innovative<br />
discipline. <br><br />
The traditional cold objectivity of science distances itself from the society.<br />
However, scientists are also being capable of feeling the beauty, sensitive to<br />
the charm of landscapes and <b>able to understand the usefulness of<br />
"useless" trees</b>…<br><br />
The design of a strategy to protect useless beauty may seem senseless but we<br />
believe that it is also the scientist’s duty. We have to remember that thinking<br />
is what distinguish <i>Homo sapiens</i> from other species on earth and this<br />
"thinking" feature allows us to understand the world and be conscious<br />
of our human Nature (Descartes: <i>Cogito ergo sum</i>). The art is an object<br />
of philosophical thought. Consciousness raises humans above all others living<br />
creatures. Thus, it is necessary to respect and protect art. And thus, it<br />
becomes essential to preserve the beauty of the Canal du Midi. <br />
</p><br />
<br />
<br />
<p class="title1" id="select2">Human<br />
intervention on Nature</p><br />
<p class="texte">Our main<br />
question is to understand the complicated relationship between man and Nature.<br />
Does mankind have the proper right to change the Nature? Is modified Nature<br />
considered as artificial?</p><br />
<p class="title2"> Mankind & Nature</p><br />
<br />
<p class="texte"> Nature deserves<br />
to be respected and loved. Mankind has always been linked to Nature as its<br />
survival depends on what comes out of the ground, the trees, the oceans… The<br />
Nature is a source of wealth for mankind. It ensures survival and development<br />
by giving men the wood, the rocks, the soil to build shelters. Being in contact<br />
with Nature can allow men to feel strong emotion, as describe by poets like<br />
Hugo and Lamartine.</p><br />
<br />
<br />
<p class="texte">Since the birth<br />
of humanity, man himself understood the importance of studying and mastering<br />
Nature to develop the civilization. Still today the most advanced technologies<br />
often try to mimic natural phenomena. With the development of civilization, men<br />
modified their environment, changing it for their own comfort depending on<br />
their own desire. With the increase of human activity, the natural environment<br />
is modified profundly. With industrialization, the<br />
natural environment suffered from waste discharges, oil slicks, intensive<br />
fishing (and many others...) but also the introduction of devastating species<br />
such as the pathogen,<i> Ceratocystis<br />
platani</i>. However, despite these negative aspects, men<br />
are capable of favorable actions to help the environment and fix their<br />
mistakes. The current trend is to limit the impact of human interventions on<br />
Nature, and hopefully this trend is not transient and will not vanish. A new<br />
desire is born, a wish to protect Nature and wilderness. Humanity can adhere to<br />
this position: human take advantage of the<br />
environment and the environment takes advantage of the reasoned human<br />
interventions. There is an adaptation of mankind to Nature. Moreover, humans<br />
can have empathy: people are capable of understanding emotions and cognitive<br />
states of other organisms. To respond to these feelings, humans have<br />
technological tools allowing them to fight against enemies. This is the case<br />
with our project: fighting <i>Ceratocystis<br />
platani</i>. <br />
</p><br />
<br />
<p class="texte">In conclusion,<br />
by destroying and hammering the Nature, we jeopardize our lives. We need<br />
Nature, we come from Nature and we depend on Nature for survival, food,<br />
discoveries and civilisation. Respecting, loving and<br />
preserving the beauty of it is also a question of<br />
survival.</p><br />
<br />
<p class="title2">Nature and artifice<br />
</p><br />
<br />
<p class="texte">Talking about<br />
the Nature refers to the whole world with an exception: all the transformations<br />
made by mankind. Nature exists regardless of men and his interventions whereas<br />
artificial is everything that exists because to humans.<br />
</p><br />
<br />
<p class="texte">However,<br />
pretending that natural and artificial are opposite does not seem to be true.<br />
Man cannot create without the various elements provided by Nature, he then justs transform Nature. Thus we may wonder if there is a<br />
true difference between natural and artificial. The border between these two<br />
notions is not as obvious as it seems. The landscapes are shaped by the hand of<br />
man, animals are domesticated, and now bacteria are considered as cell<br />
factories. A natural reserve is artificially preserved as a result of human<br />
actions. Is there still something natural since the birth of mankind? Actually,<br />
the artifice is a slight modification of Nature and couldn’t exist by itself.<br />
The distinction between natural and artificial seems sterile and we clearly<br />
understand that these notions are inextricably linked and need each other to<br />
exist. </p><br />
<br />
<p class="texte">In conclusion,<br />
isn't it our duty to use our unique position in the history of life and our human<br />
approach to try to replace the evolutive processes?</p><br />
<br />
<br />
<p class="title2">Back to our project</p><br />
<br />
<br />
<p class="texte">These<br />
inextricable links are obviously the basis of our project. We aim to<br />
artificially preserve a natural heritage shaped by Pierre Paul Riquet hundreds years ago.Fighting a<br />
naturally occurring form of life that threatens it maybe just an imitation of<br />
the natural evolution process. What is considered today as ‘non-natural’<br />
may be one day regarded differently. To the extent that everything is done not<br />
to unbalance the ecosystem, our intervention can be judged rightful, even more<br />
than the use of chemicals.</p><br />
<br />
<br />
<p class="title1" id="select3">SubtiTree</p><br />
<br />
<p class="title2"> Potential strategies discussed<br />
<br> (See more details in the <a href="https://2014.igem.org/Team:Toulouse/Project/Spreading">Spreading</a> dedicated page)<br />
</p><br />
<br />
<p class="texte">To be sure that<br />
SubtiTree will not survive and spread in the<br />
environment, many strategies were discussed to improve our bacterium: <br />
<br />
<br>- Avoid the survival in the natural environment (outside the tree) thanks to a proline auxotrophy system <br />
<br>- Prevent the sporulation of <i>B. subtilis</i> to make it annual <br />
<br>- Avoid gene transfers between SubtiTree and a wild<br />
type bacterium thanks to a toxin-antitoxin system <br />
<br>- Use an integrative plasmid to improve the genetic stability<br />
</p><br />
<br />
<br />
<br />
<p class="title2">Public perception<br />
</p><br />
<br />
<p class="texte"><I><CENTER>Political and public adhesion</I></CENTER></p><br />
<br />
<p class="texte">Due to our<br />
strong implication in preserving this magnificent work of art, our project<br />
interested several governmental services. Indeed some municipalities and<br />
regional councils supported our local engagement. Beyond that, our project<br />
interests the highest level of the “Canal du Midi” administration: the national<br />
navigation authority (VNF) and the Ministry of agriculture. Both of them funded<br />
this project. They are now looking for the continuation of the project after<br />
the iGEM competition. This is clearly a sign that we targeted the right<br />
question. </p><br />
<br />
<p class="texte">This project<br />
also received the attention of the public through several articles in<br />
newspapers, television, radio and internet. First we had just a local coverage,<br />
but days after days there were more and more media interested in SubtiTree. This mediatic coverage<br />
allowed us to contact concerned citizens who participated to the development of<br />
this project. This interaction with the public allowed us to explain and<br />
promote public knowledge of synthetic biology. </p><br />
<br />
<br />
<p class="texte"><I><CENTER>Safety principle</I></CENTER></p><br />
<br />
<p class="texte">One single tree<br />
infected by Canker, and all the trees located in an area of a couple of hundred<br />
meters around are included in the prophylactic cut. We acted to preserve the<br />
surrounding trees. The modification of the endophytic<br />
microbial fauna generated by the introduction of the engineered bacterium has<br />
to be compared to the introduction of chemicals. They contain chlorine atom and<br />
aromatic hydrocarbon, so their remediation is complicated and they represent a<br />
source of pollution. By shortening the lifespan to one season and minimizing<br />
the risks of spreading, we plan a safe and environmental-friendly way to fight<br />
Canker. <br />
<br />
<br />
<p class="title2">Feasability<br />
</p><br />
<br />
<p class="texte">We wonder about<br />
the feasibility of tree’s treatment. As we used endophytic<br />
bacteria, we can count on the natural growth of SubtiTree<br />
inside the sap. So we can inject few bacteria to be sure to have enough<br />
bacteria to protect the tree. Some researchers (Xianling<br />
Ji<sup>1</sup> et al) already injected <i>Bacillus subtilis</i> in plants and<br />
observe an increase of bacteria concentration to a maximum of 10<sup>5</sup><br />
bacteria/mL</p><br />
<br />
<p class="texte">As we aim to<br />
inject a small quantity of bacteria, this treatment remains cheaper than the<br />
injection of several liters of chemical fungicides. In addition, this injection<br />
prevents the preventive tree cutting, which is very expensive. Cutting one tree<br />
cost around € 3000. The administration in charge of the protection of the<br />
“Canal du Midi” already plans to spend 220 million euros to cut and replant all<br />
trees along the Canal. Besides the important cost of cutting trees, it will<br />
destroy one of the symbols of south-western France. </p><br />
<br />
<p class="texte">We know that SubtiTree could be improved in many ways, but in the <br />
iGEM’s circumstances we could not have the time to go<br />
deeper. First, we can improve the fixation module. Using chitin as fixation<br />
anchor is simple but not enough specific to fix just one fungus type. That’s<br />
why we first think to fix SubtiTree to one protein<br />
included in the <i>Ceratocystis<br />
platani</i>’s<br />
membrane: CP. The bacterial prototype designed this summer can be optimized to<br />
trigger the fungicides production when the binding is completed, and to be more<br />
specific changing the peptides produced.</p><br />
<br />
</div><br />
<p class="title1">References</p><br />
<br />
<li class="tree"><p class="texte"> Xianling Ji, Guobing Lu, Yingping Gai, Chengchao Zheng & Zhimei Mu.<b> Biological control against bacterialwilt and colonization of<br />
mulberry byan endophyticBacillus subtilis strain </b>. FEMS Microbiol Ecol. 65 (2008) 565–573. </p></li><br />
<br />
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{{:Team:Toulouse/template/footer}}</div>Jourdanhttp://2014.igem.org/Team:Toulouse/ethicsTeam:Toulouse/ethics2014-10-17T22:00:17Z<p>Jourdan: </p>
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<div style="margin:0 auto; width:960px;"><br />
<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Human practice&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Ethics</p> <br />
</div> <br />
</div><br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<div id="column-left"><br />
<h3 class="title2" style="margin-top:10px; color:#333;">Summary :</h3><br />
<ul class="menuleft"><br />
<li style="margin-top:25px;"><a href="#select1">Protection of the beauty</a></li><br />
<li><a href="#select2">Human intervention in the nature</a></li><br />
<li><a href="#select3">SubtiTree</a></li><br />
</ul><br />
</div><br />
<br />
<div class="column-right" style="width:75%; float:right;"><br />
<br />
<!--CITATION--><br />
<p class="citation"><br />
"Ethics is as important as laws."<br />
</p><br />
<br />
<p class="texte">The ethical<br />
questioning turned out to be one of the major starting points of our project.<br />
Acting on an established environment and modifying it is by no mean trivial and<br />
our combined technical and philosophical points of view. The actual purpose of our project also leads us to undertake an<br />
ethical questioning about the role of the scientist regarding “useless” things<br />
such as the trees lining along the Canal du Midi. </p><br />
<br />
<p class="title1" id="select1">Protection of the beauty</p><br />
<p class="title2">Is it the scientists’ role to protect beauty?<br />
</p><br />
<br />
<p class="texte"> Beauty is a<br />
feeling of satisfaction and is selfless. It is more a feeling than the property<br />
of a thing, this is not a notion we can clearly understand. Indeed, we can find<br />
something beautiful even when we don’t know the purpose of the object...</p><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2014/f/fa/Fontaine_Duchamp.jpg" width="400px"><br />
<p class="legend">Figure 1: Fontaine (Marcel Duchamp). Yes this is also art...</p></center><br />
<br />
<p class="texte">There is always<br />
a distinction between natural beauty and artistic beauty according to Hegel,<br />
the famous philosopher. The artistic beauty is born from our mind and our<br />
spirit: it is an element of signification of the work of art whereas the<br />
natural beauty of the object is external. In a way, the Canal du Midi combines<br />
both types of beauty: a natural one regarding the Nature, the centenary plane<br />
trees but also an artistic one since the Canal was built by the human hands.<br />
Usually, science judges beauty as a superficial feature not deserving to<br />
undertake any kind of scientific efforts to maintain it. The traditional role<br />
of science is to solve global issues and to elaborate complex strategies in<br />
order to find useful solutions for everyone’s life. Once made this observation,<br />
one may wonder why synthetic biology would be used only to protect the useless<br />
beauty of a local heritage such as the trees lining the Canal du Midi. <br />
</p><br />
<br />
<p class="texte"><br />
This crucial<br />
interrogation leads us to consider science and synthetic biology from a<br />
different point of view. <b>What if the role of scientists was also to make<br />
people rediscovering the beauty of Nature? What if the bases of new scientific<br />
challenges resulted from a more local scale? </b>Science does not have to be it has so much to gain opening itself to these<br />
challenges. First scientifically, as research is never<br />
useless and as we never know the impact and the scope of our results.<br />
class=GramE>Then socially, as we could measure the deep interest raised by our<br />
project within the population and the media. Adopting a new vision of synthetic<br />
biology, we will probably make people change their mind about this innovative<br />
discipline. <br><br />
The traditional cold objectivity of science distances itself from the society.<br />
However, scientists are also being capable of feeling the beauty, sensitive to<br />
the charm of landscapes and <b>able to understand the usefulness of<br />
"useless" trees</b>…<br><br />
The design of a strategy to protect useless beauty may seem senseless but we<br />
believe that it is also the scientist’s duty. We have to remember that thinking<br />
is what distinguish <i>Homo sapiens</i> from other species on earth and this<br />
"thinking" feature allows us to understand the world and be conscious<br />
of our human Nature (Descartes: <i>Cogito ergo sum</i>). The art is an object<br />
of philosophical thought. Consciousness raises humans above all others living<br />
creatures. Thus, it is necessary to respect and protect art. And thus, it<br />
becomes essential to preserve the beauty of the Canal du Midi. <br />
</p><br />
<br />
<br />
<p class="title1" id="select2">Human<br />
intervention on Nature</p><br />
<p class="texte">Our main<br />
question is to understand the complicated relationship between man and Nature.<br />
Does mankind have the proper right to change the Nature? Is modified Nature<br />
considered as artificial?</p><br />
<p class="title2"> Mankind & Nature</p><br />
<br />
<p class="texte"> Nature deserves<br />
to be respected and loved. Mankind has always been linked to Nature as its<br />
survival depends on what comes out of the ground, the trees, the oceans… The<br />
Nature is a source of wealth for mankind. It ensures survival and development<br />
by giving men the wood, the rocks, the soil to build shelters. Being in contact<br />
with Nature can allow men to feel strong emotion, as describe by poets like<br />
Hugo and Lamartine.</p><br />
<br />
<br />
<p class="texte">Since the birth<br />
of humanity, man himself understood the importance of studying and mastering<br />
Nature to develop the civilization. Still today the most advanced technologies<br />
often try to mimic natural phenomena. With the development of civilization, men<br />
modified their environment, changing it for their own comfort depending on<br />
their own desire. With the increase of human activity, the natural environment<br />
is modified profundly. With industrialization, the<br />
natural environment suffered from waste discharges, oil slicks, intensive<br />
fishing (and many others...) but also the introduction of devastating species<br />
such as the pathogen,<i> Ceratocystis<br />
platani</i>. However, despite these negative aspects, men<br />
are capable of favorable actions to help the environment and fix their<br />
mistakes. The current trend is to limit the impact of human interventions on<br />
Nature, and hopefully this trend is not transient and will not vanish. A new<br />
desire is born, a wish to protect Nature and wilderness. Humanity can adhere to<br />
this position: human take advantage of the<br />
environment and the environment takes advantage of the reasoned human<br />
interventions. There is an adaptation of mankind to Nature. Moreover, humans<br />
can have empathy: people are capable of understanding emotions and cognitive<br />
states of other organisms. To respond to these feelings, humans have<br />
technological tools allowing them to fight against enemies. This is the case<br />
with our project: fighting <i>Ceratocystis<br />
platani</i>. <br />
</p><br />
<br />
<p class="texte">In conclusion,<br />
by destroying and hammering the Nature, we jeopardize our lives. We need<br />
Nature, we come from Nature and we depend on Nature for survival, food,<br />
discoveries and civilisation. Respecting, loving and<br />
preserving the beauty of it is also a question of<br />
survival.</p><br />
<br />
<p class="title2">Nature and artifice<br />
</p><br />
<br />
<p class="texte">Talking about<br />
the Nature refers to the whole world with an exception: all the transformations<br />
made by mankind. Nature exists regardless of men and his interventions whereas<br />
artificial is everything that exists because to humans.<br />
</p><br />
<br />
<p class="texte">However,<br />
pretending that natural and artificial are opposite does not seem to be true.<br />
Man cannot create without the various elements provided by Nature, he then justs transform Nature. Thus we may wonder if there is a<br />
true difference between natural and artificial. The border between these two<br />
notions is not as obvious as it seems. The landscapes are shaped by the hand of<br />
man, animals are domesticated, and now bacteria are considered as cell<br />
factories. A natural reserve is artificially preserved as a result of human<br />
actions. Is there still something natural since the birth of mankind? Actually,<br />
the artifice is a slight modification of Nature and couldn’t exist by itself.<br />
The distinction between natural and artificial seems sterile and we clearly<br />
understand that these notions are inextricably linked and need each other to<br />
exist. </p><br />
<br />
<p class="texte">In conclusion,<br />
isn't it our duty to use our unique position in the history of life and our human<br />
approach to try to replace the evolutive processes?</p><br />
<br />
<br />
<p class="title2">Back to our project</p><br />
<br />
<br />
<p class="texte">These<br />
inextricable links are obviously the basis of our project. We aim to<br />
artificially preserve a natural heritage shaped by Pierre Paul Riquet hundreds years ago.Fighting a<br />
naturally occurring form of life that threatens it maybe just an imitation of<br />
the natural evolution process. What is considered today as ‘non-natural’<br />
may be one day regarded differently. To the extent that everything is done not<br />
to unbalance the ecosystem, our intervention can be judged rightful, even more<br />
than the use of chemicals.</p><br />
<br />
<br />
<p class="title1" id="select3">SubtiTree</p><br />
<br />
<p class="title2"> Potential strategies discussed<br />
<br> (See more details in the <a href="https://2014.igem.org/Team:Toulouse/Project/Spreading">Spreading</a> dedicated page)<br />
</p><br />
<br />
<p class="texte">To be sure that<br />
SubtiTree will not survive and spread in the<br />
environment, many strategies were discussed to improve our bacterium: <br />
<br />
<br>- Avoid the survival in the natural environment (outside the tree) thanks to a proline auxotrophy system <br />
<br>- Prevent the sporulation of <i>B. subtilis</i> to make it annual <br />
<br>- Avoid gene transfers between SubtiTree and a wild<br />
type bacterium thanks to a toxin-antitoxin system <br />
<br>- Use an integrative plasmid to improve the genetic stability<br />
</p><br />
<br />
<br />
<br />
<p class="title2">Public perception<br />
</p><br />
<br />
<p class="texte"><I><CENTER>Political and public adhesion</I></CENTER></p><br />
<br />
<p class="texte">Due to our<br />
strong implication in preserving this magnificent work of art, our project<br />
interested several governmental services. Indeed some municipalities and<br />
regional councils supported our local engagement. Beyond that, our project<br />
interests the highest level of the “Canal du Midi” administration: the national<br />
navigation authority (VNF) and the Ministry of agriculture. Both of them funded<br />
this project. They are now looking for the continuation of the project after<br />
the iGEM competition. This is clearly a sign that we targeted the right<br />
question. </p><br />
<br />
<p class="texte">This project<br />
also received the attention of the public through several articles in<br />
newspapers, television, radio and internet. First we had just a local coverage,<br />
but days after days there were more and more media interested in SubtiTree. This mediatic coverage<br />
allowed us to contact concerned citizens who participated to the development of<br />
this project. This interaction with the public allowed us to explain and<br />
promote public knowledge of synthetic biology. </p><br />
<br />
<br />
<p class="texte"><I><CENTER>Safety principle</I></CENTER></p><br />
<br />
<p class="texte">One single tree<br />
infected by Canker, and all the trees located in an area of a couple of hundred<br />
meters around are included in the prophylactic cut. We acted to preserve the<br />
surrounding trees. The modification of the endophytic<br />
microbial fauna generated by the introduction of the engineered bacterium has<br />
to be compared to the introduction of chemicals. They contain chlorine atom and<br />
aromatic hydrocarbon, so their remediation is complicated and they represent a<br />
source of pollution. By shortening the lifespan to one season and minimizing<br />
the risks of spreading, we plan a safe and environmental-friendly way to fight<br />
Canker. <br />
<br />
<br />
<p class="title2">Feasability<br />
</p><br />
<br />
<p class="texte">We wonder about<br />
the feasibility of tree’s treatment. As we used endophytic<br />
bacteria, we can count on the natural growth of SubtiTree<br />
inside the sap. So we can inject few bacteria to be sure to have enough<br />
bacteria to protect the tree. Some researchers (Xianling<br />
Ji<sup>1</sup> et al) already injected <i>Bacillus subtilis</i> in plants and<br />
observe an increase of bacteria concentration to a maximum of 10<sup>5</sup><br />
bacteria/mL</p><br />
<br />
<p class="texte">As we aim to<br />
inject a small quantity of bacteria, this treatment remains cheaper than the<br />
injection of several liters of chemical fungicides. In addition, this injection<br />
prevents the preventive tree cutting, which is very expensive. Cutting one tree<br />
cost around € 3000. The administration in charge of the protection of the<br />
“Canal du Midi” already plans to spend 220 million euros to cut and replant all<br />
trees along the Canal. Besides the important cost of cutting trees, it will<br />
destroy one of the symbols of south-western France. </p><br />
<br />
<p class="texte">We know that SubtiTree could be improved in many ways, but in the <br />
iGEM’s circumstances we could not have the time to go<br />
deeper. First, we can improve the fixation module. Using chitin as fixation<br />
anchor is simple but not enough specific to fix just one fungus type. That’s<br />
why we first think to fix SubtiTree to one protein<br />
included in the <i>Ceratocystis<br />
platani</i>’s<br />
membrane: CP. The bacterial prototype designed this summer can be optimized to<br />
trigger the fungicides production when the binding is completed, and to be more<br />
specific changing the peptides produced.</p><br />
<br />
</div><br />
<p class="title1">References</p><br />
<br />
<ul><br />
<li class="tree"><p class="texte"> Xianling Ji, Guobing Lu, Yingping Gai, Chengchao Zheng & Zhimei Mu.<b> Biological control against bacterialwilt and colonization of<br />
mulberry byan endophyticBacillus subtilis strain </b>. FEMS Microbiol Ecol. 65 (2008) 565–573. </p></li><br />
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{{:Team:Toulouse/template/footer}}</div>Jourdanhttp://2014.igem.org/Team:Toulouse/TeamTeam:Toulouse/Team2014-10-17T21:50:44Z<p>Jourdan: </p>
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<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Team&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Our Team</p> <br />
<ul class="topnav" id="topnav" style="top:15px;"><br />
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<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<br />
<p class="texte">Let us introduce you to our fabulous tree-friendly team , 100% biodegradable and certified pesticide-free.</p><br />
<br />
<p class="title1">Students</p><br />
<p class="title3"> <i>(Move your mouse over image to enlarge!)</i> </p><br />
<br></br><br />
<br />
<div class="zooma"><div style="float:left; width:215px;"><br />
<p class="title2"><br />
<img src="https://static.igem.org/mediawiki/2014/d/da/Diane_gros.png" alt="Diane Barbay" border="0" style="margin-top:5px"/><br />
</div><br />
<div style="float: right; width:720px;"> <br />
<p class="title2"><b>Diane Barbay</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Diligent (Wait a minute, I have to write down these results in my notebook!)</p><br />
<p class="textesimple"><b>Studies:</b> First year of Biochemistry Master at INSA Toulouse engineering school</p> <br />
<p class="textesimple"><b>About her:</b> Hardworking, Diane is the most conscientious in her work: her notebook is so clean and organized! She is persevering and always tries to find the answer to the problem (except when plasmids shortens between two digestions…). She talks to bacteria to encourage them taking up plasmid during transformation. She is like a babysitter for our cells :-)<br />
</p></p> <br />
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<p class="title2"><br />
<img src="https://static.igem.org/mediawiki/2014/0/0e/Emeline_gros.png" alt="Emeline Flajollet" border="0" style="margin-top:5px"/><br />
</div><br />
<div style="float:left; width:700px; margin-left:5px; margin-right:40px"> <br />
<p class="title2"><b>Emeline Flajollet</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Expert in competent cells (Oh no, I have to make competent cells again…)</p><br />
<p class="textesimple"><b>Studies:</b> First year of Master of Microbiology at Paul Sabatier University of Toulouse</p> <br />
<p class="textesimple"><b>About her:</b> Emeline looks discreet at first, but when you meet her, you discover a frank person who knows how to express her opinions and how to be understood, which is a good thing for team work. She is diligent and involved in her work, always trying to do her best, and providing advice to others. She spends a lot of time on social networks, allowing us to keep in touch with other teams.<br />
</p></p><br />
</div></div><br />
<br />
<div class="clear" style="margin-top:85px;"></div><br />
<br />
<div class="zooma"><div style="float:left; width:215px;"><br />
<p class="title2"><br />
<img src="https://static.igem.org/mediawiki/2014/8/80/Mathieu_gros.png" alt="Mathieu Fournié" border="0" style="margin-top:5px"/><br />
</div><br />
<div style="float: right; width:720px;"> <br />
<p class="title2"><b>Mathieu Fournié</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Expert in communication (I found someone else who wants to interview us!)</p><br />
<p class="textesimple"><b>Studies:</b> First year of Master of Microbiology at Paul Sabatier University of Toulouse</p> <br />
<p class="textesimple"><b>About him:</b> Initial founder of the project, Mathieu is a fan of Midi Pyrenées's region and is eager to protect its natural environment and resources. He is devoted to the project and does his best to succeed. His leadership skills help us focus on the main goals and deadlines. Even if it makes him forget to do his own tasks! His good interpersonal skills and numerous contacts helped us to capture media attention. <br />
</p></p><br />
</div></div><br />
<br />
<div class="clear" style="margin-bottom:60px;"></div><br />
<br />
<div class="zoomb"><div style="float:right; width:215px;"><br />
<p class="title2"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d2/Florie_gros.png" alt="Florie Gosseau" border="0" style="margin-top:5px"/><br />
</div><br />
<div style="float:left; width:700px; margin-left:5px; margin-right:40px"> <br />
<p class="title2"><b>Florie Gosseau</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Our IT expert (Modeling? Ok let’s do it!)</p><br />
<p class="textesimple"><b>Studies:</b> First year of Bioinformatics and Biological Systems Master at Paul Sabatier University of Toulouse</p> <br />
<p class="textesimple"><b>About her:</b> Florie is a smiling and spontaneous girl. She is this kind of person capable of giving a true smile when nothing works, and she can disentangle conflicts thanks to her patience and her kindness. She is really helpful and never says no when you ask for service. She is also frank and reports problems when there are some. Finally, she is the only one good at informatics, which makes her indispensable for us! When she wears her lab coat, she is able to fly like Batman or being the queen of cats' kingdom. Really, it is wonderful to see her!<br />
</p></p><br />
</div></div><br />
<br />
<div class="clear" style="margin-top:85px;"></div><br />
<br />
<div class="zooma"><div style="float:left; width:215px;"><br />
<p class="title2"><br />
<img src="https://static.igem.org/mediawiki/2014/9/92/Camille_gros.png" alt="Camille Jourdan" border="0" style="margin-top:5px"/><br />
</div><br />
<div style="float: right; width:720px;"><br />
<p class="title2"><b>Camille Jourdan</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Expert in cloning (Camille, which enzyme should I take?)</p><br />
<p class="textesimple"><b>Studies:</b> First year of Biochemistry Master at INSA Toulouse engineering school</p> <br />
<p class="textesimple"><b>About him:</b> Jovial and funny, Camille has sometimes eruptions of madness which makes him so special. Conversely, he is capable of incredible moments of intelligence and concentration (primers and plasmids hold no secret for him) and he is really diligent. He is sarcastic but definitely not nasty. He is the athlete of the team and never misses an opportunity to make gymnastics exercises or Plasmid Dance!<br />
</p></p><br />
</div></div><br />
<br />
<div class="clear" style="margin-bottom:60px;"></div><br />
<br />
<div class="zoomb"><div style="float:right; width:215px;"><br />
<p class="title2"><br />
<img src="https://static.igem.org/mediawiki/2014/c/c2/Aur%C3%A9lie_gros.png" alt="Aurélie Kanitzer" border="0" style="margin-top:5px"/><br />
</div><br />
<div style="float:left; width:700px; margin-left:5px; margin-right:40px"><br />
<p class="title2"><b>Aurélie Kanitzer</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Distracted (Oh I forgot my keys! I have to go back home, see you in 15 minutes!)</p><br />
<p class="textesimple"><b>Studies:</b> First year of Biochemistry Master at INSA Toulouse engineering school</p> <br />
<p class="textesimple"><b>About her:</b> Aurélie is a joyful and spontaneous person, which makes you laugh and smile. Often distracted, she can make blunders… She is natural and honest and does not try to play a role. She is open-minded and helpful: you can count on her. Strong and sensitive at the same time, she is able to face difficulties. Don’t go by appearances, this tiny girl has personality and she does not let others walk over her!<br />
</p></p><br />
</div></div><br />
<br />
<div class="clear" style="margin-top:85px;"></div><br />
<br />
<div class="zooma"><div style="float:left; width:215px;"><br />
<p class="title2"><br />
<img src="https://static.igem.org/mediawiki/2014/2/25/Laureen_gros.png" alt="Laureen Mirassou" border="0" style="margin-top:5px"/><br />
</div><br />
<div style="float: right; width:720px;"><br />
<p class="title2"><b>Laureen Mirassou</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Our best negotiator (Don’t worry about prices for flight tickets, I deal with that)</p><br />
<p class="textesimple"><b>Studies:</b> First year of Biochemistry Master at INSA Toulouse engineering school</p> <br />
<p class="textesimple"><b>About her:</b> Laureen is a mix of kindness, helpfulness and generosity. Always trying to help people, she is essential to ease tensions and to solve social issues: she says what is wrong gently and calmly. Her good interpersonal abilities helped us to make good business and to solve many administrative problems. Her high level of English proficiency is also something precious for the team.<br />
</p></p><br />
</div></div><br />
<br />
<div class="clear" style="margin-bottom:60px;"></div><br />
<br />
<div class="zoomb"><div style="float:right; width:215px;"><br />
<p class="title2"><br />
<img src="https://static.igem.org/mediawiki/2014/8/8d/Manon_gros.png" alt="Manon Molina" border="0" style="margin-top:5px"/><br />
</div><br />
<div style="float:left; width:700px; margin-left:5px; margin-right:40px"><br />
<p class="title2"><b>Manon Molina</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Organized (Let’s make a to-do-list!)</p><br />
<p class="textesimple"><b>Studies:</b> First year of Biochemistry Master at INSA Toulouse engineering school </p> <br />
<p class="textesimple"><b>About her:</b> Manon is organized: she always makes to-do-lists to not forget anything and do tasks in time. Thank goodness, she is here to monitor the budget and do the accounting! She makes her work conscientiously and meticulously, not allowing mistakes. She is kind and lenient but can also be frank when she does not endorse your methods. In short: serious and devoted to the project.<br />
</p></p><br />
</div></div><br />
<br />
<div class="clear" style="margin-top:85px;"></div><br />
<br />
<div class="zooma"><div style="float:left; width:215px;"><br />
<p class="title2"><br />
<img src="https://static.igem.org/mediawiki/2014/5/5c/Fanny_gros.png" alt="Fanny Pineau" border="0" style="margin-top:5px"/><br />
</div><br />
<div style="float: right; width:720px;"><br />
<p class="title2"><b>Fanny Pineau</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Absent-minded (which cloning are we doing?)</p><br />
<p class="textesimple"><b>Studies:</b> First year of Biochemistry Master at INSA Toulouse engineering school </p> <br />
<p class="textesimple"><b>About her:</b> In the lab, Fanny is often lost in her works: too many cloning at the same time, you should not ask too much to her blond brain! Not enough concentrated, she can sometimes make blunders. Besides that, she is applied and perfectionist when she plunges into her work. Finally, she is a joyful and smiling person; also honest and frank, telling it like it is.<br />
</p></p><br />
</div></div><br />
<br />
<div class="clear" style="margin-bottom:60px;"></div><br />
<br />
<div class="zoomb"><div style="float:right; width:215px;"><br />
<p class="title2"><br />
<img src="https://static.igem.org/mediawiki/2014/0/0d/Pierre_gros.png" alt="Pierre Reitzer" border="0" style="margin-top:5px"/><br />
</div><br />
<div style="float:left; width:700px; margin-left:5px; margin-right:40px"><br />
<p class="title2"><b>Pierre Reitzer</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Relaxed (Make a presentation barefoot, why not?)</p><br />
<p class="textesimple"><b>Studies:</b> First year of Biochemistry Master at INSA Toulouse engineering school </p> <br />
<p class="textesimple"><b>About him:</b> Pierre is the co-founder of the project. He is really friendly and is always willing to discuss and help people with their experiments. Moreover, he greets us with a smile each time we see him in the lab. He is spontaneous and forthright: we know what he thinks. Sometimes, he has his head in the clouds and fails the same experiment three times… But he is involved in his work and does not count hours. <br />
</p></p><br />
</div></div><br />
<br />
<div class="clear" style="margin-top:85px;"></div><br />
<br />
<div class="zooma"><div style="float:left; width:215px;"><br />
<p class="title2"><br />
<img src="https://static.igem.org/mediawiki/2014/f/f6/Abdel_gros.png" alt="Abdel Touré" border="0" style="margin-top:5px"/><br />
</div><br />
<div style="float: right; width:720px;"><br />
<p class="title2"><b>Abdel Touré</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Luxury tastes (Why would we go elsewhere than the Hilton?)</p><br />
<p class="textesimple"><b>Studies:</b> First year of Master at ENSAT Toulouse agronomic school</p> <br />
<p class="textesimple"><b>About him:</b> Abdel is this kind of person that you remember: he is always jovial and running everywhere. He is a funny guy who likes making jokes. But, be careful! He is also sensitive, your jokes might not make him laugh at all. He takes care of his appearance, and likes smoking his e-cigarette. He always swears in English, but we like it because this is funny, and he is a good English speaker!<br />
</p></p><br />
</div></div><br />
<br />
<div class="clear" style="margin-top:85px;"></div><br />
<br />
<p class="title1">Instructors</p><br />
<br />
<div style="float:left; width:215px;"><br />
<img src="https://static.igem.org/mediawiki/2014/b/b6/Advisor_Gilles.jpg" style="margin-top:5px"/><br />
</div> <br />
<br />
<div style="float: right; width:700px; margin-left:44px;"> <br />
<p class="title2"><b>Gilles Truan</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Our amazing instructor!</p><br />
<p class="textesimple"><b>Job:</b> Senior scientist at CNRS (Centre National de la Recherche Scientifique) and at LISBP (Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés).</p> <br />
<p class="textesimple"><b>About him:</b> He is with us from the beginning to the end as a father (and already a grandfather!!), our instructor is the master of cloning and the captain of PCR! He provided us valuable help and good tips he has known since the time he was supervising Marie Curie's doctoral thesis.</p><br />
</div><br />
<br />
<div class="clear" style="margin-bottom:60px;"></div><br />
<br />
<div style="float:right; width:215px;"><br />
<img src="https://static.igem.org/mediawiki/2014/8/81/Advisor_Brice.jpg" style="margin-top:5px" /><br />
</div> <br />
<br />
<div style="float:left; width:700px; margin-left:5px; margin-right:40px"><br />
<p class="title2"><b>Brice Enjalbert</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> The right-hand man of our amazing instructor!</p><br />
<p class="textesimple"><b>Job:</b> Assistant professor at LISBP (Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés).</p> <br />
</div><br />
<br />
<div class="clear" style="margin-bottom:60px;"></div><br />
<br />
<div style="float:left; width:215px;"><br />
<img src="https://static.igem.org/mediawiki/2014/b/bd/Advisor_Florence.jpg" style="margin-top:5px" /><br />
</div> <br />
<br />
<div style="float: right; width:700px; margin-left:44px;"> <br />
<p class="title2"><b>Florence Bordes</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> The only female advisor! </p><br />
<p class="textesimple"><b>Job:</b> Assistant professor at LISBP (Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés).</p> <br />
</div><br />
<br />
<div class="clear" style="margin-bottom:60px;"></div><br />
<br />
<div style="float:right; width:215px;"><br />
<img src="https://static.igem.org/mediawiki/2014/2/2d/Advisor_Cam.jpg" style="margin-top:5px" /><br />
</div> <br />
<br />
<div style="float:left; width:700px; margin-left:5px; margin-right:40px"><br />
<p class="title2"><b>Kaymeuang Cam</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Our bacteriologist advisor! </p><br />
<p class="textesimple"><b>Job:</b> Teacher-Researcher at IPBS (Institut de Pharmacologie et de Biologie Structurale)</p> <br />
</div><br />
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{{:Team:Toulouse/template/footer}}</div>Jourdanhttp://2014.igem.org/Team:Toulouse/Project/SpreadingTeam:Toulouse/Project/Spreading2014-10-17T21:48:24Z<p>Jourdan: </p>
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<h2>Spreading</h2><br />
<p>How to keep control on SubtiTree?</p><br />
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<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Project&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Spreading</p> <br />
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<br />
<p class="texte">Our engineered bacterium is designed to be inoculated in a tree and to cure fungal diseases. To avoid the environmental issues <br />
resulting from the use of a modified organism on the trees lining the Canal du Midi, our team worked on different aspects to ensure a safe use of<br />
SubtiTree. <br />
The first objective is to avoid the spreading of our smart bacterium outside of the tree. In other words, the purpose is to ensure that once SubtiTree <br />
is in the tree, it is unable to live anywhere else. Another issue concerns the horizontal transfers of the genetic material between different bacteria. <br />
Taking into account these key points, we elaborate different strategies.<br />
</p><br />
<br />
<div id="Spreading"><br />
<center style="margin-bottom:80px;"><img alt="schema" style="width:700px; z-index:2; " src="https://static.igem.org/mediawiki/2014/2/27/Spreading_sch%C3%A9ma.jpg"></img></center><br />
<a class="Auxotro" HREF="#Auxotrophy"></a><br />
<a class="NSporing" HREF="#NonSporing"></a><br />
<a class="Tox" HREF="#Toxin"></a><br />
</div><br />
<br />
<p class="title1">Survival in the environment: <i>B. subtilis</i> that is proline auxotrophic</p><br />
<p class="texte">SubtiTree will live in sap tree, thus we will use an endophyte <I>B. subtilis</I> strain. In order to contain our bacteria in this <br />
area during a short period of time, we think about modifying some of its survival characteristics. To turn the bacterium growth dependant on the presence <br />
inside the tree (and therefore avoid spreading in the environment), we planned to use a <i>B.subtilis</i> strain that is proline auxotrophic. The bacterium<br />
should then be unable to synthesize this essential amino acid. Proline is the most abundant amino acid in the phloem sap. If the bacterium is in the sap,<br />
it should grow normally without any deficiency, but if it escapes from the tree and <i>a fortiori</i> from the sap, it will not be able to survive for a <br />
long time as proline is present only in very low quantities in the soil. <br />
<br/> <br />
Auxotrophic <i>B.subtilis</i> strains already exist and are indexed in the databases BGSC (Bacillus Genetic Stock Center).</p><br />
<br />
<p class="title1">Preventing sporulation of <i>B. subtilis</i></p><br />
<p class="texte"><br />
It is known that endophyte bacteria must sporulate to survive during the winter. We planned to limit Subtitree's lifespan to only one season.<br />
The bacteria should be injected in spring, grow during the summer and finally should<br />
die in the fall.<br\> <br />
<i>B. subtilis</i> is a spore-forming bacterium: sporulation enables the microorganism to resist to very harsh conditions and <br />
to spread from tree to tree.<br/> <br />
To control any unwanted long-term development of SubtiTree, our strain should therefore be unable to sporulate. Thus, during fall, when the sap become <br />
less nutritious and the temperature is lower, the engineered bacterium will die and not pass through the following winter. This question has been studied in our<br />
<a= href "https://2014.igem.org/Team:Toulouse/Modelling" Modeling part> </a>.<br/><br />
In addition, deleting all the engineered bacterial community every year puts a brake on the evolution due to random mutations,<br />
thus allowing a better faith on the genetic constructions.<br />
</p><br />
<br />
<p class="texte">These two strategies aim to make <i>B. subtilis</i> an annual bacterium, growing only in the sap tree. By combining them, <br />
they should prevent any long term colonization of any other ecological niche than plane trees.</p><br />
<br />
<br />
<p class="title1">Gene transfer: toxin-antitoxin system</p><br />
<br />
<p class="texte">We also wondered about horizontal gene transfers. The goal of this module is to prevent horizontal transfers between bacteria <br />
and any exchange of synthetic genetic material that could be dangerous between wild type organisms and optimized organisms.<br />
<br>We thought about a system limiting such transfers: a toxin-antitoxin module. It involves the addition of two genes to the bacterium: a gene encoding <br />
for a toxin (for example <i>tse2</i>, placed next to the engineered genetic modules) and a gene encoding for the antitoxin (<i>tsi1</i>), placing them far away from each other in the genome. The large intergenic region<br />
between them prevents simultaneous transfers: if the optimized bacterium transfers the gene encoding for the toxin, the probability that the gene <br />
encoding for the antitoxin may be transferred simultaneously is very low.<br/><br />
Therefore, if another host bacterium receives the gene encoding for the toxin, it will be unable to survive since it will not possess the antitoxin. <br />
If it receives the antitoxin only, it will not be useful for the bacterium, and will not affect it.<br/><br />
In summary, since a simultaneous transfer is dimly probable, the bacterium will either die because of the toxin or live while expressing the antitoxin. <br />
</p><br />
<br />
<p class="title1">Using integrative plasmids</p><br />
<p class="texte"><br />
One of the side effects of our cloning method is the persistence of antibiotic resistance genes. This is incompatible with the introduction <br />
in the tree, and with the stability of our constructs. To avoid this, all our constructions are carried by integrative plasmids. Consequently, our different<br />
genetic modules should be integrated into the bacterium genome. The integration in the genome is more stable as the constructions are less likely to be <br />
transferred to other microorganisms. In addition to that, the expression of our genetic modules would not be dependent on a selective pressure, <br />
allowing a high level of transcription <i>in planta</i>. <br />
</p><br />
<br />
<p class="texte"><br />
<br><br />
While we have not constructed yet these modules, we definitely think that the elaborated strategies we designed should render the use of SubtiTree acceptable <br />
in real conditions. <br />
</p><br />
<p class="title1">References</p><br />
<br />
<ul><br />
<li class="tree"><p class="texte">S. Dinant, J.L. Bonnemain, C. Girousse, J. Kehr. <b> Phloem sap intricacy and interplay with aphid feeding.</b>C R Biol. 2010 Jun-Jul;333(6-7):504-15. doi: 10.1016/j.crvi.2010.03.008. Epub 2010 May 14.</p></li><br />
<li class="tree"><p class="texte"> Z.N. Senwo and M.A. Tabatabai. <b> Amino acid composition of soil organic matter.</b> Biology and Fertility of SoilsFebruary 1998, Volume 26, Issue 3, pp 235-242 </p></li><br />
<li class="tree"><p class="texte">A.M. Guérout-Fleury, N. Frandsen and P. Stragier <b> Plasmids for ectopic integration in Bacillus subtilis.</b> Gene. 1996 Nov 21;180(1-2):57-61.</p></li><br />
<li class="tree"><p class="texte">G. Shang, X. Liu, D. Lu, J. Zhang, N. Li, C. Zhu, S. Liu, Q. Yu, Y. Zhao and L. Gu. <b> Structural insight into how Pseudomonas aeruginosa peptidoglycanhydrolase Tse1 and its immunity protein Tsi1 function.</b> Biochem J. 2012 Dec 1;448(2):201-11. doi: 10.1042/BJ20120668.</p></li><br />
<li class="tree"><p class="texte">W.Z. Hua, C. S. Yong and X.T. Ren<b>Biology and chemistry of endophytes.</b> Nat. Prod. Rep., 2006, 23, 753–771, 753</p></li><br />
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{{:Team:Toulouse/template/footer}}</div>Jourdanhttp://2014.igem.org/Team:Toulouse/Project/SpreadingTeam:Toulouse/Project/Spreading2014-10-17T21:47:38Z<p>Jourdan: </p>
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<h2>Spreading</h2><br />
<p>How to keep control on SubtiTree?</p><br />
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<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Project&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Spreading</p> <br />
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<p class="texte">Our engineered bacterium is designed to be inoculated in a tree and to cure fungal diseases. To avoid the environmental issues <br />
resulting from the use of a modified organism on the trees lining the Canal du Midi, our team worked on different aspects to ensure a safe use of<br />
SubtiTree. <br />
The first objective is to avoid the spreading of our smart bacterium outside of the tree. In other words, the purpose is to ensure that once SubtiTree <br />
is in the tree, it is unable to live anywhere else. Another issue concerns the horizontal transfers of the genetic material between different bacteria. <br />
Taking into account these key points, we elaborate different strategies.<br />
</p><br />
<br />
<div id="Spreading"><br />
<center style="margin-bottom:80px;"><img alt="schema" style="width:700px; z-index:2; " src="https://static.igem.org/mediawiki/2014/2/27/Spreading_sch%C3%A9ma.jpg"></img></center><br />
<a class="Auxotro" HREF="#Auxotrophy"></a><br />
<a class="NSporing" HREF="#NonSporing"></a><br />
<a class="Tox" HREF="#Toxin"></a><br />
</div><br />
<br />
<p class="title1">Survival in the environment: <i>B. subtilis</i> that is proline auxotrophic</p><br />
<p class="texte">SubtiTree will live in sap tree, thus we will use an endophyte <I>B. subtilis</I> strain. In order to contain our bacteria in this <br />
area during a short period of time, we think about modifying some of its survival characteristics. To turn the bacterium growth dependant on the presence <br />
inside the tree (and therefore avoid spreading in the environment), we planned to use a <i>B.subtilis</i> strain that is proline auxotrophic. The bacterium<br />
should then be unable to synthesize this essential amino acid. Proline is the most abundant amino acid in the phloem sap. If the bacterium is in the sap,<br />
it should grow normally without any deficiency, but if it escapes from the tree and <i>a fortiori</i> from the sap, it will not be able to survive for a <br />
long time as proline is present only in very low quantities in the soil. <br />
<br/> <br />
Auxotrophic <i>B.subtilis</i> strains already exist and are indexed in the databases BGSC (Bacillus Genetic Stock Center).</p><br />
<br />
<p class="title1">Preventing sporulation of <i>B. subtilis</i></p><br />
<p class="texte"><br />
It is known that endophyte bacteria must sporulate to survive during the winter. We planned to limit Subtitree's lifespan to only one season.<br />
The bacteria should be injected in spring, grow during the summer and finally should<br />
die in the fall.<br\> <br />
<i>B. subtilis</i> is a spore-forming bacterium: sporulation enables the microorganism to resist to very harsh conditions and <br />
to spread from tree to tree.<br/> <br />
To control any unwanted long-term development of SubtiTree, our strain should therefore be unable to sporulate. Thus, during fall, when the sap become <br />
less nutritious and the temperature is lower, the engineered bacterium will die and not pass through the following winter. This question has been studied in our<br />
<a= href "https://2014.igem.org/Team:Toulouse/Modelling" Modeling part> </a>.<br/><br />
In addition, deleting all the engineered bacterial community every year puts a brake on the evolution due to random mutations,<br />
thus allowing a better faith on the genetic constructions.<br />
</p><br />
<br />
<p class="texte">These two strategies aim to make <i>B. subtilis</i> an annual bacterium, growing only in the sap tree. By combining them, <br />
they should prevent any long term colonization of any other ecological niche than plane trees.</p><br />
<br />
<br />
<p class="title1">Gene transfer: toxin-antitoxin system</p><br />
<br />
<p class="texte">We also wondered about horizontal gene transfers. The goal of this module is to prevent horizontal transfers between bacteria <br />
and any exchange of synthetic genetic material that could be dangerous between wild type organisms and optimized organisms.<br />
<br>We thought about a system limiting such transfers: a toxin-antitoxin module. It involves the addition of two genes to the bacterium: a gene encoding <br />
for a toxin (for example <i>tse2</i>, placed next to the engineered genetic modules) and a gene encoding for the antitoxin (<i>tsi1</i>), placing them far away from each other in the genome. The large intergenic region<br />
between them prevents simultaneous transfers: if the optimized bacterium transfers the gene encoding for the toxin, the probability that the gene <br />
encoding for the antitoxin may be transferred simultaneously is very low.<br/><br />
Therefore, if another host bacterium receives the gene encoding for the toxin, it will be unable to survive since it will not possess the antitoxin. <br />
If it receives the antitoxin only, it will not be useful for the bacterium, and will not affect it.<br/><br />
In summary, since a simultaneous transfer is dimly probable, the bacterium will either die because of the toxin or live while expressing the antitoxin. <br />
</p><br />
<br />
<p class="title1">Using integrative plasmids</p><br />
<p class="texte"><br />
One of the side effects of our cloning method is the persistence of antibiotic resistance genes. This is incompatible with the introduction <br />
in the tree, and with the stability of our constructs. To avoid this, all our constructions are carried by integrative plasmids. Consequently, our different<br />
genetic modules should be integrated into the bacterium genome. The integration in the genome is more stable as the constructions are less likely to be <br />
transferred to other microorganisms. In addition to that, the expression of our genetic modules would not be dependent on a selective pressure, <br />
allowing a high level of transcription <i>in planta</i>. <br />
</p><br />
<br />
<p class="texte"><br />
<br><br />
While we have not constructed yet these modules, we definitely think that the measures that we designed should render the use of SubtiTree acceptable <br />
in real conditions. <br />
</p><br />
<p class="title1">References</p><br />
<br />
<ul><br />
<li class="tree"><p class="texte">S. Dinant, J.L. Bonnemain, C. Girousse, J. Kehr. <b> Phloem sap intricacy and interplay with aphid feeding.</b>C R Biol. 2010 Jun-Jul;333(6-7):504-15. doi: 10.1016/j.crvi.2010.03.008. Epub 2010 May 14.</p></li><br />
<li class="tree"><p class="texte"> Z.N. Senwo and M.A. Tabatabai. <b> Amino acid composition of soil organic matter.</b> Biology and Fertility of SoilsFebruary 1998, Volume 26, Issue 3, pp 235-242 </p></li><br />
<li class="tree"><p class="texte">A.M. Guérout-Fleury, N. Frandsen and P. Stragier <b> Plasmids for ectopic integration in Bacillus subtilis.</b> Gene. 1996 Nov 21;180(1-2):57-61.</p></li><br />
<li class="tree"><p class="texte">G. Shang, X. Liu, D. Lu, J. Zhang, N. Li, C. Zhu, S. Liu, Q. Yu, Y. Zhao and L. Gu. <b> Structural insight into how Pseudomonas aeruginosa peptidoglycanhydrolase Tse1 and its immunity protein Tsi1 function.</b> Biochem J. 2012 Dec 1;448(2):201-11. doi: 10.1042/BJ20120668.</p></li><br />
<li class="tree"><p class="texte">W.Z. Hua, C. S. Yong and X.T. Ren<b>Biology and chemistry of endophytes.</b> Nat. Prod. Rep., 2006, 23, 753–771, 753</p></li><br />
<br />
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{{:Team:Toulouse/template/footer}}</div>Jourdanhttp://2014.igem.org/Team:Toulouse/Project/FungicidesTeam:Toulouse/Project/Fungicides2014-10-17T20:43:28Z<p>Jourdan: </p>
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<h2>Fungicides</h2><br />
<p>To eradicate fungal diseases</p><br />
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<center><img style="width:700px; " src="https://static.igem.org/mediawiki/2014/0/0c/Recap_fungicides.jpg"><br />
<br><br />
<p class="legend">Figure 1: Schema of the fungicide module</p></center><br />
<br />
<p class="textesimple">The main objective of SubtiTree is to ensure the <b> destruction of the pathogenic fungi </b> inside the tree.<br />
In order to achieve this goal, we built a genetic module to produce three different peptides with antifungal activities. This triple therapy provides <br />
the advantage to minimize the resistance phenomenon.</p> <br><br />
<br />
<p class="textesimple">Originally from plants, these peptides have different targets thus increasing the lethality on <i>C. platani</i>.</p><br />
<br></br> <br />
<ul><br />
<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<br />
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><br />
<br />
<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<br />
originating from the Asiatic orchid <i>Gastrodia elata</i>, a traditional Chinese medicinal herb cultured for thousands of years. <br />
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 <br />
ascomycete and basidiomycete fungal plant pathogens.</p></li><br />
<br />
<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<br />
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.<br />
EcAMP-1 exhibits high activity against fungi of the genus <i>Fusarium</i>.</p></li><br />
</ul><br />
</p><br />
<br><br />
<p class="title1" style="margin-top:30px;">More information about this module </p><br />
<p class="texte"><br />
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<br />
antifungal peptides are placed under the control of the constitutive promoter P<sub>veg</sub> in <i>Bacillus subtilis</i>.</p><br />
<br />
<center><img style="width:930px; float:left; margin: 30px 0 45px;" src="https://static.igem.org/mediawiki/parts/d/d0/Fungicideprod.jpg"><br />
<p class="legend">Figure 2: Fungicide operon</p></center><br />
<br />
<p class="title2">Added parts</p><br />
<p class="title3">EcAMP-1</p><br />
<p class="texte">EcAMP-1 was already present in the Registry, added by the Utah State 2013 iGEM team <br />
(<a href="http://parts.igem.org/Part:BBa_K1162001"_blank">BBa_K1162001</a>). This part has been modified and improved by our team <br />
(<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><br />
<p class="title3">D4E1 and GAFP-1</p><br />
<p class="texte">We added D4E1 and GAFP-1 to the Registry of Standard Biological Parts <br />
(See <a href="https://2014.igem.org/Team:Toulouse/Result/parts/Submitted_parts"_blank">Submitted parts</a>).<br />
<br> These new BioBricks were designed in order to be expressed and secreted with <i>Bacillus subtilis</i>. </p><br />
<br><br />
<p class="title2">Secretion</p><br />
<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<br />
a signal peptide (amyE signal peptide) followed by a pro peptide, cleaved during the secretion process.</p><br><br />
<br />
<br />
<center><img style="width:400px; " src="https://static.igem.org/mediawiki/2014/2/2e/Secretion.jpg"><br />
<img style="width:500px; " src="https://static.igem.org/mediawiki/2014/d/d7/Fongpep.jpg"><br />
<br><p class="legend">Figure 3: Design of GAFP-1 and D4E1</p></center><br />
<br><br />
<br />
<br />
<p class="title1">References</p><br />
<br />
<ul><br />
<li class="tree"><p class="texte">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. </p></li><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><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><br />
<li class="tree"><p class="texte">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</p></li><br />
<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><br />
<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 (Echinochloa crus-galli)</b>. The journal of Biological Chemistry. 2011, Vol. 286, 25145–25153</p></li><br />
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{{:Team:Toulouse/template/footer}}</div>Jourdanhttp://2014.igem.org/Team:Toulouse/Project/FungicidesTeam:Toulouse/Project/Fungicides2014-10-17T20:41:05Z<p>Jourdan: </p>
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<h2>Fungicides</h2><br />
<p>To eradicate fungal diseases</p><br />
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<br />
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<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Project&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Fungicides</p> <br />
</div><br />
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<br />
<!--Short description : à changer!!!--><br />
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<center><img style="width:700px; " src="https://static.igem.org/mediawiki/2014/0/0c/Recap_fungicides.jpg"><br />
<br><br />
<p class="legend">Figure 1: Schema of the fungicide module</p></center><br />
<br />
<p class="textesimple">The main objective of SubtiTree is to ensure the <b> destruction of the pathogenic fungi </b> inside the tree.<br />
In order to achieve this goal, we built a genetic module to produce three different peptides with antifungal activities. This triple therapy provides <br />
the advantage to minimize the resistance phenomenon.</p> <br><br />
<br />
<p class="textesimple">Originally from plants, these peptides have different targets thus increasing the lethality on <i>C. platani</i>.</p><br />
<br></br> <br />
<ul><br />
<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<br />
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><br />
<br />
<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<br />
originating from the Asiatic orchid <i>Gastrodia elata</i>, a traditional Chinese medicinal herb cultured for thousands of years. <br />
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 <br />
ascomycete and basidiomycete fungal plant pathogens.</p></li><br />
<br />
<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<br />
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.<br />
EcAMP-1 exhibits high activity against fungi of the genus <i>Fusarium</i>.</p></li><br />
</ul><br />
</p><br />
<br><br />
<p class="title1" style="margin-top:30px;">More information about this module </p><br />
<p class="texte"><br />
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<br />
antifungal peptides are placed under the control of the constitutive promoter P<sub>veg</sub> in <i>Bacillus subtilis</i>.</p><br />
<br />
<center><img style="width:930px; float:left; margin: 30px 0 45px;" src="https://static.igem.org/mediawiki/parts/d/d0/Fungicideprod.jpg"><br />
<p class="legend">Figure 2: Fungicide operon</p></center><br />
<br />
<p class="title2">Added parts</p><br />
<p class="title3">EcAMP-1</p><br />
<p class="texte">EcAMP-1 was already present in the Registry, added by the Utah State 2013 iGEM team <br />
(<a href="http://parts.igem.org/Part:BBa_K1162001"_blank">BBa_K1162001</a>). This part has been modified and improved by our team <br />
(<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><br />
<p class="title3">D4E1 and GAFP-1</p><br />
<p class="texte">We added D4E1 and GAFP-1 to the Registry of Standard Biological Parts (See <a href="https://2014.igem.org/Team:Toulouse/Result/parts/Submitted_parts"_blank">Submitted parts</a>). <br>We ordered the genes to a synthesis company and did cloning. These new BioBricks were designed in order to be expressed and secreted with <i>Bacillus subtilis</i>. </p><br />
<br><br />
<p class="title2">Secretion</p><br />
<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<br />
a signal peptide (amyE signal peptide) followed by a pro peptide, cleaved during the secretion process.</p><br><br />
<br />
<br />
<center><img style="width:400px; " src="https://static.igem.org/mediawiki/2014/2/2e/Secretion.jpg"><br />
<img style="width:500px; " src="https://static.igem.org/mediawiki/2014/d/d7/Fongpep.jpg"><br />
<br><p class="legend">Figure 3: Design of GAFP-1 and D4E1</p></center><br />
<br><br />
<br />
<br />
<p class="title1">References</p><br />
<br />
<ul><br />
<li class="tree"><p class="texte">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. </p></li><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><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><br />
<li class="tree"><p class="texte">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</p></li><br />
<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><br />
<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 (Echinochloa crus-galli)</b>. The journal of Biological Chemistry. 2011, Vol. 286, 25145–25153</p></li><br />
</ul><br />
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{{:Team:Toulouse/template/footer}}</div>Jourdanhttp://2014.igem.org/Team:Toulouse/Project/bindingTeam:Toulouse/Project/binding2014-10-17T20:27:29Z<p>Jourdan: </p>
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<h2>Binding</h2><br />
<p>To be attached to the fungal cell wall</p><br />
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<!--Short description : à changer!!!--><br />
<center><img style="width:700px; " src="https://static.igem.org/mediawiki/2014/e/e1/Bindingresume.png"></center><br />
<p class="legend">Figure 1: Schema of the binding module</p><br />
<br />
<p class="texte"> In order to be highly efficient in the fight against the pythopathogen <i>Ceratocystis platani</i>, our optimized bacterium has to be anchored to the fungus. <br />
Thus, we designed a chimeric protein (<a href="http://parts.igem.org/Part:BBa_K1364005"target="_blank">BBa_K1364005</a>) capable of building <br />
a <B>bridge between the bacterial peptidoglycan and the fungal chitin</B>, the main component of the pathogen’s cell wall. <br />
According to the work of <a href="https://2010.igem.org/Team:Imperial_College_London"target="_blank">the Imperial College 2010</a> iGEM team, <br />
we used the Cell Wall Binding (CWB) domain of the LytC protein (coding for a N-acetylmuramoyl-L-alanine amidase) to attach our chimeric protein <br />
to the <I>B. subtilis</I> cell wall. On the other side of our protein, we added the domain 4 of GbpA from <I>Vibrio cholerae</I>, which is known to <br />
recognize chitin.<br />
</p><br />
<br />
<br><br />
<p class="title1"> More information about this module </p><br />
<p class="texte"> <br />
The open reading frame of the Binding Module is composed of 3 sections:</p><br />
<br />
<ul><br />
<li class="tree"><p class="texte"><B>Anchor section</B>: the CWB (Cell Wall Binding) domain is extracted from LytC gene and composes the 5' side of our binding module. As previously described by the Imperial College of London 2010 iGEM team, we kept the first 318 bp. We can note the presence of the signal peptide at the beginning of the sequence, from 1 to 24 bp.</p></li><br />
<li class="tree"><p class="texte"><B>Chitin Binding Domain (CBD) section</b>: the domain 4 of GbpA from <I>Vibrio cholerae </I> is able to bind to N-Acetyl Glucosamine oligosacchararides. Also, the 3' side of our gene is composed by a part of the GbpA sequence (from 423 to 484 bp).</p></li><br />
<li class="tree"><p class="texte"><B>Helical Linker</B>: According to the work of the 2010 Imperial College of London iGEM team, we used the same six amino acids sequence (SRGSRA) to make a bridge between the Anchor section and the Chitin Binding section.<br />
</p></li></ul><br />
<center style="margin:-30px;"><img style="width:500px; " src="https://static.igem.org/mediawiki/2014/a/a0/Binding.png"></center><br />
<p class="legend">Figure 2: Binding gene composition</p><br />
<br></br><br />
<p class="texte"> <br />
The sequence has been designed <i>in silico</i> and codon optimized for the transcription in <i>B. subtilis</i>. <br />
</p><br />
<br />
<B class="title1">Final construction</B> <br />
<br />
<B class="title2">(More details about the intermediate parts <a href="https://2014.igem.org/Team:Toulouse/Result/parts#select2"target="_blank">Here</a>)</B> <br />
<br />
<p class="texte"><br />
<br>The binding module has been placed under the control of Pveg (<a href="http://parts.igem.org/Part:BBa_K143012"target="_blank">BBa_K143012</a>),<br />
a strong constitutive promoter and we used a consensus RBS (<a href="http://parts.igem.org/Part:BBa_K090505"target="_blank">BBa_K090505</a>) as well as a<br />
double terminator (<a href="http://parts.igem.org/Part:BBa_B0015"target="_blank">B0015</a>). <br />
</p> <br />
<br />
<center style="margin:20px;"><img style="width:500px; " src="https://static.igem.org/mediawiki/2014/f/f5/BBa_K1364005.png"></center><br />
<p class="legend">Figure 3: Binding gene construction</p><br />
<br />
<p class="title1">References</p><br />
<br />
<ul><br />
<li class="tree"><p class="texte">M. Desvaux, E. Dumas, I. Chafsey and M. Hébraud.<b> Protein cell surface display in Gram-positive bacteria: from single protein to macromolecular protein structure </b>. FEMS Microbiol. Lett. 256, 1–15 (2006). </p></li><br />
<li class="tree"><p class="texte">E. Wong, G. Vaaje-Kolstad, A. Ghosh, R. Hurtado-Guerrero, PV. Konarev, AF. Ibrahim, DI. Svergun, VG. Eijsink, NS. Chatterjee and DM. van Aalten.<b>The Vibrio cholerae colonization factor GbpA possesses a modular structure that governs binding to different host surfaces</b>. PLoS Pathog. 8, e1002373 (2012).</p></li><br />
<br />
<li class="tree"><p class="texte">H. Yamamoto, S. Kurosawa and J. Sekiguchi. <b>Localization of the vegetative cell wall hydrolases LytC, LytE, and LytF on the Bacillus subtilis cell surface and stability of these enzymes to cell wall-bound or extracellular proteases</b>. J. Bacteriol. 185, 6666–6677 (2003).</p></li><br />
</ul><br />
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{{:Team:Toulouse/template/footer}}</div>Jourdanhttp://2014.igem.org/Team:Toulouse/Project/ChemotaxisTeam:Toulouse/Project/Chemotaxis2014-10-17T20:16:23Z<p>Jourdan: </p>
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<h2>Chemotaxis</h2><br />
<p>To target the pathogenic fungus</p><br />
</div><br />
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<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Project&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Chemotaxis</p> <br />
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<br />
<center><img style="width:420px; " src="https://static.igem.org/mediawiki/parts/e/e9/Recap_chemotax.jpg"></center><br />
<p class="legend">Figure 1: Schema of the chemotaxis module</p><br />
<br />
<p class="title1">What is chemotaxis?</p><br />
<br />
<p class="texte"><br />
Chemotaxis is a bacterial function which induces a movement toward a gradient of concentration of a molecule of interest. With this system the bacteria are able to swim to a location containing higher concentrations of molecules such as sugar, amino acid, vitamins... <br />
Chemotactic-signal transducers respond to changes in the concentration of attractants and repellents in the environment, transduce the signal from the outside to the inside of the cell, and facilitate sensory adaptation through the variation of the level of methylation. <br />
<br />
<p class="title1">More information on this module</p><br />
<p class="texte"><br />
Chemotaxis is used as a way to detect and come close to the location of fungi infection. During its growth, fungi release N-acetylglucosamine (NAG), the basic unit of chitin which composed its cell wall. Thus, there should exist a gradient of the concentration of NAG around the fungi.</p><br />
<p class="texte"><br />
It is known that <i>B. subtilis</i> is able to detect and to swim towards glucose using the Methyl-accepting chemotaxis protein, henceforth called <b>McpA</b> (<a href="http://www.uniprot.org/uniprot/P39214"_blanck">MCPA_BACSU</a>).<br><br />
Some bacteria are attracted by NAG, like <i>Vibrio cholerae</i> which has a NAG regulated methyl-accepting chemotaxis protein: <b>VCD</b> (<a href="http://www.uniprot.org/uniprot/C3NYT2"_blank">VCD_000306</a>).</p><br />
<br />
<center><img width="500px" SRC="https://static.igem.org/mediawiki/2014/4/47/Chimio1.png" alt="schema" style="margin-bottom:60px;"></center><br />
<p class="legend">Figure 2: Chimeric protein of chemotaxis</p><br />
<br />
<p class="texte"><br />
Therefore, our idea is to switch the natural glucose specificity of <i>B. subtilis'</i>, mediated by McpA, to a NAG specificity. To achieve this, we need to change the extracellular domain of McpA, responsible for the specificity, by the extracellular domain of VCD.<br />
The whole sequence has been designed <i>in silico</i> and codon optimized for the transcription in <i>B. subtilis</i> before its synthesis.</p><br />
<br />
<center><img width="600px" SRC="https://static.igem.org/mediawiki/2014/e/e4/Chimio2.png" alt="gene construct" style="margin-bottom:40px;"></center><br />
<p class="legend">Figure 3: Construction of the chemotaxis gene</p><br />
<br />
<p class="title1">References</p><br />
<ul><br />
<br />
<li class="tree"><p class="texte">K. Meibom,L. Xibing, A. Nielsen, CY. Wu, S. Roseman and G. Schoolnik.<b> The Vibrio cholerae chitin utilization program </b>. The National Academy of Sciences of the USA (2004).</p></li><br />
<li class="tree"><p class="texte">C. Kristich and GW. Ordal. <b><i>Bacillus subtilis</i> CheD is a chemoreceptor modification enzyme required for chemotaxis</b>. J Biol Chem. 2002 Jul 12;277(28):25356-62. Epub 2002 May 13.<br></p></li><br />
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{{:Team:Toulouse/template/footer}}</div>Jourdanhttp://2014.igem.org/Team:Toulouse/Project/ChemotaxisTeam:Toulouse/Project/Chemotaxis2014-10-17T20:14:27Z<p>Jourdan: </p>
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<h2>Chemotaxis</h2><br />
<p>To target the pathogenic fungus</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Project&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Chemotaxis</p> <br />
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<br />
<center><img style="width:420px; " src="https://static.igem.org/mediawiki/parts/e/e9/Recap_chemotax.jpg"></center><br />
<p class="legend">Figure 1: Schema of the chemotaxis module</p><br />
<br />
<p class="title1">What is chemotaxis?</p><br />
<br />
<p class="texte"><br />
Chemotaxis is a bacterial function which induces a movement toward a gradient of concentration of a molecule of interest. With this system the bacteria are able to swim to a location containing higher concentrations of molecules such as sugar, amino acid, vitamins... <br />
Chemotactic-signal transducers respond to changes in the concentration of attractants and repellents in the environment, transduce the signal from the outside to the inside of the cell, and facilitate sensory adaptation through the variation of the level of methylation. <br />
<br />
<p class="title1">More information on this module</p><br />
<p class="texte"><br />
Chemotaxis is used as a way to detect and come close to the location of fungi infection. During its growth, fungi release N-acetylglucosamine (NAG), the basic unit of chitin which composed its cell wall. Thus, there should exist a gradient of the concentration of NAG around the fungi.</p><br />
<p class="texte"><br />
It is known that <i>B. subtilis</i> is able to detect and to swim towards glucose using the Methyl-accepting chemotaxis protein, henceforth called <b>McpA</b>(<a> href="MCPA_BACSU"_blanck">MCPA_BACSU).<br><br />
Some bacteria are attracted by NAG, like <i>Vibrio cholerae</i> which has a NAG regulated methyl-accepting chemotaxis protein: <b>VCD</b> (<a href="http://www.uniprot.org/uniprot/C3NYT2"_blank">VCD_000306</a>).</p><br />
<br />
<center><img width="500px" SRC="https://static.igem.org/mediawiki/2014/4/47/Chimio1.png" alt="schema" style="margin-bottom:60px;"></center><br />
<p class="legend">Figure 2: Chimeric protein of chemotaxis</p><br />
<br />
<p class="texte"><br />
Therefore, our idea is to switch the natural glucose specificity of <i>B. subtilis'</i>, mediated by McpA, to a NAG specificity. To achieve this, we need to change the extracellular domain of McpA, responsible for the specificity, by the extracellular domain of VCD.<br />
The whole sequence has been designed <i>in silico</i> and codon optimized for the transcription in <i>B. subtilis</i> before its synthesis.</p><br />
<br />
<center><img width="600px" SRC="https://static.igem.org/mediawiki/2014/e/e4/Chimio2.png" alt="gene construct" style="margin-bottom:40px;"></center><br />
<p class="legend">Figure 3: Construction of the chemotaxis gene</p><br />
<br />
<p class="title1">References</p><br />
<ul><br />
<br />
<li class="tree"><p class="texte">K. Meibom,L. Xibing, A. Nielsen, CY. Wu, S. Roseman and G. Schoolnik.<b> The Vibrio cholerae chitin utilization program </b>. The National Academy of Sciences of the USA (2004).</p></li><br />
<li class="tree"><p class="texte">C. Kristich and GW. Ordal. <b><i>Bacillus subtilis</i> CheD is a chemoreceptor modification enzyme required for chemotaxis</b>. J Biol Chem. 2002 Jul 12;277(28):25356-62. Epub 2002 May 13.<br></p></li><br />
</ul><br />
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{{:Team:Toulouse/template/footer}}</div>Jourdanhttp://2014.igem.org/Team:Toulouse/Project/OverviewsTeam:Toulouse/Project/Overviews2014-10-17T19:57:21Z<p>Jourdan: </p>
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<p>SubtiTree, a bacterium to save our trees</p><br />
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Among many other things, Southern France is well-known for the gorgeousness of its landscapes. Plane trees (<i>Platanus sp.</i>) are widely present and participate to the charm of this area, especially along the famous “Canal du Midi”. It is impossible to imagine this UNESCO World Heritage masterpiece without its trees. Unfortunately, these trees are threatened by a severe fungal infection called Canker Stain, and today the only treatment consists in a costly preventive tree-cutting and implies significant ecological troubles.<br />
</p><br />
<br />
<p class="texte"><br />
Facing this emergency, the students from the iGEM Toulouse Team, who enjoy chilling in this peaceful place, decided to be committed to the protection of their local heritage. Using a bacterium vector naturally present in the trees, our team offers an alternative solution originated from synthetic biology. Using different genetic modules, we engineered a bacterium (SubtiTree) capable of heading towards the pathogen, binding to its cell wall and finally delivering different fungicides to save the tree from its invaders. Our team also began to elaborate different strategies to prevent any accidental spreading of the optimized microorganism, thus limiting the ecological and ethical footprints of SubtiTree. Although our project originated from a very local and specific tree disease, it could be transposable to other plant diseases.<br />
</p><br />
<br />
<br />
<p class="title1">Choice of the chassis </p><br />
<p class="texte"><i>Bacillus subtilis</i> has been reported to be an endophyte bacterium of a large variety of plants and trees. Therefore, this model organism is a perfect chassis for our project. Already used to treat plant diseases and fungal pathogens, we aim to engineer <i>Bacillus subtilis</i> to fight Canker Stain from the inside of the tree. Injected directly in the tree sap, our smart bacterium will act as a curative and preventive drug.</p><br />
<br />
<center><img style="width:700px; margin: -10px 0 55px 130px;" ; src="https://static.igem.org/mediawiki/parts/2/2b/Overview_.jpg"></center><br />
<p class="legend">Figure 1: Schema of our general strategy</p><br />
<br />
<p class="title1">Chemotaxis<a href="https://2014.igem.org/Team:Toulouse/Project/Chemotaxis"; style="font-size: 13px; cursor: pointer; color: #888; margin-left: 10px;"> Show more</a></p><br />
<br />
<p class="texte">First, the bacterium targets the pathogen with a chemotaxis module which recognizes the soluble molecules released by the fungus' cell wall (N-acetylglucosamine).</p><br />
<br />
<br />
<p class="title1">Binding<a href="https://2014.igem.org/Team:Toulouse/Project/binding"; style="font-size: 13px; cursor: pointer; color: #888; margin-left: 10px;"> Show more</a></p><br />
<br />
<p class="texte">Then, SubtiTree binds onto the pathogen using a chimeric protein anchored to the bacterium peptidoglycan and which can make a bridge between bacterial cell wall and fungal chitin, the main component of the pathogen's cell wall.</p><br />
<br />
<p class="title1">Fungicides<a href="https://2014.igem.org/Team:Toulouse/Project/Fungicides"; style="font-size: 13px; cursor: pointer; color: #888; margin-left: 10px;"> Show more</a></p><br />
<br />
<p class="texte">In the third step, our designed bacterium fights the pathogen by producing a powerful treatment of three different fungicides.</p><br />
<br />
<p class="title1">Spreading <a href="https://2014.igem.org/Team:Toulouse/Project/Spreading"; style="font-size: 13px; cursor: pointer; color: #888; margin-left: 10px;">Show more</a></p><br />
<p class="texte">Our team worked on different aspects to control SubtiTree's spreading. The aim is to prevent horizontal transfers between different bacteria and to limit the growth and the survival of the engineered bacterium inside the tree during one season.</p><br />
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{{:Team:Toulouse/template/footer}}</div>Jourdanhttp://2014.igem.org/Team:Toulouse/Result/experimental-resultsTeam:Toulouse/Result/experimental-results2014-10-17T18:32:57Z<p>Jourdan: </p>
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<h2>Experimental results</h2><br />
<p> Are our modules functionnal? </p><br />
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<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Results&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Experimental results</p> <br />
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<p class="texte">How did we validate the three modules and improve our new protocols? Click below to find out…</p><br />
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<p style="font-size:1.3em; margin: 0 0 30px 0;"><a href="#" onClick="ddaccordion.expandall('technology'); return false">Expand all</a> | <a href="#" onClick="ddaccordion.collapseall('technology'); return false">Collapse all</a></p><br />
<br />
<div class="technology">Chemotaxis</div><br />
<div class="thelanguage"><br />
<br />
<br />
<p class="texte"> We used and developed several protocols to demonstrate the existence of chemotaxis in <i>B. subtilis</i> wild type (WT) strain and SubtiTree bacterium towards N-Acetylglucosamine. <br />
</p><br />
<p class="title2">1. Petri Dishes Test </p><br />
<br />
<p class="texte">We first wanted to visualize chemotaxis on Petri dishes. We hoped to obtain pictures with bacteria halos directed or around attractive components and thus tried different protocols. The first protocol was adapted from the one published by <a href="https://2011.igem.org/Team:Imperial_College_London/Protocols_Chemotaxis">the Imperial College 2011 iGEM team</a>. They put the attractive compound on a paper disk in the middle of a Petri dish containing a medium with 0.3% agar. Cells are loaded in this medium (Figure 1).<br />
</br><br />
</br><br />
<b>We first tried to test chemotaxis onto Petri Dishes filled with a 0.3% agar medium. This semi-solid medium allows the bacterial motility. A paper disk containing an attractive compound is placed in the middle of the dish and cells are then loaded in the medium (see Figure 1). This protocol was taken from the <a href="https://2011.igem.org/Team:Imperial_College_London/Protocols_Chemotaxis">the Imperial College 2011 iGEM team</a></b>.</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/0/05/Schema_1.png" alt="schema Figure 1" style="width:500px"></center><br />
<p class="legend">Figure 1: Scheme showing how cells are filled into the medium. (A) A pipet tip is used to deposit cells in the gelose. (B) Bacteria should move toward the attractive compound which diffuses.</p><br />
<br />
<p class="texte">We did not have any result with WT <i>Bacillus subtilis</i> and glucose as attractive compound (Figure 2-A). <i>B. subtilis</i> is attracted by many other glucides and amino-acids, so we also tried to test diluted glucose in LB medium attractant (Figure 2-B).</p><br />
<br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/f/ff/Fig2_AetB.png" alt="Figure 2" style="width:750px"></center><br />
<p class="legend">Figure 2: Chemotaxis test with Glucose as attractive compound (A) and Glucose added to LB medium as attractant (B).</p><br />
<br />
<p class="texte"> We could not notice any difference between the petri dish with or without glucose. With an addition of LB medium to sugar, a large halo around the paper disk was noticeable. This halo may correspond to cells attracted by the solution, as it is not noticeable when cells are not added (data not shown). Anyway we did not have enough reproducible and reliable results to be satisfied with this test.<br> <br />
Furthermore, with the addition of LB medium, it is hard to make the distinction between the attractive effects and the simple growth resulting from random diffusion.</br><br />
We have started new tries using different protocols.</p><br />
<br />
<p class="title2">2. Plug in Pond system<br />
</p><br />
<br />
<p class="texte"><br />
This protocol we worked on is taken from a thesis (see references [1]). . A solution of <i>B.subtilis</i> is grown overnight so as to obtain a cell density of 8x10⁸ cells/mL. 10mL of the solution is mixed with 15mL of LB medium with 1.5 % agar kept at 45°.The final concentration of the obtained medium is 0.9% agar. Tetracycline is aded at 25µg/mL, in order to inhibit growth and to only observe the chemotaxis phenomenon. Plates are cooled and dried, before digging wells with a punch or 1mL tips. The wells are filled with attractive compounds (Figure 3). After one hour at room temperature, photos of the plates are taken and the results are analyzed.<br />
</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/c/cd/Fig3.png" alt="Figure 3" style="width:400px"></center><br />
<p class="legend">Figure 3: Schema showing how are made plug-in-pond tests.</p><br />
<br />
<p class="texte"><br />
On our first try with <i>B. subtilis</i>, we made three wells per plate (Figure 4).The wells were filled with glucose at different concentrations and tetracycline was not added in one of the plates.<br />
</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/c/ce/Fig4.png" alt="Figure 4" style="width:400px"></center><br />
<p class="legend">Figure 4: Plates after 12h at room temperature.</p><br />
<br />
<p class="texte"><br />
After an hour, no tangible results were obtained. It is only after 12hours that we were able to observe halos around the wells with glucose at 1M in the plates without tetracycline. Tetracycline concentration seems to be too high and inhibits any bacterial activity. Therfore, we have worked with tetracycline at 15µg/mL.<br />
We tried this protocol again with this new condition. We made two wells per plate (Figure 5), one with either Glucose or N-acetyl-glucosamine and one with LB medium. As previsously, no results were achieved after 1h, but after 12hours we could notice halos.<br />
</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/c/c3/Bsubtilis_result.png" alt="Figure 5" style="width:750px"></center><br />
<p class="legend">Figure 5: Chemotaxis test with <i>Bacillus subtilis</i> WT. The upper wells contain attractive compound and the lower contain medium without attractive compound. </p><br />
<br />
<p class="texte"><br />
Results are not as clear as the first time, but we observed halos around the well with glucose at 250mM with and without tetracycline. We have then tried the same experiment with N-acetyl-glucosamine and we did not see any halo in the tested conditions. Thus we assumed that our <i>B. subtilis</i> 168 strain was not attracted to N-acetyl-glucosamine.<br />
However, the results are not clear, reliable and reproducible enough with the plug-in-pond protocol. Another testing protocol was then adopted. <br />
<br />
</p><br />
<br />
<br />
<p class="texte"><br />
<b>References:</b></br><br />
[1]: Etude de la réponse adaptative à l'oxyde de triméthylamine et de son mécanisme de détection chez <i>Escherichia coli</i> et <i>Shewanella oneidensis</i>, 2008, Claudine Baraquet, Université de la Méditerranée Aix-Marseille II<br />
</p><br />
<br />
<br />
<br />
<br />
<p class="title2">4. Capillary test between two tubes also called the tubes test</p><br />
<p class="texte">After the experiment of the plug-in-pond, we decided to construct a system by welding two Eppendorf tubes with a capillary thanks to an electric burner.</p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/f/fb/Chemotaxis_-_eppendorf.png"></center><br />
<p class="legend">Figure 6: Photography of the first tubes system</p><br />
<br />
<p class="texte">We tested this system with a fuchsin dye and water and we were able to observe the diffusion of fuchsin towards water. However this construction had a leakage next to the weld seam that we could not stop. <br />
Thus, we asked the help from the INSA glass blower, Patrick Chekroun. He designed two systems composed of two tubes linked by a capillary.</p><br />
<br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/2/2b/Chemotaxis_-_tubes.png"><center><br />
<p class="legend">Figure 7: Scheme of the tubes system</p><br />
<br />
<p class="texte">As we did previously, we tested this new system with fuchsin. This experiment was made with WT <i>Bacillus subtilis</i> and N-Acetylglucosamine.<br />
<br><br><br />
<i>NB: We could not see the diffusion from one tube to the other. We made the hypothesis that it was not visible by sight because of the small diameter of the capillary. <br />
</i><br><br />
<br><br />
The following strategy was used to avoid disturbance due to pressure and liquid movement through the capillary:<br><br />
- The first step was the addition of Wash Buffer until the capillary was full to avoid the presence of air bubbles which could lead to diffusion problems.<br><br />
- Then, the tube 2 was plugged with the thumb while another person was adding the bacterial solution of WT <i>Bacillus subtilis</i> in the tube 1. <br><br />
- The tube 1 was also plugged and only after the thumb could be removed from the tube 2. <br><br />
- In the same way, the N-Acetylglucosamine was added in the tube 2. <br><br />
- The same process was made with a xylose positive control.<br><br />
<br><br />
<i>NB: According to the article Chemotaxis towards sugars by </i>Bacillus subtilis, (George W. Ordal et al., 1979), <i>glucose and xylose have the same attractant power. We prefer a positive control instead of a negative one because we were not sure that this system was efficient.</i><br><br />
<br><br />
- The system was kept straight for 2hours. Every 40 minutes, we took a sample of each tube and spread it on an agar plate (dilution 1/1,000).</p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/1/1b/Chemotaxis_-_tubes_photo.png"></center><br />
<p class="legend">Figure 8: Photography of the tubes system</p><br />
<br />
<br />
<p class="texte">Unfortunately, the dilution was too high to detect any chemotaxis movement and the time was too short. We did not find any information in the literature.<br><br />
As we did not have the time to optimize this protocol we preferred using the protocol of the 2011 Imperial college iGEM team : the tips capillary test.</br><br />
</p><br />
<br />
<p class="title2"> 5. Tips capillary system</p><br />
<p class="title3">First tips capillary system</p><br />
<p class="texte">This protocol comes from 2011 Imperial College iGEM team and was adapted by our team in several steps (See <a href="https://2014.igem.org/Team:Toulouse/Notebook/Protocols#select8">chemotaxis protocol</a>).<br />
<br><br />
In the first tips capillary system, we used parafilm to avoid any kind of air disturbance in the tips. The different steps are described below:<br><br />
- 15µL of each chemo-attractant was pipetted. <br><br />
- The bottom of tip with the pipette was then put on a piece of parafilm and the pipette was removed from the top of the tip.<br><br />
- The top of the tip was then sealed with a piece of parafilm. By this way, the sterility can be assured and the liquid stays inside the tip. <br><br />
- To finish, the level of the solution in the tip was marked.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/9/94/Chemotaxis_-_tip.png"></center><br />
<p class="legend">Figure 9: Sealing of a tip with parafilm</p><br />
<br />
<p class="texte">- After all the attractants were added in the tips, we put them on a green base to carry them. The whole process can be seen on Figure 10.<br><br />
- Each tip was immersed in 300 µL of a bacterial solution in the wells of an Elisa plate.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/0/05/Chemotaxis_-_tip_and_support.png"></center><br />
<p class="legend">Figure 10: First tips capillary system</p><br />
<br />
<p class="texte"><i>NB: the yellow carton was used to stabilize the system and keep it straight.</i><br><br />
<br><br />
- After one hour, the tips were removed from the bacteria solutions and the content of the tips was observed with a Thoma cell under the microscope.<br><br />
<br><br />
We had several problems with this system:<br><br />
- The liquid level decreased during the experiment and we did not have enough liquid to fill the Thoma cell. Thus, it was not possible to count.<br><br />
- The bacteria were moving and therefore, we could not proceed to a bacteria count.<br><br />
<br><br />
Regarding these observations we decided to spread the tips content on agar plates instead of using Thoma cell and microscopy.<br><br />
<p class="title3">Second tips capillary system<br />
</p><br />
<p class="texte"And then the revolution came! We found a multichannel pipette :D The same protocol was performed except that the parafilm was used to avoid the air entrance between the tips and the pipette and therefore the loss of liquid.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/e/e4/Chemotaxis_-_pipette.png"></center><br />
<p class="legend">Figure 11: Second tips capillary system</p><br />
<br />
<p class="title3">Improvement of the second tips capillary system</p><br />
<p class="texte">However this system was not optimal it is why we decided to use blu tack instead of parafilm: <br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/4/42/Chemotaxis_-_pipette_and_blu_tack.png"></center><br />
<p class="legend">Figure 12: Improvement of the second tips capillary system</p><br />
<br />
<p class="texte"><b>At that point, the protocol was approved and the final test could finally start! :-)</b><br><br />
<br><br />
There was just one tiny problem… we did not have our optimized bacterium transformed with the chemotaxis module!!! That is why we concentrated our efforts on WT <i>Bacillus subtilis</i> strain.<br><br />
<br><br />
The main goal was to find an optimized control and to analyze the eventual chemotaxis of the WT strain. To avoid osmolality bias, we wanted to find a molecule which was non-attractant and with a similar molecular weight than the N-Acetylglucosamine (221.21 g/mol). Our first idea was to use fuchsin (Molecular weight: 337.85 g/mol).<br><br />
<br><br />
At the beginning, the experiment was conducted with only one negative contraol, the fuchsin and different NAG concentrations: 25mM, 250mM and 500mM. The tested strain was <i>Bacillus subtilis </i>168:<br><br />
<br></p><br />
<center><br />
<table align="center"><br />
<tr><td align=center><img src="https://static.igem.org/mediawiki/2014/8/8c/Chemotaxis_-_results_fuch.png"></td><br />
<td align=center><img src="https://static.igem.org/mediawiki/2014/f/fd/Chemotaxis_-_results_fuchsin.png"></td></tr><br />
<tr><td align=center><p class="legend">Figure 13: Fuchsin - negative control (dilution 1/50)</p></td><br />
<td align=center><p class="legend">Figure 14: NAG (25mM) (dilution 1/50)</p></td></tr><br />
</table></center><br><br />
<p class="texte">The average number of colonies with the negative control is 121. On the contrary, a cell layer is observed for the NAG plates with every concentration.<br><br />
<br><br />
Thus, we assumed that WT <i>Bacillus subtilis</i> was more attracted by NAG than fuchsin. Indeed we can neglect the bacterial growth because the test only lasts one hour. We also neglect diffusion and osmolality phenomena for the previous reasons. <br><br />
<br><br />
Unfortunately for us we forgot one major effect… Can you believe that fuchsin solution contains about 15% of ethanol?!!! This concentration can lead to the death of some cells which probably happened to our results.<br><br />
<br><br />
<b><p class="texte">This incredible and dramatic discovery destroyed all of our hopes about the God of chemotaxis! :-(</b><br><br />
<br><br />
However, our team did not give up on synthetic biology ! :-) Indeed, after days of disappointment and no time left for lab work, we raised from ashes and tried to find another negative control.<br><br />
<br><br />
Hopefully, we managed to find a negative control: galactose (25mM). The article Chemotaxis towards sugars by <i>Bacillus subtilis</i> (<i>George W. Ordal et al., 1979</i>) proved that it was a poor attractant.<br><br />
<br><br />
We made our tests again with this new molecule and glucose (25mM) as positive control.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/8/86/Chemotaxis_-_final_results.png"></center><br />
<p class="legend">Figure 15: Final results (dilution : 1/10,000)</p><br />
<p class="texte"> The miracle arrived! We managed to prove that our WT <i>Bacillus subtilis</i> was indeed naturally attracted to NAG.</p><br />
<p class="texte"><i>NB: It was our last experiment. Unfortunately we were running out of time and we could not do much more test. We would like to do the experiment with a lower dilution and repeat it several times.</i><br><br />
<br><br />
<b><p class="texte">Our results are not statistically significant however this result has been proved in literature.</p></b><br></p><br />
<br />
</br><br />
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<br />
<div class="technology">Binding module</div><br />
<div class="thelanguage"><br />
<br />
<br />
<p class="title2">1. Preliminary experiments</p><br />
<p class="title3">Purpose</p><br />
<p class="texte">The first experiment deals with the culture conditions to see if <i>Bacillus subtilis</i> can resist to a low temperature and with the Chitin Beads Buffer (CBB) buffer. To do that, several bacterial concentrations have been tested starting with an OD of 0.1 and diluting this solution to get estimated ODs of 0.05, 0.025, 0.01. These different <i>Bacillus subtilis</i> solutions were incubated 1 hour at 4°C with 500µL of CBB or water. Finally a cell count on Thoma cell counting chamber was performed.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">We could not count bacteria either because of the high number of bacteria with the 0.1 OD solution or the low number of bacteria with the 0.01 OD solution. Thus, the study is mostly focused on the intermediate values (Figure 16).<br />
<br/>First of all, cells do not seem affected by the presence of CBB or water with estimated ODs of 0.05 or 0.025. Moreover, twice less cells can be found in the lowest concentrations in bacteria comparing to the 0.05 OD concentration which is in agreement with the dilution ratio.<br />
</br>Thus the the experimental conditions regarding the presence of CBB and the incubation temperature at 4°C are compatible with the bacterial life. <br />
<br/>Thus, the experimental conditions regarding the presence of CBB and the incubation temperature at 4°C do not harm the cell surviving.<br />
</p><br />
<br />
</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/c/ce/Graphe_binding_1.png" width="45%"></center><br />
<br />
</br><br />
<p class="legend">Figure 16: CBB presence has no effect on bacterial survival. The bacterial concentration was measured in <span style="color:#0000FF">the presence</span> or <span style="color:#FF0000">the absence </span> of CBB for the observed OD (0.1) or estimated ODs (0.05, 0.025, 0.01).</p><br />
<br />
<p class="title2">2. Binding test using engineered <i>B. subtilis</i></p><br />
<br />
<p class="title3">Purpose</p><br />
<p class="texte"><i>B. subtilis</i> transformed with the binding module should produce a chimeric protein composed of the Cell Wall Binding Domain of LycT to attach the chimeric protein to the cell wall, and the GbpA domain 4 of <i>Vibrio cholerae</i> to bind chitin. The synthetic bacterium is put in contact with chitin beads (chitin: polymer present on the fungal pathogen wall). After several washes, bacteria remaining on the beads are counted.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">The first observation is that both bacterial solutions of wild type <i>Bacillus subtilis</i> and SubtiTree have the same concentration: 10<sup>5</sup> bacteria/mL (Figure 17). Even though there is no significant difference between both strains after the first wash, the second wash has a major effect since it removes 40 times more wild-type bacteria than SubtiTree. This result correlates to the number of Subtitree bound to the beads. <br />
<br/>Thus, the binding system is validated: SubtiTree binds efficiently to chitin.</p><br />
<br />
</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/e/ea/Graphe_binding_2.png" width="60%"></center><br />
</br><br />
<br />
<p class="legend">Figure 17: Attachment of WT <i>B. subtilis</i> and Subtitree the to chitin. The <span style="color:#0000FF">WT bacteria</span> or <span style="color:#FF0000">the bacteria with the binding system</span> concentrations have been determined during the different steps of the binding test. The stars represent a significant difference observed with a Student test with p<0.05.<br />
</p><br />
<br />
<p class="title2">3. Microscopic observations</p><br />
<br />
<p class="title3">Purpose</p><br />
<p class="texte">We wanted to observe SubtiTree bound on the chitin coated beads. In order to perform a 3D reconstruction showing this interaction, we used confocal laser scanning microscope. Through the use of a fluorochrome (Syto9), we highlighted the presence of bacteria on the surface of the beads (individualized by phase-contrast). A first calibration step determined the minimum threshold to remove the background noise and the natural fluorescence.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">First, we can notice that SubtiTree is sitting (well, sort off!!) on the surface of beads coated with chitin. These images seem to highlight their interactions.</br></p><br />
<center><img src="https://static.igem.org/mediawiki/2014/archive/5/53/20141013073044!Photo_billes_microscopie.png" width="45%"></center><br />
</br><br />
<p class="legend">Figure 18: Microscopic view of beads surfaces coated with chitin</p><br />
<br />
<p class="texte">Using ImageJ software, we are able to create 3D pictures and movies of those comments.</br></p><br />
<center><img src="https://static.igem.org/mediawiki/2014/5/53/Photo_billes_microscopie.png" width="45%" style="float:left;"><iframe width="380" height="315" src="//www.youtube.com/embed/ztIHIKQr3g0" frameborder="0" allowfullscreen></iframe></center><br />
</br><br />
<p class="legend">Figure 19: A short movie of 3D bead surfaces coated with chitin and Subtitree (emotional sequence for Subtitree: first movie apparition, before Cannes…)</p><br />
<br />
<p class="texte">We then performed a wash step on the chitin beads. We measured the release of bacteria on the washing solution. When our bacterium has the binding module, there is less release and therefore, SubtiTree is retained by the beads.</p><br />
<center><img src="https://static.igem.org/mediawiki/2014/9/97/Photo_lavage_microscopie.png" width="45%"></center><br />
<p class="legend">Figure 20: Microscopic view of bacteria after washing <br />
</p><br />
<br />
<p class="texte">Finally, all results are consistent with the presence of functional binding system. We thus validate the second module.</p><br />
<br />
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<br />
<div class="technology">Fungicides module</div><br />
<div class="thelanguage"><br />
<br />
<br />
<p class="title2"> 1. Preliminary experiments</p><br />
<p class="title3">Tests with commercial peptides and controls</p><br />
<p class="texte">The first tests were accomplished with commercial GAFP-1 and D4E1 peptides at different concentrations (12.5µM, 25µM, 100µM). These tests were performed on different fungal strains sharing the same phylum with <i>Ceratocystis Platani</i>.<br />
As <i>Ceratocystis Platani</i> is pathogenic, we could not perform tests directly with this fungus.</br><br />
After several days at 30°C, the PDA (Potato Dextrose Agar) plates covered with fungus and commercial peptides were analyzed.</p></p><br />
<p class="texte">An inhibition halo was noticeable with commercial D4E1 peptide at 100µM on <i>Aspergillus brasiliensis</i>. Less bright halos were also present with lower concentrations. Concerning commercial GAFP-1, we did not notice any effect in the tested conditions. As positive control, a well-known chemical fungicide was used: the Copper Sulfate. The inhibition of the fungal growth was complete at 20mg/ml, and at 10mg/ml a darker halo appeared around the pad filled with Copper Sulfate as we can see on the figure below. This corresponds to a sporulating halo in response to the stress generated by the fungicide.<br />
</p><br />
<br />
<br />
<center><img style="width:450px; " src="https://static.igem.org/mediawiki/parts/a/a8/Prelim_tests_fung.jpg"><br />
<img style="width:450px; " src="https://static.igem.org/mediawiki/parts/f/f8/Controls_fung.jpg"></center><br />
<p class="legend"> Figure 21: Results of the preliminary tests</p><br />
<br />
<br />
</br><br />
<br />
<p class="texte">Regarding these results, we concluded that very high fungicide concentrations are required to inhibit the fungal growth in the tested conditions. Following these tests, new conditions were adopted in order not to encourage too much fungal growth over bacterial growth. The culture medium was adjusted to fit our objective and to approximate the conditions found in the trees: a 'sap-like' medium was elaborated. The incubations were then carried at room temperature.<br><br />
We also concluded that turning blue the Canal du Midi using high copper sulfate concentrations is not such a good idea... Thereby strengthening our faith in SubtiTree :-) ! <br />
</p><br />
<br />
<p class="title2">2. Test with SubtiTree</p><br />
<br />
<p class="texte">In order to test <i>Bacillus subtilis</i> mutants, it was essential to find the right balance between the fungal growth and the bacterial one. This condition was necessary to get a high concentration of peptides. In our genetic constructions, these peptides are designed to be exported in the extracellular medium.</br><br />
</br><br />
The transformed <i>Bacillus subtilis</i> strains grew at 37°C during 72h and were tested. After centrifugation, the supernatant and the resuspended pellet were placed on pads and disposed on plates previously seeded with a defined number of conidia (see protocols to have more details). After several days at room temperature, an inhibition halo of <i>Trichoderma reesei</i>'s growth was clearly observable for the strain expressing D4E1 gene. The inhibition was even more noticeable with the strain carrying the GAFP-1 + D4E1 operon (see the photos below).</br><br />
However, no effect was detected for the strain expressing the GAFP-1 gene, supposing a synergistic effect between these two peptides.</br><br />
Regarding EcAMP and the triple-fungicides operon, no effect has been detected on the fungal growth. Several factors can explain these results: a number of post-transcriptional modifications are required to have a functional EcAMP and in addition to that, sequencing results of these constructs showed some differences with the original designed sequence.<br />
<p class="texte">Inhibition halos are not visible with supernatants, probably because of their low concentrations in the extracellular medium. <br />
Another effect was noted with the same strains expressing D4E1 and GAFP-1 + D4E1 on another fungus <i>Aspergillus brasiliensis</i>. This effect is comparable to the one previously noted with a low concentration of sulfate copper. </br><br />
</p><br />
<br />
</br><br />
<img style="width:400px; " src="https://static.igem.org/mediawiki/parts/c/c2/Resultfong.jpg"><br />
<img style="width:400px; " src="https://static.igem.org/mediawiki/parts/9/92/Results_fong_2.jpg"> <p class="legend">Figure 22: Results with transformed bacteria.</p><br />
<br />
<p class="texte"><br />
The choice of our chassis appears to be optimal as we noted that wild type <i>Bacillus subtilis</i> disturbs the hyphae growth of the fungi. Some strains of <i>Bacillus subtilis</i> (qst 713) are already used as Biofungicides for use on several minor crops to treat a variety of plant diseases and fungal pathogens.</br><br />
After this set of experiments, the strains expressing D4E1 and expressing GAFP-1 + D4E1 have shown to be the best candidates to play a major role in the fight against fungal diseases such as Canker stain. Keeping in mind our objective, <b> we decided to tests these strains in model plants</b>: <i>Nicotiana benthamiana</i> and <i>Arabidopsis thaliana</i>.</br><br />
These tests were performed in the National Institute for the Agronomic Research by experts in this domain. <br />
<br />
<br />
<p class="title2">3. <i>In planta</i> tests with SubtiTree</p><br />
<br />
<br />
<center><img style="width:215px;" img src="https://static.igem.org/mediawiki/parts/a/af/In_planta.jpg" style="margin-top:5px"/></center><br />
<p class="legend"> Figure 23: Injection of SubtiTree in a model plant</p><br />
<br />
<p class="texte"><br />
The goal of the project is to introduce the transformed bacteria in a diseased tree. So it is necessary to perform <i> in planta </i> tests to judge the fungus-killing abilities of the two strains selected after the previous set of experiments. </br><br />
SubtiTree is first inoculated in two model plants (<i>Arabidopsis thaliana</i> and <i>Nicotiana benthamiana</i>). After this step, a phytopathogenic fungus (<i>Sclerotinia sclerotiorum</i>) is placed on the leaves. </br><br />
These tests were made in association with Sylvain Raffaële and Marielle Barascud of the National Institute for the Agronomic Research laboratory. </br><br />
</p><br />
<br />
<br />
<br />
<p class="texte">Twenty-four hours after SubtiTree inoculation, no phenotypic modification of the leaves can be detected. We can conclude that our bacterium, its introduction and the fungicides production in plants do not have deleterious effects.</br><br />
Without proper treatment, the drop of the pyhtopathogenic fungus on <i>Nicotiana benthamiana</i>'s leaves causes a necrosis halo which can be measured after 40h. The lesion size and the number of inoculated sites seem to be reduced by <i>B. subtilis</i> expressing DE41 or GAFP1-D4E1, unlike with the WT bacterium. A second set of experiments is expected to be more statistically precise.</br><br></br><br />
We did not observe any significant results for <i>Arabidopsis thaliana</i> because of the use of two plants batches with different ages.</br><br />
<br />
We can therefore conclude that when SubtiTree is in plant's physiological conditions, <b> it is harmless to the plant, and that the production of fungicides is effective, reducing the leaves necrosis. </b><br />
</p><br />
<br />
<center><img style="width:860px;" img src="https://static.igem.org/mediawiki/parts/f/f1/Results_d4%2B_gafp1.jpg"></center> <p class="legend">Figure 24: Results of <i>in planta</i> test</p><br />
<br />
<br />
<p class="texte">Thanks to the diversity of anti-fungal peptides, this strategy can be adapted to different types of diseases, with different degree of specifity, etc.</p><br />
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<h2>Experimental results</h2><br />
<p> Are our modules functionnal? </p><br />
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<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Results&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Experimental results</p> <br />
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<br />
<p class="texte">How did we validate the three modules and improve our new protocols? Click below to find out…</p><br />
<br />
<p style="font-size:1.3em; margin: 0 0 30px 0;"><a href="#" onClick="ddaccordion.expandall('technology'); return false">Expand all</a> | <a href="#" onClick="ddaccordion.collapseall('technology'); return false">Collapse all</a></p><br />
<br />
<div class="technology">Chemotaxis</div><br />
<div class="thelanguage"><br />
<br />
<br />
<p class="texte"> We used and developed several protocols to demonstrate the existence of chemotaxis in <i>B. subtilis</i> wild type (WT) strain and SubtiTree bacterium towards N-Acetylglucosamine. <br />
</p><br />
<p class="title2">1. Petri Dishes Test </p><br />
<br />
<p class="texte">We first wanted to visualize chemotaxis on Petri dishes. We hoped to obtain pictures with bacteria halos directed or around attractive components and thus tried different protocols. The first protocol was adapted from the one published by <a href="https://2011.igem.org/Team:Imperial_College_London/Protocols_Chemotaxis">the Imperial College 2011 iGEM team</a>. They put the attractive compound on a paper disk in the middle of a Petri dish containing a medium with 0.3% agar. Cells are loaded in this medium (Figure 1).<br />
</br><br />
</br><br />
<b>We first tried to test chemotaxis onto Petri Dishes filled with a 0.3% agar medium. This semi-solid medium allows the bacterial motility. A paper disk containing an attractive compound is placed in the middle of the dish and cells are then loaded in the medium (see Figure 1). This protocol was taken from the <a href="https://2011.igem.org/Team:Imperial_College_London/Protocols_Chemotaxis">the Imperial College 2011 iGEM team</a></b>.</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/0/05/Schema_1.png" alt="schema Figure 1" style="width:500px"></center><br />
<p class="legend">Figure 1: Scheme showing how cells are filled into the medium. (A) A pipet tip is used to deposit cells in the gelose. (B) Bacteria should move toward the attractive compound which diffuses.</p><br />
<br />
<p class="texte">We did not have any result with WT <i>Bacillus subtilis</i> and glucose as attractive compound (Figure 2-A). <i>B. subtilis</i> is attracted by many other glucides and amino-acids, so we also tried to test diluted glucose in LB medium attractant (Figure 2-B).</p><br />
<br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/f/ff/Fig2_AetB.png" alt="Figure 2" style="width:750px"></center><br />
<p class="legend">Figure 2: Chemotaxis test with Glucose as attractive compound (A) and Glucose added to LB medium as attractant (B).</p><br />
<br />
<p class="texte"> We could not notice any difference between the petri dish with or without glucose. With an addition of LB medium to sugar, a large halo around the paper disk was noticeable. This halo may correspond to cells attracted by the solution, as it is not noticeable when cells are not added (data not shown). Anyway we did not have enough reproducible and reliable results to be satisfied with this test.<br> <br />
Furthermore, with the addition of LB medium, it is hard to make the distinction between the attractive effects and the simple growth resulting from random diffusion.</br><br />
We have started new tries using different protocols.</p><br />
<br />
<p class="title2">2. Plug in Pond system<br />
</p><br />
<br />
<p class="texte"><br />
This protocol we worked on is taken from a thesis (see references [1]). . A solution of <i>B.subtilis</i> is grown overnight so as to obtain a cell density of 8x10⁸ cells/mL. 10mL of the solution is mixed with 15mL of LB medium with 1.5 % agar kept at 45°.The final concentration of the obtained medium is 0.9% agar. Tetracycline is aded at 25µg/mL, in order to inhibit growth and to only observe the chemotaxis phenomenon. Plates are cooled and dried, before digging wells with a punch or 1mL tips. The wells are filled with attractive compounds (Figure 3). After one hour at room temperature, photos of the plates are taken and the results are analyzed.<br />
</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/c/cd/Fig3.png" alt="Figure 3" style="width:400px"></center><br />
<p class="legend">Figure 3: Schema showing how are made plug-in-pond tests.</p><br />
<br />
<p class="texte"><br />
On our first try with <i>B. subtilis</i>, we made three wells per plate (Figure 4).The wells were filled with glucose at different concentrations and tetracycline was not added in one of the plates.<br />
</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/c/ce/Fig4.png" alt="Figure 4" style="width:400px"></center><br />
<p class="legend">Figure 4: Plates after 12h at room temperature.</p><br />
<br />
<p class="texte"><br />
After an hour, no tangible results were obtained. It is only after 12hours that we were able to observe halos around the wells with glucose at 1M in the plates without tetracycline. Tetracycline concentration seems to be too high and inhibits any bacterial activity. Therfore, we have worked with tetracycline at 15µg/mL.<br />
We tried this protocol again with this new condition. We made two wells per plate (Figure 5), one with either Glucose or N-acetyl-glucosamine and one with LB medium. As previsously, no results were achieved after 1h, but after 12hours we could notice halos.<br />
</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/c/c3/Bsubtilis_result.png" alt="Figure 5" style="width:750px"></center><br />
<p class="legend">Figure 5: Chemotaxis test with <i>Bacillus subtilis</i> WT. The upper wells contain attractive compound and the lower contain medium without attractive compound. </p><br />
<br />
<p class="texte"><br />
Results are not as clear as the first time, but we observed halos around the well with glucose at 250mM with and without tetracycline. We have then tried the same experiment with N-acetyl-glucosamine and we did not see any halo in the tested conditions. Thus we assumed that our <i>B. subtilis</i> 168 strain was not attracted to N-acetyl-glucosamine.<br />
However, the results are not clear, reliable and reproducible enough with the plug-in-pond protocol. Another testing protocol was then adopted. <br />
<br />
</p><br />
<br />
<br />
<p class="texte"><br />
<b>References:</b></br><br />
[1]: Etude de la réponse adaptative à l'oxyde de triméthylamine et de son mécanisme de détection chez <i>Escherichia coli</i> et <i>Shewanella oneidensis</i>, 2008, Claudine Baraquet, Université de la Méditerranée Aix-Marseille II<br />
</p><br />
<br />
<br />
<br />
<br />
<p class="title2">4. Capillary test between two tubes also called the tubes test</p><br />
<p class="texte">After the experiment of the plug-in-pond, we decided to construct a system by welding two Eppendorf tubes with a capillary thanks to an electric burner.</p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/f/fb/Chemotaxis_-_eppendorf.png"></center><br />
<p class="legend">Figure 6: Photography of the first tubes system</p><br />
<br />
<p class="texte">We tested this system with a fuchsin dye and water and we were able to observe the diffusion of fuchsin towards water. However this construction had a leakage next to the weld seam that we could not stop. <br />
Thus, we asked the help from the INSA glass blower, Patrick Chekroun. He designed two systems composed of two tubes linked by a capillary.</p><br />
<br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/2/2b/Chemotaxis_-_tubes.png"><center><br />
<p class="legend">Figure 7: Scheme of the tubes system</p><br />
<br />
<p class="texte">As we did previously, we tested this new system with fuchsin. This experiment was made with WT <i>Bacillus subtilis</i> and N-Acetylglucosamine.<br />
<br><br><br />
<i>NB: We could not see the diffusion from one tube to the other. We made the hypothesis that it was not visible by sight because of the small diameter of the capillary. <br />
</i><br><br />
<br><br />
The following strategy was used to avoid disturbance due to pressure and liquid movement through the capillary:<br><br />
- The first step was the addition of Wash Buffer until the capillary was full to avoid the presence of air bubbles which could lead to diffusion problems.<br><br />
- Then, the tube 2 was plugged with the thumb while another person was adding the bacterial solution of WT <i>Bacillus subtilis</i> in the tube 1. <br><br />
- The tube 1 was also plugged and only after the thumb could be removed from the tube 2. <br><br />
- In the same way, the N-Acetylglucosamine was added in the tube 2. <br><br />
- The same process was made with a xylose positive control.<br><br />
<br><br />
<i>NB: According to the article Chemotaxis towards sugars by </i>Bacillus subtilis, (George W. Ordal et al., 1979), <i>glucose and xylose have the same attractant power. We prefer a positive control instead of a negative one because we were not sure that this system was efficient.</i><br><br />
<br><br />
- The system was kept straight for 2hours. Every 40 minutes, we took a sample of each tube and spread it on an agar plate (dilution 1/1,000).</p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/1/1b/Chemotaxis_-_tubes_photo.png"></center><br />
<p class="legend">Figure 8: Photography of the tubes system</p><br />
<br />
<br />
<p class="texte">Unfortunately, the dilution was too high to detect any chemotaxis movement and the time was too short. We did not find any information in the literature.<br><br />
As we did not have the time to optimize this protocol we preferred using the protocol of the 2011 Imperial college iGEM team : the tips capillary test.</br><br />
</p><br />
<br />
<p class="title2"> 5. Tips capillary system</p><br />
<p class="title3">First tips capillary system</p><br />
<p class="texte">This protocol comes from 2011 Imperial College iGEM team and was adapted by our team in several steps (See <a href="https://2014.igem.org/Team:Toulouse/Notebook/Protocols#select8">chemotaxis protocol</a>).<br />
<br><br />
In the first tips capillary system, we used parafilm to avoid any kind of air disturbance in the tips. The different steps are described below:<br><br />
- 15µL of each chemo-attractant was pipetted. <br><br />
- The bottom of tip with the pipette was then put on a piece of parafilm and the pipette was removed from the top of the tip.<br><br />
- The top of the tip was then sealed with a piece of parafilm. By this way, the sterility can be assured and the liquid stays inside the tip. <br><br />
- To finish, the level of the solution in the tip was marked.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/9/94/Chemotaxis_-_tip.png"></center><br />
<p class="legend">Figure 9: Sealing of a tip with parafilm</p><br />
<br />
<p class="texte">- After all the attractants were added in the tips, we put them on a green base to carry them. The whole process can be seen on Figure 10.<br><br />
- Each tip was immersed in 300 µL of a bacterial solution in the wells of an Elisa plate.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/0/05/Chemotaxis_-_tip_and_support.png"></center><br />
<p class="legend">Figure 10: First tips capillary system</p><br />
<br />
<p class="texte"><i>NB: the yellow carton was used to stabilize the system and keep it straight.</i><br><br />
<br><br />
- After one hour, the tips were removed from the bacteria solutions and the content of the tips was observed with a Thoma cell under the microscope.<br><br />
<br><br />
We had several problems with this system:<br><br />
- The liquid level decreased during the experiment and we did not have enough liquid to fill the Thoma cell. Thus, it was not possible to count.<br><br />
- The bacteria were moving and therefore, we could not proceed to a bacteria count.<br><br />
<br><br />
Regarding these observations we decided to spread the tips content on agar plates instead of using Thoma cell and microscopy.<br><br />
<p class="title3">Second tips capillary system<br />
</p><br />
<p class="texte"And then the revolution came! We found a multichannel pipette :D The same protocol was performed except that the parafilm was used to avoid the air entrance between the tips and the pipette and therefore the loss of liquid.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/e/e4/Chemotaxis_-_pipette.png"></center><br />
<p class="legend">Figure 11: Second tips capillary system</p><br />
<br />
<p class="title3">Improvement of the second tips capillary system</p><br />
<p class="texte">However this system was not optimal it is why we decided to use blu tack instead of parafilm: <br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/4/42/Chemotaxis_-_pipette_and_blu_tack.png"></center><br />
<p class="legend">Figure 12: Improvement of the second tips capillary system</p><br />
<br />
<p class="texte"><b>At that point, the protocol was approved and the final test could finally start! :-)</b><br><br />
<br><br />
There was just one tiny problem… we did not have our optimized bacterium transformed with the chemotaxis module!!! That is why we concentrated our efforts on WT <i>Bacillus subtilis</i> strain.<br><br />
<br><br />
The main goal was to find an optimized control and to analyze the eventual chemotaxis of the WT strain. To avoid osmolality bias, we wanted to find a molecule which was non-attractant and with a similar molecular weight than the N-Acetylglucosamine (221.21 g/mol). Our first idea was to use fuchsin (Molecular weight: 337.85 g/mol).<br><br />
<br><br />
At the beginning, the experiment was conducted with only one negative contraol, the fuchsin and different NAG concentrations: 25mM, 250mM and 500mM. The tested strain was <i>Bacillus subtilis </i>168:<br><br />
<br></p><br />
<center><br />
<table align="center"><br />
<tr><td align=center><img src="https://static.igem.org/mediawiki/2014/8/8c/Chemotaxis_-_results_fuch.png"></td><br />
<td align=center><img src="https://static.igem.org/mediawiki/2014/f/fd/Chemotaxis_-_results_fuchsin.png"></td></tr><br />
<tr><td align=center><p class="legend">Figure 13: Fuchsin - negative control (dilution 1/50)</p></td><br />
<td align=center><p class="legend">Figure 14: NAG (25mM) (dilution 1/50)</p></td></tr><br />
</table></center><br><br />
<p class="texte">The average number of colonies with the negative control is 121. On the contrary, a cell layer is observed for the NAG plates with every concentration.<br><br />
<br><br />
Thus, we assumed that WT <i>Bacillus subtilis</i> was more attracted by NAG than fuchsin. Indeed we can neglect the bacterial growth because the test only lasts one hour. We also neglect diffusion and osmolality phenomena for the previous reasons. <br><br />
<br><br />
Unfortunately for us we forgot one major effect… Can you believe that fuchsin solution contains about 15% of ethanol?!!! This concentration can lead to the death of some cells which probably happened to our results.<br><br />
<br><br />
<b><p class="texte">This incredible and dramatic discovery destroyed all of our hopes about the God of chemotaxis! :-(</b><br><br />
<br><br />
However, our team did not give up on synthetic biology ! :-) Indeed, after days of disappointment and no time left for lab work, we raised from ashes and tried to find another negative control.<br><br />
<br><br />
Hopefully, we managed to find a negative control: galactose (25mM). The article Chemotaxis towards sugars by <i>Bacillus subtilis</i> (<i>George W. Ordal et al., 1979</i>) proved that it was a poor attractant.<br><br />
<br><br />
We made our tests again with this new molecule and glucose (25mM) as positive control.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/8/86/Chemotaxis_-_final_results.png"></center><br />
<p class="legend">Figure 15: Final results (dilution : 1/10,000)</p><br />
<p class="texte"> The miracle arrived! We managed to prove that our WT <i>Bacillus subtilis</i> was indeed naturally attracted to NAG.</p><br />
<p class="texte"><i>NB: It was our last experiment. Unfortunately we were running out of time and we could not do much more test. We would like to do the experiment with a lower dilution and repeat it several times.</i><br><br />
<br><br />
<b><p class="texte">Our results are not statistically significant however this result has been proved in literature.</p></b><br></p><br />
<br />
</br><br />
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<br />
<div class="technology">Binding module</div><br />
<div class="thelanguage"><br />
<br />
<br />
<p class="title2">1. Preliminary experiments</p><br />
<p class="title3">Purpose</p><br />
<p class="texte">The first experiment deals with the culture conditions to see if <i>Bacillus subtilis</i> can resist to a low temperature and with the Chitin Beads Buffer (CBB) buffer. To do that, several bacterial concentrations have been tested starting with an OD of 0.1 and diluting this solution to get estimated ODs of 0.05, 0.025, 0.01. These different <i>Bacillus subtilis</i> solutions were incubated 1 hour at 4°C with 500µL of CBB or water. Finally a cell count on Thoma cell counting chamber was performed.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">We could not count bacteria either because of the high number of bacteria with the 0.1 OD solution or the low number of bacteria with the 0.01 OD solution. Thus, the study is mostly focused on the intermediate values (Figure 16).<br />
<br/>First of all, cells do not seem affected by the presence of CBB or water with estimated ODs of 0.05 or 0.025. Moreover, twice less cells can be found in the lowest concentrations in bacteria comparing to the 0.05 OD concentration which is in agreement with the dilution ratio.<br />
</br>Thus the the experimental conditions regarding the presence of CBB and the incubation temperature at 4°C are compatible with the bacterial life. <br />
<br/>Thus, the experimental conditions regarding the presence of CBB and the incubation temperature at 4°C do not harm the cell surviving.<br />
</p><br />
<br />
</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/c/ce/Graphe_binding_1.png" width="45%"></center><br />
<br />
</br><br />
<p class="legend">Figure 16: CBB presence has no effect on bacterial survival. The bacterial concentration was measured in <span style="color:#0000FF">the presence</span> or <span style="color:#FF0000">the absence </span> of CBB for the observed OD (0.1) or estimated ODs (0.05, 0.025, 0.01).</p><br />
<br />
<p class="title2">2. Binding test using engineered <i>B. subtilis</i></p><br />
<br />
<p class="title3">Purpose</p><br />
<p class="texte"><i>B. subtilis</i> transformed with the binding module should produce a chimeric protein composed of the Cell Wall Binding Domain of LycT to attach the chimeric protein to the cell wall, and the GbpA domain 4 of <i>Vibrio cholerae</i> to bind chitin. The synthetic bacterium is put in contact with chitin beads (chitin: polymer present on the fungal pathogen wall). After several washes, bacteria remaining on the beads are counted.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">The first observation is that both bacterial solutions of wild type <i>Bacillus subtilis</i> and SubtiTree have the same concentration: 10^5 bacteria/mL (Figure 17). Even though there is no significant difference between both strains after the first wash, the second wash has a major effect since it removes 40 times more wild-type bacteria than SubtiTree. This result correlates to the number of Subtitree bound to the beads. <br />
<br/>Thus, the binding system is validated: SubtiTree binds efficiently to chitin.</p><br />
<br />
</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/e/ea/Graphe_binding_2.png" width="60%"></center><br />
</br><br />
<br />
<p class="legend">Figure 17: Attachment of WT <i>B. subtilis</i> and Subtitree the to chitin. The <span style="color:#0000FF">WT bacteria</span> or <span style="color:#FF0000">the bacteria with the binding system</span> concentrations have been determined during the different steps of the binding test. The stars represent a significant difference observed with a Student test with p<0.05.<br />
</p><br />
<br />
<p class="title2">3. Microscopic observations</p><br />
<br />
<p class="title3">Purpose</p><br />
<p class="texte">We wanted to observe SubtiTree bound on the chitin coated beads. In order to perform a 3D reconstruction showing this interaction, we used confocal laser scanning microscope. Through the use of a fluorochrome (Syto9), we highlighted the presence of bacteria on the surface of the beads (individualized by phase-contrast). A first calibration step determined the minimum threshold to remove the background noise and the natural fluorescence.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">First, we can notice that SubtiTree is sitting (well, sort off!!) on the surface of beads coated with chitin. These images seem to highlight their interactions.</br></p><br />
<center><img src="https://static.igem.org/mediawiki/2014/archive/5/53/20141013073044!Photo_billes_microscopie.png" width="45%"></center><br />
</br><br />
<p class="legend">Figure 18: Microscopic view of beads surfaces coated with chitin</p><br />
<br />
<p class="texte">Using ImageJ software, we are able to create 3D pictures and movies of those comments.</br></p><br />
<center><img src="https://static.igem.org/mediawiki/2014/5/53/Photo_billes_microscopie.png" width="45%" style="float:left;"><iframe width="380" height="315" src="//www.youtube.com/embed/ztIHIKQr3g0" frameborder="0" allowfullscreen></iframe></center><br />
</br><br />
<p class="legend">Figure 19: A short movie of 3D bead surfaces coated with chitin and Subtitree (emotional sequence for Subtitree: first movie apparition, before Cannes…)</p><br />
<br />
<p class="texte">We then performed a wash step on the chitin beads. We measured the release of bacteria on the washing solution. When our bacterium has the binding module, there is less release and therefore, SubtiTree is retained by the beads.</p><br />
<center><img src="https://static.igem.org/mediawiki/2014/9/97/Photo_lavage_microscopie.png" width="45%"></center><br />
<p class="legend">Figure 20: Microscopic view of bacteria after washing <br />
</p><br />
<br />
<p class="texte">Finally, all results are consistent with the presence of functional binding system. We thus validate the second module.</p><br />
<br />
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<br />
<div class="technology">Fungicides module</div><br />
<div class="thelanguage"><br />
<br />
<br />
<p class="title2"> 1. Preliminary experiments</p><br />
<p class="title3">Tests with commercial peptides and controls</p><br />
<p class="texte">The first tests were accomplished with commercial GAFP-1 and D4E1 peptides at different concentrations (12.5µM, 25µM, 100µM). These tests were performed on different fungal strains sharing the same phylum with <i>Ceratocystis Platani</i>.<br />
As <i>Ceratocystis Platani</i> is pathogenic, we could not perform tests directly with this fungus.</br><br />
After several days at 30°C, the PDA (Potato Dextrose Agar) plates covered with fungus and commercial peptides were analyzed.</p></p><br />
<p class="texte">An inhibition halo was noticeable with commercial D4E1 peptide at 100µM on <i>Aspergillus brasiliensis</i>. Less bright halos were also present with lower concentrations. Concerning commercial GAFP-1, we did not notice any effect in the tested conditions. As positive control, a well-known chemical fungicide was used: the Copper Sulfate. The inhibition of the fungal growth was complete at 20mg/ml, and at 10mg/ml a darker halo appeared around the pad filled with Copper Sulfate as we can see on the figure below. This corresponds to a sporulating halo in response to the stress generated by the fungicide.<br />
</p><br />
<br />
<br />
<center><img style="width:450px; " src="https://static.igem.org/mediawiki/parts/a/a8/Prelim_tests_fung.jpg"><br />
<img style="width:450px; " src="https://static.igem.org/mediawiki/parts/f/f8/Controls_fung.jpg"></center><br />
<p class="legend"> Figure 21: Results of the preliminary tests</p><br />
<br />
<br />
</br><br />
<br />
<p class="texte">Regarding these results, we concluded that very high fungicide concentrations are required to inhibit the fungal growth in the tested conditions. Following these tests, new conditions were adopted in order not to encourage too much fungal growth over bacterial growth. The culture medium was adjusted to fit our objective and to approximate the conditions found in the trees: a 'sap-like' medium was elaborated. The incubations were then carried at room temperature.<br><br />
We also concluded that turning blue the Canal du Midi using high copper sulfate concentrations is not such a good idea... Thereby strengthening our faith in SubtiTree :-) ! <br />
</p><br />
<br />
<p class="title2">2. Test with SubtiTree</p><br />
<br />
<p class="texte">In order to test <i>Bacillus subtilis</i> mutants, it was essential to find the right balance between the fungal growth and the bacterial one. This condition was necessary to get a high concentration of peptides. In our genetic constructions, these peptides are designed to be exported in the extracellular medium.</br><br />
</br><br />
The transformed <i>Bacillus subtilis</i> strains grew at 37°C during 72h and were tested. After centrifugation, the supernatant and the resuspended pellet were placed on pads and disposed on plates previously seeded with a defined number of conidia (see protocols to have more details). After several days at room temperature, an inhibition halo of <i>Trichoderma reesei</i>'s growth was clearly observable for the strain expressing D4E1 gene. The inhibition was even more noticeable with the strain carrying the GAFP-1 + D4E1 operon (see the photos below).</br><br />
However, no effect was detected for the strain expressing the GAFP-1 gene, supposing a synergistic effect between these two peptides.</br><br />
Regarding EcAMP and the triple-fungicides operon, no effect has been detected on the fungal growth. Several factors can explain these results: a number of post-transcriptional modifications are required to have a functional EcAMP and in addition to that, sequencing results of these constructs showed some differences with the original designed sequence.<br />
<p class="texte">Inhibition halos are not visible with supernatants, probably because of their low concentrations in the extracellular medium. <br />
Another effect was noted with the same strains expressing D4E1 and GAFP-1 + D4E1 on another fungus <i>Aspergillus brasiliensis</i>. This effect is comparable to the one previously noted with a low concentration of sulfate copper. </br><br />
</p><br />
<br />
</br><br />
<img style="width:400px; " src="https://static.igem.org/mediawiki/parts/c/c2/Resultfong.jpg"><br />
<img style="width:400px; " src="https://static.igem.org/mediawiki/parts/9/92/Results_fong_2.jpg"> <p class="legend">Figure 22: Results with transformed bacteria.</p><br />
<br />
<p class="texte"><br />
The choice of our chassis appears to be optimal as we noted that wild type <i>Bacillus subtilis</i> disturbs the hyphae growth of the fungi. Some strains of <i>Bacillus subtilis</i> (qst 713) are already used as Biofungicides for use on several minor crops to treat a variety of plant diseases and fungal pathogens.</br><br />
After this set of experiments, the strains expressing D4E1 and expressing GAFP-1 + D4E1 have shown to be the best candidates to play a major role in the fight against fungal diseases such as Canker stain. Keeping in mind our objective, <b> we decided to tests these strains in model plants</b>: <i>Nicotiana benthamiana</i> and <i>Arabidopsis thaliana</i>.</br><br />
These tests were performed in the National Institute for the Agronomic Research by experts in this domain. <br />
<br />
<br />
<p class="title2">3. <i>In planta</i> tests with SubtiTree</p><br />
<br />
<br />
<center><img style="width:215px;" img src="https://static.igem.org/mediawiki/parts/a/af/In_planta.jpg" style="margin-top:5px"/></center><br />
<p class="legend"> Figure 23: Injection of SubtiTree in a model plant</p><br />
<br />
<p class="texte"><br />
The goal of the project is to introduce the transformed bacteria in a diseased tree. So it is necessary to perform <i> in planta </i> tests to judge the fungus-killing abilities of the two strains selected after the previous set of experiments. </br><br />
SubtiTree is first inoculated in two model plants (<i>Arabidopsis thaliana</i> and <i>Nicotiana benthamiana</i>). After this step, a phytopathogenic fungus (<i>Sclerotinia sclerotiorum</i>) is placed on the leaves. </br><br />
These tests were made in association with Sylvain Raffaële and Marielle Barascud of the National Institute for the Agronomic Research laboratory. </br><br />
</p><br />
<br />
<br />
<br />
<p class="texte">Twenty-four hours after SubtiTree inoculation, no phenotypic modification of the leaves can be detected. We can conclude that our bacterium, its introduction and the fungicides production in plants do not have deleterious effects.</br><br />
Without proper treatment, the drop of the pyhtopathogenic fungus on <i>Nicotiana benthamiana</i>'s leaves causes a necrosis halo which can be measured after 40h. The lesion size and the number of inoculated sites seem to be reduced by <i>B. subtilis</i> expressing DE41 or GAFP1-D4E1, unlike with the WT bacterium. A second set of experiments is expected to be more statistically precise.</br><br></br><br />
We did not observe any significant results for <i>Arabidopsis thaliana</i> because of the use of two plants batches with different ages.</br><br />
<br />
We can therefore conclude that when SubtiTree is in plant's physiological conditions, <b> it is harmless to the plant, and that the production of fungicides is effective, reducing the leaves necrosis. </b><br />
</p><br />
<br />
<center><img style="width:860px;" img src="https://static.igem.org/mediawiki/parts/f/f1/Results_d4%2B_gafp1.jpg"></center> <p class="legend">Figure 24: Results of <i>in planta</i> test</p><br />
<br />
<br />
<p class="texte">Thanks to the diversity of anti-fungal peptides, this strategy can be adapted to different types of diseases, with different degree of specifity, etc.</p><br />
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{{:Team:Toulouse/template/footer}}</div>Jourdanhttp://2014.igem.org/Team:Toulouse/Result/experimental-resultsTeam:Toulouse/Result/experimental-results2014-10-17T18:29:41Z<p>Jourdan: </p>
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<h2>Experimental results</h2><br />
<p> Are our modules functionnal? </p><br />
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<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Results&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Experimental results</p> <br />
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<p class="texte">How did we validate the three modules and improve our new protocols? Click below to find out…</p><br />
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<p style="font-size:1.3em; margin: 0 0 30px 0;"><a href="#" onClick="ddaccordion.expandall('technology'); return false">Expand all</a> | <a href="#" onClick="ddaccordion.collapseall('technology'); return false">Collapse all</a></p><br />
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<div class="technology">Chemotaxis</div><br />
<div class="thelanguage"><br />
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<p class="texte"> We used and developed several protocols to demonstrate the existence of chemotaxis in <i>B. subtilis</i> wild type (WT) strain and SubtiTree bacterium towards N-Acetylglucosamine. <br />
</p><br />
<p class="title2">1. Petri Dishes Test </p><br />
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<p class="texte">We first wanted to visualize chemotaxis on Petri dishes. We hoped to obtain pictures with bacteria halos directed or around attractive components and thus tried different protocols. The first protocol was adapted from the one published by <a href="https://2011.igem.org/Team:Imperial_College_London/Protocols_Chemotaxis">the Imperial College 2011 iGEM team</a>. They put the attractive compound on a paper disk in the middle of a Petri dish containing a medium with 0.3% agar. Cells are loaded in this medium (Figure 1).<br />
</br><br />
</br><br />
<b>We first tried to test chemotaxis onto Petri Dishes filled with a 0.3% agar medium. This semi-solid medium allows the bacterial motility. A paper disk containing an attractive compound is placed in the middle of the dish and cells are then loaded in the medium (see Figure 1). This protocol was taken from the <a href="https://2011.igem.org/Team:Imperial_College_London/Protocols_Chemotaxis">the Imperial College 2011 iGEM team</a></b>.</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/0/05/Schema_1.png" alt="schema Figure 1" style="width:500px"></center><br />
<p class="legend">Figure 1: Scheme showing how cells are filled into the medium. (A) A pipet tip is used to deposit cells in the gelose. (B) Bacteria should move toward the attractive compound which diffuses.</p><br />
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<p class="texte">We did not have any result with WT <i>Bacillus subtilis</i> and glucose as attractive compound (Figure 2-A). <i>B. subtilis</i> is attracted by many other glucides and amino-acids, so we also tried to test diluted glucose in LB medium attractant (Figure 2-B).</p><br />
<br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/f/ff/Fig2_AetB.png" alt="Figure 2" style="width:750px"></center><br />
<p class="legend">Figure 2: Chemotaxis test with Glucose as attractive compound (A) and Glucose added to LB medium as attractant (B).</p><br />
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<p class="texte"> We could not notice any difference between the petri dish with or without glucose. With an addition of LB medium to sugar, a large halo around the paper disk was noticeable. This halo may correspond to cells attracted by the solution, as it is not noticeable when cells are not added (data not shown). Anyway we did not have enough reproducible and reliable results to be satisfied with this test.<br> <br />
Furthermore, with the addition of LB medium, it is hard to make the distinction between the attractive effects and the simple growth resulting from random diffusion.</br><br />
We have started new tries using different protocols.</p><br />
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<p class="title2">2. Plug in Pond system<br />
</p><br />
<br />
<p class="texte"><br />
This protocol we worked on is taken from a thesis (see references [1]). . A solution of <i>B.subtilis</i> is grown overnight so as to obtain a cell density of 8x10⁸ cells/mL. 10mL of the solution is mixed with 15mL of LB medium with 1.5 % agar kept at 45°.The final concentration of the obtained medium is 0.9% agar. Tetracycline is aded at 25µg/mL, in order to inhibit growth and to only observe the chemotaxis phenomenon. Plates are cooled and dried, before digging wells with a punch or 1mL tips. The wells are filled with attractive compounds (Figure 3). After one hour at room temperature, photos of the plates are taken and the results are analyzed.<br />
</p><br />
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<center><img SRC="https://static.igem.org/mediawiki/2014/c/cd/Fig3.png" alt="Figure 3" style="width:400px"></center><br />
<p class="legend">Figure 3: Schema showing how are made plug-in-pond tests.</p><br />
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<p class="texte"><br />
On our first try with <i>B. subtilis</i>, we made three wells per plate (Figure 4).The wells were filled with glucose at different concentrations and tetracycline was not added in one of the plates.<br />
</p><br />
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<center><img SRC="https://static.igem.org/mediawiki/2014/c/ce/Fig4.png" alt="Figure 4" style="width:400px"></center><br />
<p class="legend">Figure 4: Plates after 12h at room temperature.</p><br />
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<p class="texte"><br />
After an hour, no tangible results were obtained. It is only after 12hours that we were able to observe halos around the wells with glucose at 1M in the plates without tetracycline. Tetracycline concentration seems to be too high and inhibits any bacterial activity. Therfore, we have worked with tetracycline at 15µg/mL.<br />
We tried this protocol again with this new condition. We made two wells per plate (Figure 5), one with either Glucose or N-acetyl-glucosamine and one with LB medium. As previsously, no results were achieved after 1h, but after 12hours we could notice halos.<br />
</p><br />
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<center><img SRC="https://static.igem.org/mediawiki/2014/c/c3/Bsubtilis_result.png" alt="Figure 5" style="width:750px"></center><br />
<p class="legend">Figure 5: Chemotaxis test with <i>Bacillus subtilis</i> WT. The upper wells contain attractive compound and the lower contain medium without attractive compound. </p><br />
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<p class="texte"><br />
Results are not as clear as the first time, but we observed halos around the well with glucose at 250mM with and without tetracycline. We have then tried the same experiment with N-acetyl-glucosamine and we did not see any halo in the tested conditions. Thus we assumed that our <i>B. subtilis</i> 168 strain was not attracted to N-acetyl-glucosamine.<br />
However, the results are not clear, reliable and reproducible enough with the plug-in-pond protocol. Another testing protocol was then adopted. <br />
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</p><br />
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<p class="texte"><br />
<b>References:</b></br><br />
[1]: Etude de la réponse adaptative à l'oxyde de triméthylamine et de son mécanisme de détection chez <i>Escherichia coli</i> et <i>Shewanella oneidensis</i>, 2008, Claudine Baraquet, Université de la Méditerranée Aix-Marseille II<br />
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<p class="title2">4. Capillary test between two tubes also called the tubes test</p><br />
<p class="texte">After the experiment of the plug-in-pond, we decided to construct a system by welding two Eppendorf tubes with a capillary thanks to an electric burner.</p><br />
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<center><img src="https://static.igem.org/mediawiki/2014/f/fb/Chemotaxis_-_eppendorf.png"></center><br />
<p class="legend">Figure 6: Photography of the first tubes system</p><br />
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<p class="texte">We tested this system with a fuchsin dye and water and we were able to observe the diffusion of fuchsin towards water. However this construction had a leakage next to the weld seam that we could not stop. <br />
Thus, we asked the help from the INSA glass blower, Patrick Chekroun. He designed two systems composed of two tubes linked by a capillary.</p><br />
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<center><img src="https://static.igem.org/mediawiki/2014/2/2b/Chemotaxis_-_tubes.png"><center><br />
<p class="legend">Figure 7: Scheme of the tubes system</p><br />
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<p class="texte">As we did previously, we tested this new system with fuchsin. This experiment was made with WT <i>Bacillus subtilis</i> and N-Acetylglucosamine.<br />
<br><br><br />
<i>NB: We could not see the diffusion from one tube to the other. We made the hypothesis that it was not visible by sight because of the small diameter of the capillary. <br />
</i><br><br />
<br><br />
The following strategy was used to avoid disturbance due to pressure and liquid movement through the capillary:<br><br />
- The first step was the addition of Wash Buffer until the capillary was full to avoid the presence of air bubbles which could lead to diffusion problems.<br><br />
- Then, the tube 2 was plugged with the thumb while another person was adding the bacterial solution of WT <i>Bacillus subtilis</i> in the tube 1. <br><br />
- The tube 1 was also plugged and only after the thumb could be removed from the tube 2. <br><br />
- In the same way, the N-Acetylglucosamine was added in the tube 2. <br><br />
- The same process was made with a xylose positive control.<br><br />
<br><br />
<i>NB: According to the article Chemotaxis towards sugars by </i>Bacillus subtilis, (George W. Ordal et al., 1979), <i>glucose and xylose have the same attractant power. We prefer a positive control instead of a negative one because we were not sure that this system was efficient.</i><br><br />
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- The system was kept straight for 2hours. Every 40 minutes, we took a sample of each tube and spread it on an agar plate (dilution 1/1,000).</p><br />
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<center><img src="https://static.igem.org/mediawiki/2014/1/1b/Chemotaxis_-_tubes_photo.png"></center><br />
<p class="legend">Figure 8: Photography of the tubes system</p><br />
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<p class="texte">Unfortunately, the dilution was too high to detect any chemotaxis movement and the time was too short. We did not find any information in the literature.<br><br />
As we did not have the time to optimize this protocol we preferred using the protocol of the 2011 Imperial college iGEM team : the tips capillary test.</br><br />
</p><br />
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<p class="title2"> 5. Tips capillary system</p><br />
<p class="title3">First tips capillary system</p><br />
<p class="texte">This protocol comes from 2011 Imperial College iGEM team and was adapted by our team in several steps (See <a href="https://2014.igem.org/Team:Toulouse/Notebook/Protocols#select8">chemotaxis protocol</a>).<br />
<br><br />
In the first tips capillary system, we used parafilm to avoid any kind of air disturbance in the tips. The different steps are described below:<br><br />
- 15µL of each chemo-attractant was pipetted. <br><br />
- The bottom of tip with the pipette was then put on a piece of parafilm and the pipette was removed from the top of the tip.<br><br />
- The top of the tip was then sealed with a piece of parafilm. By this way, the sterility can be assured and the liquid stays inside the tip. <br><br />
- To finish, the level of the solution in the tip was marked.<br></p><br />
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<center><img src="https://static.igem.org/mediawiki/2014/9/94/Chemotaxis_-_tip.png"></center><br />
<p class="legend">Figure 9: Sealing of a tip with parafilm</p><br />
<br />
<p class="texte">- After all the attractants were added in the tips, we put them on a green base to carry them. The whole process can be seen on Figure 10.<br><br />
- Each tip was immersed in 300 µL of a bacterial solution in the wells of an Elisa plate.<br></p><br />
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<center><img src="https://static.igem.org/mediawiki/2014/0/05/Chemotaxis_-_tip_and_support.png"></center><br />
<p class="legend">Figure 10: First tips capillary system</p><br />
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<p class="texte"><i>NB: the yellow carton was used to stabilize the system and keep it straight.</i><br><br />
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- After one hour, the tips were removed from the bacteria solutions and the content of the tips was observed with a Thoma cell under the microscope.<br><br />
<br><br />
We had several problems with this system:<br><br />
- The liquid level decreased during the experiment and we did not have enough liquid to fill the Thoma cell. Thus, it was not possible to count.<br><br />
- The bacteria were moving and therefore, we could not proceed to a bacteria count.<br><br />
<br><br />
Regarding these observations we decided to spread the tips content on agar plates instead of using Thoma cell and microscopy.<br><br />
<p class="title3">Second tips capillary system<br />
</p><br />
<p class="texte"And then the revolution came! We found a multichannel pipette :D The same protocol was performed except that the parafilm was used to avoid the air entrance between the tips and the pipette and therefore the loss of liquid.<br></p><br />
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<center><img src="https://static.igem.org/mediawiki/2014/e/e4/Chemotaxis_-_pipette.png"></center><br />
<p class="legend">Figure 11: Second tips capillary system</p><br />
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<p class="title3">Improvement of the second tips capillary system</p><br />
<p class="texte">However this system was not optimal it is why we decided to use blu tack instead of parafilm: <br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/4/42/Chemotaxis_-_pipette_and_blu_tack.png"></center><br />
<p class="legend">Figure 12: Improvement of the second tips capillary system</p><br />
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<p class="texte"><b>At that point, the protocol was approved and the final test could finally start! :-)</b><br><br />
<br><br />
There was just one tiny problem… we did not have our optimized bacterium transformed with the chemotaxis module!!! That is why we concentrated our efforts on WT <i>Bacillus subtilis</i> strain.<br><br />
<br><br />
The main goal was to find an optimized control and to analyze the eventual chemotaxis of the WT strain. To avoid osmolality bias, we wanted to find a molecule which was non-attractant and with a similar molecular weight than the N-Acetylglucosamine (221.21 g/mol). Our first idea was to use fuchsin (Molecular weight: 337.85 g/mol).<br><br />
<br><br />
At the beginning, the experiment was conducted with only one negative contraol, the fuchsin and different NAG concentrations: 25mM, 250mM and 500mM. The tested strain was <i>Bacillus subtilis </i>168:<br><br />
<br></p><br />
<center><br />
<table align="center"><br />
<tr><td align=center><img src="https://static.igem.org/mediawiki/2014/8/8c/Chemotaxis_-_results_fuch.png"></td><br />
<td align=center><img src="https://static.igem.org/mediawiki/2014/f/fd/Chemotaxis_-_results_fuchsin.png"></td></tr><br />
<tr><td align=center><p class="legend">Figure 13: Fuchsin - negative control (dilution 1/50)</p></td><br />
<td align=center><p class="legend">Figure 14: NAG (25mM) (dilution 1/50)</p></td></tr><br />
</table></center><br><br />
<p class="texte">The average number of colonies with the negative control is 121. On the contrary, a cell layer is observed for the NAG plates with every concentration.<br><br />
<br><br />
Thus, we assumed that WT <i>Bacillus subtilis</i> was more attracted by NAG than fuchsin. Indeed we can neglect the bacterial growth because the test only lasts one hour. We also neglect diffusion and osmolality phenomena for the previous reasons. <br><br />
<br><br />
Unfortunately for us we forgot one major effect… Can you believe that fuchsin solution contains about 15% of ethanol?!!! This concentration can lead to the death of some cells which probably happened to our results.<br><br />
<br><br />
<b><p class="texte">This incredible and dramatic discovery destroyed all of our hopes about the God of chemotaxis! :-(</b><br><br />
<br><br />
However, our team did not give up on synthetic biology ! :-) Indeed, after days of disappointment and no time left for lab work, we raised from ashes and tried to find another negative control.<br><br />
<br><br />
Hopefully, we managed to find a negative control: galactose (25mM). The article Chemotaxis towards sugars by <i>Bacillus subtilis</i> (<i>George W. Ordal et al., 1979</i>) proved that it was a poor attractant.<br><br />
<br><br />
We made our tests again with this new molecule and glucose (25mM) as positive control.<br></p><br />
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<center><img src="https://static.igem.org/mediawiki/2014/8/86/Chemotaxis_-_final_results.png"></center><br />
<p class="legend">Figure 15: Final results (dilution : 1/10,000)</p><br />
<p class="texte"> The miracle arrived! We managed to prove that our WT <i>Bacillus subtilis</i> was indeed naturally attracted to NAG.</p><br />
<p class="texte"><i>NB: It was our last experiment. Unfortunately we were running out of time and we could not do much more test. We would like to do the experiment with a lower dilution and repeat it several times.</i><br><br />
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<b><p class="texte">Our results are not statistically significant however this result has been proved in literature.</p></b><br></p><br />
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<div class="technology">Binding module</div><br />
<div class="thelanguage"><br />
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<p class="title2">1. Preliminary experiments</p><br />
<p class="title3">Purpose</p><br />
<p class="texte">The first experiment deals with the culture conditions to see if <i>Bacillus subtilis</i> can resist to a low temperature and with the Chitin Beads Buffer (CBB) buffer. To do that, several bacterial concentrations have been tested starting with an OD of 0.1 and diluting this solution to get estimated ODs of 0.05, 0.025, 0.01. These different <i>Bacillus subtilis</i> solutions were incubated 1 hour at 4°C with 500µL of CBB or water. Finally a cell count on Thoma cell counting chamber was performed.</p><br />
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<p class="title3">Results</p><br />
<p class="texte">We could not count bacteria either because of the high number of bacteria with the 0.1 OD solution or the low number of bacteria with the 0.01 OD solution. Thus, the study is mostly focused on the intermediate values (Figure 16).<br />
<br/>First of all, cells do not seem affected by the presence of CBB or water with estimated ODs of 0.05 or 0.025. Moreover, twice less cells can be found in the lowest concentrations in bacteria comparing to the 0.05 OD concentration which is in agreement with the dilution ratio.<br />
</br>Thus the the experimental conditions regarding the presence of CBB and the incubation temperature at 4°C are compatible with the bacterial life. <br />
<br/>Thus, the experimental conditions regarding the presence of CBB and the incubation temperature at 4°C do not harm the cell surviving.<br />
</p><br />
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</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/c/ce/Graphe_binding_1.png" width="45%"></center><br />
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</br><br />
<p class="legend">Figure 16: CBB presence has no effect on bacterial survival. The bacterial concentration was measured in <span style="color:#0000FF">the presence</span> or <span style="color:#FF0000">the absence </span> of CBB for the observed OD (0.1) or estimated ODs (0.05, 0.025, 0.01).</p><br />
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<p class="title2">2. Binding test using engineered <i>B. subtilis</i></p><br />
<br />
<p class="title3">Purpose</p><br />
<p class="texte"><i>B. subtilis</i> transformed with the binding module should produce a chimeric protein composed of the Cell Wall Binding Domain of LycT to attach the chimeric protein to the cell wall, and the GbpA domain 4 of <i>Vibrio cholerae</i> to bind chitin. The synthetic bacterium is put in contact with chitin beads (chitin: polymer present on the fungal pathogen wall). After several washes, bacteria remaining on the beads are counted.</p><br />
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<p class="title3">Results</p><br />
<p class="texte">The first observation is that both bacterial solutions of wild type <i>Bacillus subtilis</i> and SubtiTree have the same concentration: 10^5 bacteria/mL (Figure 17). Even though there is no significant difference between both strains after the first wash, the second wash has a major effect since it removes 40 times more wild-type bacteria than SubtiTree. This result correlates to the number of Subtitree bound to the beads. <br />
<br/>Thus, the binding system is validated: SubtiTree binds efficiently to chitin.</p><br />
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</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/e/ea/Graphe_binding_2.png" width="60%"></center><br />
</br><br />
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<p class="legend">Figure 17: Attachment of WT <i>B. subtilis</i> and Subtitree the to chitin. The <span style="color:#0000FF">WT bacteria</span> or <span style="color:#FF0000">the bacteria with the binding system</span> concentrations have been determined during the different steps of the binding test. The stars represent a significant difference observed with a Student test with p<0.05.<br />
</p><br />
<br />
<p class="title2">3. Microscopic observations</p><br />
<br />
<p class="title3">Purpose</p><br />
<p class="texte">We wanted to observe SubtiTree bound on the chitin coated beads. In order to perform a 3D reconstruction showing this interaction, we used confocal laser scanning microscope. Through the use of a fluorochrome (Syto9), we highlighted the presence of bacteria on the surface of the beads (individualized by phase-contrast). A first calibration step determined the minimum threshold to remove the background noise and the natural fluorescence.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">First, we can notice that SubtiTree is sitting (well, sort off!!) on the surface of beads coated with chitin. These images seem to highlight their interactions.</br></p><br />
<center><img src="https://static.igem.org/mediawiki/2014/archive/5/53/20141013073044!Photo_billes_microscopie.png" width="45%"></center><br />
</br><br />
<p class="legend">Figure 18: Microscopic view of beads surfaces coated with chitin</p><br />
<br />
<p class="texte">Using ImageJ software, we are able to create 3D pictures and movies of those comments.</br></p><br />
<center><img src="https://static.igem.org/mediawiki/2014/5/53/Photo_billes_microscopie.png" width="45%" style="float:left;"><iframe width="380" height="315" src="//www.youtube.com/embed/ztIHIKQr3g0" frameborder="0" allowfullscreen></iframe></center><br />
</br><br />
<p class="legend">Figure 19: A short movie of 3D bead surfaces coated with chitin and Subtitree (emotional sequence for Subtitree: first movie apparition, before Cannes…)</p><br />
<br />
<p class="texte">We then performed a wash step on the chitin beads. We measured the release of bacteria on the washing solution. When our bacterium has the binding module, there is less release and therefore, SubtiTree is retained by the beads.</p><br />
<center><img src="https://static.igem.org/mediawiki/2014/9/97/Photo_lavage_microscopie.png" width="45%"></center><br />
<p class="legend">Figure 20: Microscopic view of bacteria after washing <br />
</p><br />
<br />
<p class="texte">Finally, all results are consistent with the presence of functional binding system. We thus validate the second module.</p><br />
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<div class="technology">Fungicides module</div><br />
<div class="thelanguage"><br />
<br />
<br />
<p class="title2"> 1. Preliminary experiments</p><br />
<p class="title3">Tests with commercial peptides and controls</p><br />
<p class="texte">The first tests were accomplished with commercial GAFP-1 and D4E1 peptides at different concentrations (12.5µM, 25µM, 100µM). These tests were performed on different fungal strains sharing the same phylum with <i>Ceratocystis Platani</i>.<br />
As <i>Ceratocystis Platani</i> is pathogenic, we could not perform tests directly with this fungus.</br><br />
After several days at 30°C, the PDA (Potato Dextrose Agar) plates covered with fungus and commercial peptides were analyzed.</p></p><br />
<p class="texte">An inhibition halo was noticeable with commercial D4E1 peptide at 100µM on <i>Aspergillus brasiliensis</i>. Less bright halos were also present with lower concentrations. Concerning commercial GAFP-1, we did not notice any effect in the tested conditions. As positive control, a well-known chemical fungicide was used: the Copper Sulfate. The inhibition of the fungal growth was complete at 20mg/ml, and at 10mg/ml a darker halo appeared around the pad filled with Copper Sulfate as we can see on the figure below. This corresponds to a sporulating halo in response to the stress generated by the fungicide.<br />
</p><br />
<br />
<br />
<center><img style="width:450px; " src="https://static.igem.org/mediawiki/parts/a/a8/Prelim_tests_fung.jpg"><br />
<img style="width:450px; " src="https://static.igem.org/mediawiki/parts/f/f8/Controls_fung.jpg"></center><br />
<p class="legend"> Figure 21: Results of the preliminary tests</p><br />
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<br />
</br><br />
<br />
<p class="texte">Regarding these results, we concluded that very high fungicide concentrations are required to inhibit the fungal growth in the tested conditions. Following these tests, new conditions were adopted in order not to encourage too much fungal growth over bacterial growth. The culture medium was adjusted to fit our objective and to approximate the conditions found in the trees: a 'sap-like' medium was elaborated. The incubations were then carried at room temperature.<br><br />
We also concluded that turning blue the Canal du Midi using high copper sulfate concentrations is not such a good idea... Thereby strengthing our faith in SubtiTree :-) ! <br />
</p><br />
<br />
<p class="title2">2. Test with SubtiTree</p><br />
<br />
<p class="texte">In order to test <i>Bacillus subtilis</i> mutants, it was essential to find the right balance between the fungal growth and the bacterial one. This condition was necessary to get a high concentration of peptides. In our genetic constructions, these peptides are designed to be exported in the extracellular medium.</br><br />
</br><br />
The transformed <i>Bacillus subtilis</i> strains grew at 37°C during 72h and were tested. After centrifugation, the supernatant and the resuspended pellet were placed on pads and disposed on plates previously seeded with a defined number of conidia (see protocols to have more details). After several days at room temperature, an inhibition halo of <i>Trichoderma reesei</i>'s growth was clearly observable for the strain expressing D4E1 gene. The inhibition was even more noticeable with the strain carrying the GAFP-1 + D4E1 operon (see the photos below).</br><br />
However, no effect was detected for the strain expressing the GAFP-1 gene, supposing a synergistic effect between these two peptides.</br><br />
Regarding EcAMP and the triple-fungicides operon, no effect has been detected on the fungal growth. Several factors can explain these results: a number of post-transcriptional modifications are required to have a functional EcAMP and in addition to that, sequencing results of these constructs showed some differences with the original designed sequence.<br />
<p class="texte">Inhibition halos are not visible with supernatants, probably because of their low concentrations in the extracellular medium. <br />
Another effect was noted with the same strains expressing D4E1 and GAFP-1 + D4E1 on another fungus <i>Aspergillus brasiliensis</i>. This effect is comparable to the one previously noted with a low concentration of sulfate copper. </br><br />
</p><br />
<br />
</br><br />
<img style="width:400px; " src="https://static.igem.org/mediawiki/parts/c/c2/Resultfong.jpg"><br />
<img style="width:400px; " src="https://static.igem.org/mediawiki/parts/9/92/Results_fong_2.jpg"> <p class="legend">Figure 22: Results with transformed bacteria.</p><br />
<br />
<p class="texte"><br />
The choice of our chassis appears to be optimal as we noted that wild type <i>Bacillus subtilis</i> disturbs the hyphae growth of the fungi. Some strains of <i>Bacillus subtilis</i> (qst 713) are already used as Biofungicides for use on several minor crops to treat a variety of plant diseases and fungal pathogens.</br><br />
After this set of experiments, the strains expressing D4E1 and expressing GAFP-1 + D4E1 have shown to be the best candidates to play a major role in the fight against fungal diseases such as Canker stain. Keeping in mind our objective, <b> we decided to tests these strains in model plants</b>: <i>Nicotiana benthamiana</i> and <i>Arabidopsis thaliana</i>.</br><br />
These tests were performed in the National Institute for the Agronomic Research by experts in this domain. <br />
<br />
<br />
<p class="title2">3. <i>In planta</i> tests with SubtiTree</p><br />
<br />
<br />
<center><img style="width:215px;" img src="https://static.igem.org/mediawiki/parts/a/af/In_planta.jpg" style="margin-top:5px"/></center><br />
<p class="legend"> Figure 23: Injection of SubtiTree in a model plant</p><br />
<br />
<p class="texte"><br />
The goal of the project is to introduce the transformed bacteria in a diseased tree. So it is necessary to perform <i> in planta </i> tests to judge the fungus-killing abilities of the two strains selected after the previous set of experiments. </br><br />
SubtiTree is first inoculated in two model plants (<i>Arabidopsis thaliana</i> and <i>Nicotiana benthamiana</i>). After this step, a phytopathogenic fungus (<i>Sclerotinia sclerotiorum</i>) is placed on the leaves. </br><br />
These tests were made in association with Sylvain Raffaële and Marielle Barascud of the National Institute for the Agronomic Research laboratory. </br><br />
</p><br />
<br />
<br />
<br />
<p class="texte">Twenty-four hours after SubtiTree inoculation, no phenotypic modification of the leaves can be detected. We can conclude that our bacterium, its introduction and the fungicides production in plants do not have deleterious effects.</br><br />
Without proper treatment, the drop of the pyhtopathogenic fungus on <i>Nicotiana benthamiana</i>'s leaves causes a necrosis halo which can be measured after 40h. The lesion size and the number of inoculated sites seem to be reduced by <i>B. subtilis</i> expressing DE41 or GAFP1-D4E1, unlike with the WT bacterium. A second set of experiments is expected to be more statistically precise.</br><br></br><br />
We did not observe any significant results for <i>Arabidopsis thaliana</i> because of the use of two plants batches with different ages.</br><br />
<br />
We can therefore conclude that when SubtiTree is in plant's physiological conditions, <b> it is harmless to the plant, and that the production of fungicides is effective, reducing the leaves necrosis. </b><br />
</p><br />
<br />
<center><img style="width:860px;" img src="https://static.igem.org/mediawiki/parts/f/f1/Results_d4%2B_gafp1.jpg"></center> <p class="legend">Figure 24: Results of <i>in planta</i> test</p><br />
<br />
<br />
<p class="texte">Thanks to the diversity of anti-fungal peptides, this strategy can be adapted to different types of diseases, with different degree of specifity, etc.</p><br />
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{{:Team:Toulouse/template/footer}}</div>Jourdanhttp://2014.igem.org/Team:Toulouse/Result/parts/Submitted_partsTeam:Toulouse/Result/parts/Submitted parts2014-10-17T09:56:49Z<p>Jourdan: </p>
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<h2>Parts</h2><br />
<p>What did we send to the Registry?</p><br />
</div><br />
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<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Results&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Parts</p> <br />
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<div class="centering" style="padding-top: 20px; padding-bottom:40px;"><br />
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<br />
<p class="title1">Submitted parts</p><br />
<br />
<p class="texte"><br />
We have deposed 16 new BioBrick parts to the Registry. <br />
All of them were cloned into the standard plasmid pSB1C3, tested and sequenced.<br><br />
<!--à vérifier et confirmer--><br />
</p><br />
<br />
<p class="title2">I. Chemotaxis</p><br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</a>: N-acetylglucosamine based chemotaxis for <i>Bacillus subtilis</i></div><br />
<div class="thelanguage"><br />
<br />
<p class="texte">This part is designed to enable a N-acetylglucosamine <br />
based chemotaxis in <i>Bacillus subtilis</i>.<br />
<p class="title4">Design</p><br />
<p class="texte">The encoded protein is a chimera of two proteins:<br><br />
- the methyl accepting Chemotaxis protein (McpA from <i>Bacillus subtilis</i> which is required for taxis towards glucose.<br><br />
- the N-acetylglucosamine regulated methyl-accepting chemotaxis protein from <i>Vibrio cholerae</i> (VCD).<br><br />
<br><br />
The chimeric protein contains the intracellular domains of McpA from <i>Bacillus subtilis</i> to enable the transduction of the signal: <br />
these domains correspond to the amino acids 1-37 and 282-610. <br />
The extracellular domain of VCD is inserted between the intracellular regions of McpA <br />
to sense N-acetylglucosamine and corresponds to the amino acids 31-310.<br></p><br />
<img src="https://static.igem.org/mediawiki/2014/a/a2/BBa_K1364000.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Translation unit</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part is not tested yet.</p><br />
<p class="title4">References</p><br />
<p class="texte">Uniprot:<br><br />
- <a href="http://www.uniprot.org/uniprot/P39214">McpA</a><br><br />
- <a href="http://www.uniprot.org/uniprot/C3NYT2">N-acetylglucosamine regulated methyl-accepting chemotaxis protein</a><br></p><br />
<!--à compléter--><br />
</p><br />
<br><br />
<br />
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</div><br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364004">BBa_K1364004</a>: P<sub>veg</sub> + N-acetylatedglucosamine based chemotaxis for <i>B. subtilis</i></div><br />
<div class="thelanguage"><br />
<br />
<br><br />
<p class="texte">This part is designed to enable a N-acetylglucosamine based chemotaxis in <i>Bacillus subtilis</i>.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This expression cassette is designed for the expression of an antifungal peptide, <br />
D4E1 and for its secretion in <i>Bacillus subtilis</i>.<br />
It is composed of the strong, constitutive promoter of <i>B. subtilis</i> <br />
P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">K823003</a>),<br />
strong RBS for <i>B. subtilis</i> (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>),<br />
the open reading frame of D4E1<br />
and a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>)</p><br />
<img src="https://static.igem.org/mediawiki/2014/3/35/BBa_K1364004.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was not tested yet.</p><br />
<p class="title4">References</p><br />
<p class="texte">Uniprot:<br><br />
- <a href="http://www.uniprot.org/uniprot/P39214">McpA</a><br><br />
- <a href="http://www.uniprot.org/uniprot/C3NYT2">N-acetylglucosamine regulated methyl-accepting chemotaxis protein</a></p><br />
<br />
<br />
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<br />
<br></br><br />
<br />
<p class="title2">II. Binding</p><br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364005">BBa_K1364005</a>: P<sub>veg</sub> + Chitin Binding Cell Wall protein</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte">This part is designed to enable the binding of <i>Bacillus subtilis</i> to the fungi wall made of chitin.<p/><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a Cell Wall Binding (CWB) sequence <br />
and a Chitin Binding Domain (CBD) linked with a 6 amino acids linker.<br><br />
<br><br />
The CWB domain has been extracted from LytC (<a href="http://parts.igem.org/Part:BBa_K316030">BBa_K316030</a>) of the iGEM 2010 Imperial College team.<br><br />
The CBD is composed of the Domain 4 of the GbpA protein of <i>Vibrio cholerae</i>,<br />
a protein reported to mediate bacterial attachment to chitin. <br />
It has been shown that the Domain 4 was essential to bind chitin.</p><br />
<img src="https://static.igem.org/mediawiki/2014/f/f5/BBa_K1364005.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested with chitin beads. (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Binding module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)<br><br />
- The Vibrio cholerae Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373<br><br />
</p><br />
<br />
<br><br />
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<br />
<br></br><br />
<br />
<p class="title2"> III. Fungicides</p><br />
<p class="title3">GAFP-1</p><br />
<br />
<br><br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364002">BBa_K1364002</a>: RBS - Antifungal GAFP-1</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte">The <i>Gastrodia</i> anti-fungal protein(GAFP-1), also known as gastrodianin, is a mannose and chitin binding lectin<br />
originating from the Asiatic orchid Gastrodia elata, a traditional Chinese medicinal herb cultured for thousands of years.<br />
GAFP-1 is composed of 15 amino acids LDSLSFSYNNFEEDD and is able to inhibit the growth of multiples species of plant pathogenic fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a Strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>) and<br />
the open reading frame of the <i>Gastrodia</i> anti-fungal protein 1 (GAFP-1)<br />
optimized for its expression and its secretion in <i>Bacillus subtilis</i>.<br />
The codon optimization was made thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/5/5f/BBa_K1364002.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Translational unit</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)<br><br />
- The Vibrio cholerae Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373<br></p><br />
<br><br />
<br />
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</div><br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364007">BBa_K1364007</a>: RBS + Antifungal GAFP-1 + Double terminator</div><br />
<div class="thelanguage"><br />
<br />
<br />
<p class="texte">The <i>Gastrodia</i> anti-fungal protein(GAFP-1), also known as gastrodianin, is a mannose and chitin binding lectin<br />
originating from the Asiatic orchid Gastrodia elata, a traditional Chinese medicinal herb cultured for thousands of years.<br />
GAFP-1 is composed of 15 amino acids LDSLSFSYNNFEEDD and is able to inhibit the growth of multiples species of plant pathogenic fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a Strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>),<br />
the open reading frame of the <i>Gastrodia</i> anti-fungal protein 1 (GAFP-1) <br />
and a double terminator <a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>.<br />
optimized for its expression and its secretion in <i>Bacillus subtilis</i>.<br />
The codon optimization was made thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/4/42/BBa_K1364007.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Composite part</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)<br><br />
- The Vibrio cholerae Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373<br></p><br />
<br><br />
<br />
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<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364008">BBa_K1364008</a>: P<sub>veg</sub> - strong RBS - Antifungal GAFP-1 - Double terminator</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte">This expression cassette is designed for the expression and secretion of the <i>Gastrodia</i> anti-fungal protein(GAFP-1), also known as gastrodianin. GAFP-1 is a mannose and chitin binding lectin<br />
originating from the Asiatic orchid Gastrodia elata, a traditional Chinese medicinal herb cultured for thousands of years.<br />
GAFP-1 is composed of 15 amino acids LDSLSFSYNNFEEDD and is able to inhibit the growth of multiples species of plant pathogenic fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of the strong, constitutive promoter of <i>Bacillus subtilis</i> P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">K823003</a>),<br />
strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>),<br />
the open reading frame of the <i>Gastrodia</i> anti-fungal protein 1 (GAFP-1) <br />
and a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>).<br />
optimized for its expression and its secretion in <i>Bacillus subtilis</i>.<br />
The codon optimization was made thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/6/67/BBa_K1364008.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)<br><br />
- The Vibrio cholerae Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373<br><br />
</p><br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 5); return false">Collapse</a></p><br />
</div><br />
<br />
<br></br><br />
<br />
<p class="title3">D4E1</p><br />
<br />
<br><br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364003">BBa_K1364003</a>:RBS - Antifungal D4E1 - Double terminator</div><br />
<div class="thelanguage"><br />
<br />
<br />
<p class="texte">D4E1 is a linear synthetic peptide of 17 amino acids <br />
which has shown to have antifungal activities <br />
by complexing with a sterol present in conodial wall of a varety of fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a Strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>),<br />
the open reading frame of D4E1 and <br />
a Double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>)<br />
optimized for its expression and its secretion in <i>Bacillus subtilis</i>.<br />
This part was optimized for the expression and its secretion in <i>Bacillus subtilis </i><br />
thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/b/bb/BBa_K1364003.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Translational unit</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- De Lucca AJ, Bland JM, Grimm C, Jacks TJ, Cary JW, Jaynes JM, Cleveland TE, Walsh TJ. Fungicidal properties, sterol binding, and proteolytic resistance of the synthetic peptide D4E1.<br><br />
- Can J Microbiol. 1998 Jun;44(6):514-20.</p><br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 6); return false">Collapse</a></p><br />
</div><br />
<br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364009">BBa_K1364009</a>:P<sub>veg</sub> - RBS - Antifungal D4E1 - Double Terminator</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte"><br />
D4E1 is a linear synthetic peptide of 17 amino acids <br />
which has shown to have antifungal activities <br />
by complexing with a sterol present in conodial wall of a varety of fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of the strong, constitutive promoter of <i>B. subtilis</i> <br />
P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">K823003</a>),<br />
a strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>),<br />
the open reading frame of D4E1 and <br />
a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>).<br />
This part was optimized for the expression and its secretion in <i>Bacillus subtilis </i><br />
thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/4/47/BBa_K1364009.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Translational unit</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- De Lucca AJ, Bland JM, Grimm C, Jacks TJ, Cary JW, Jaynes JM, Cleveland TE, Walsh TJ. Fungicidal properties, sterol binding, and proteolytic resistance of the synthetic peptide D4E1.<br><br />
- Can J Microbiol. 1998 Jun;44(6):514-20.</p><br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 7); return false">Collapse</a></p><br />
</div><br />
<br />
<br></br><br />
<br />
<p class="title3">EcAMP-1</p><br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364010">BBa_K1364010</a>: P<sub>veg</sub> - SpoVG - Antifungal EcAMP-1</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte"><br />
EcAMP-1 is an antimicrobial peptide of 37 amino acids <br />
originated from the specie <i>Echinochloa crus-galli</i>, <br />
a type of wild grass. <br />
This peptide has a particular structure : it is helical because of two disulfide bonds.<br><br />
EcAMP-1 has shown to have antifungal activities. Its mode of action may be the prevention of hyphae elongation.<br><br />
This part was added to the Registry by the iGEM Utah State team in 2013.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of the constitutive promoter P<sub>veg</sub> and<br />
strong RBS for <i>B. subtilis</i><br />
(<a href="http://parts.igem.org/Part:BBa_K733013">K733013</a>) and<br />
the open reading frame of EcAMP-1.<br><br />
The EcAMP-1 part was codon optimized for <i>E. coli</i> by the iGEM Utah State team<br />
and thanks to the Life Technologies GeneArt software program. </p><br />
<img src="https://static.igem.org/mediawiki/2014/5/5f/BBa_K1364010.png"><br />
<p class="title4">Type</p><br />
<p class="texte"> Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part is not tested yet.</p><br />
<p class="title4">References</p><br />
<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 (Echinochloa crus-galli)</b>. The journal of Biological Chemistry. 2011, Vol. 286, 25145–25153</p><br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 8); return false">Collapse</a></p><br />
</div><br />
<br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364011">BBa_K1364011</a>: P<sub>veg</sub> - SpoVG - Antifungal EcAMP-1 - Double terminator</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte" id="select10"><br />
EcAMP-1 is an antimicrobial peptide of 37 amino acids <br />
originated from the specie <i>Echinochloa crus-galli</i>, <br />
a type of wild grass. <br />
This peptide has a particular structure : it is helical because of two disulfide bonds.<br><br />
EcAMP-1 has shown to have antifungal activities. Its mode of action may be the prevention of hyphae elongation.<br><br />
This part was added to the Registry by the iGEM Utah State team in 2013.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of the constitutive promoter P<sub>veg</sub> and<br />
strong RBS for <i>B. subtilis</i><br />
(<a href="http://parts.igem.org/Part:BBa_K733013">K733013</a>),<br />
the open reading frame of EcAMP-1 and <br />
a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>).<br><br />
The EcAMP-1 part was codon optimized for <i>E. coli</i> by the iGEM Utah State team<br />
and thanks to the Life Technologies GeneArt software program. </p><br />
<img src="https://static.igem.org/mediawiki/2014/7/74/BBa_K1364011.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part is not tested yet.</p><br />
<p class="title4">References</p><br />
<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 (Echinochloa crus-galli)</b>. The journal of Biological Chemistry. 2011, Vol. 286, 25145–25153</p><br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 9); return false">Collapse</a></p><br />
</div><br />
<br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364019">BBa_K1364019</a>: P<sub>veg</sub> - RBS - Antifungal EcAMP (revised with a stop codon) - Double terminator</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte">EcAMP-1 is an antimicrobial peptide of 37 amino acids <br />
originated from the specie <i>Echinochloa crus-galli</i>, <br />
a type of wild grass. <br />
This peptide has a particular structure : it is helical because of two disulfide bonds.<br><br />
EcAMP-1 has shown to have antifungal activities. Its mode of action may be the prevention of hyphae elongation.<br><br />
This part was added to the Registry by the iGEM Utah State team in 2013.</br><br />
We observed the presence of the stop codon in the suffix. When we have digested this Biobrick, it disappeared. So we added this codon in upstream of the suffix and now, we can reuse this Biobrick.<br />
</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of the constitutive promoter P<sub>veg</sub> and<br />
strong RBS for <i>B. subtilis</i><br />
(<a href="http://parts.igem.org/Part:BBa_K733013">K733013</a>),<br />
<b>the revised open reading frame of EcAMP-1 (with a stop codon) </b> and <br />
a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>).<br><br />
The EcAMP-1 part was codon optimized for <i>E. coli</i> by the iGEM Utah State team<br />
and thanks to the Life Technologies GeneArt software program. </p><br />
<img src="https://static.igem.org/mediawiki/2014/7/74/BBa_K1364011.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Composite</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was not tested yet</p><br />
<p class="title4">References</p><br />
<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 (Echinochloa crus-galli)</b>. The journal of Biological Chemistry. 2011, Vol. 286, 25145–25153</p><br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 13); return false">Collapse</a></p><br />
</div><br />
<br />
<br></br><br />
<br />
<p class="title3">Fungicide operons</p><br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364013">BBa_K1364013</a>: P<sub>veg</sub> - SpoVG - Antifungals GAFP-1 and D4E1 - Double terminator</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte"><br />
<p class="title4">Design</p><br />
<p class="texte">This part is designed for the co-expression of two different peptides <br />
with anti-fungal activities : D4E1 and GAFP-1. <br />
It is composed of the strong, constitutive promoter of <i>B. subtilis</i> <br />
P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">K823003</a>) <br />
and the translation unit with GAFP-1 and D4E1 (BBa_K1364012).</p><br />
<img src="https://static.igem.org/mediawiki/2014/4/40/BBa_K1364013.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
See <a href="http://parts.igem.org/Part:BBa_K1364002">BBa_K1364002</a> and <a href="http://parts.igem.org/Part:BBa_K1364003">BBa_K1364003</a>.</p><br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 10); return false">Collapse</a></p><br />
</div><br />
<br />
<br></br><br />
<br />
<p class="title2">Basic tools</p><br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364015">BBa_K1364015</a>: P<sub>veg</sub> + RFP</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte"><!--Compléter--><br />
This part is designed to enable the expression of a Red Fluorescent Protein in Bacillus subtilis under the control of a constitutive promoter Pveg. This construction has been checked by sequencing and has shown to work also in E. coli<br />
</p><br />
<img src="https://static.igem.org/mediawiki/parts/6/69/Pveg%2BRFP.jpg"><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a constitutive promoter Pveg (K823003), spoVG RBS (K143021), the coding sequence of the RFP (E1010) and a double terminator (B0015) </p><br />
<p class="title4">Type</p><br />
<p class="texte">Reporter</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested in both E.coli and B. subtilis. The sequences was verified by sequencing. </p><br />
<br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 10); return false">Collapse</a></p><br />
</div><br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364016">BBa_K1364016</a>: P<sub>lepA</sub> + RFP</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte"><!--Compléter--><br />
This part is designed to enable the expression of a Red Fluorescent Protein in Bacillus subtilis under the control of a constitutive promoter PlepA. This construction has been checked by sequencing and has shown to work also in E. coli<br />
</p><br />
<img src="https://static.igem.org/mediawiki/parts/1/12/PlepA%2BRFP.jpg"><br />
<br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a constitutive promoter PlepA (K823002), spoVG RBS (K143021), the coding sequence of the RFP (E1010) and a double terminator (B0015) </p><br />
<p class="title4">Type</p><br />
<p class="texte">Reporter</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested in both E.coli and B. subtilis. The sequences was verified by sequencing.</p><br />
<br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 11); return false">Collapse</a></p><br />
</div><br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364017">BBa_K1364017</a>: P<sub>lepA</sub> + RBS SpoVG</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte"><!--Compléter--><br />
PlepA is a constitutive promoter in Bacillus subtilis (BBa_K823002) coupled with a RBS spoVG (BBa_K143021). To get the highest level of translation from this Promoter-RBS combination it must be connected to a coding region preceded by a coding region prefix. A standard prefix will increase the distance between the RBS and the start codon, reducing translational efficiency. <br />
This construction is also working with E. coli and has been verified by sequencing.<br />
</p><br />
<img src="https://static.igem.org/mediawiki/parts/d/da/PlepA%2BSpoVG.jpg"><br />
<p class="title4">Type</p><br />
<p class="texte">Composite</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was checked by sequencing</p><br />
<br />
<br />
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</div><br />
<br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364021">BBa_K1364021</a>: Integrative plasmid for <i>Bacillus subtilis</i> (pSBbs4E)</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte">This part is an empty backbone vector for the usage in <i>Bacillus subtilis</i>. It integrates in the thrC locus and can be selected with Erythromycin . It has an Ampicillin resistance for cloning in E.coli. The backbones contains an RFP in the BioBrick site (J04450) to facilitate the cloning in coli. <br><br />
The handling of this type of vector is described <a href="https://2014.igem.org/Team:Toulouse/Notebook/Protocols#select6">here</a>.<br />
<br />
</p><br />
<p class="title4">Design</p><br />
<p class="texte">We digested pKL190 plasmid and PCR products of BBa_J04450 with BamHI and EcoRI. We proceded to ligation.</p><br />
<p class="title4">Type</p><br />
<p class="texte">Backbone</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested by transforming <i>B. subtilis</i>. Clones obtained were Threonine-dependant and resistant to Erythromycin (10µg/ml).</p><br />
<p class="title4">References</p><br />
<p class="texte"><br />
R. Bernard, K.A. Marquis, and D.Z. Rudner. <b>Nucleoid occlusion prevents cell division during replication fork arrest in Bacillus subtilis</b>Mol Microbiol. Nov 2010; 78(4): 866–882.</p><br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 13); return false">Collapse</a></p><br />
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{{:Team:Toulouse/template/footer}}</div>Jourdanhttp://2014.igem.org/Team:Toulouse/Result/parts/Submitted_partsTeam:Toulouse/Result/parts/Submitted parts2014-10-17T09:53:24Z<p>Jourdan: </p>
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<h2>Parts</h2><br />
<p>What did we send to the Registry?</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Results&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Parts</p> <br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 20px; padding-bottom:40px;"><br />
<br />
<br />
<p class="title1">Submitted parts</p><br />
<br />
<p class="texte"><br />
We have deposed 16 new BioBrick parts to the Registry. <br />
All of them were cloned into the standard plasmid pSB1C3, tested and sequenced.<br><br />
<!--à vérifier et confirmer--><br />
</p><br />
<br />
<p class="title2">I. Chemotaxis</p><br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</a>: N-acetylglucosamine based chemotaxis for <i>Bacillus subtilis</i></div><br />
<div class="thelanguage"><br />
<br />
<p class="texte">This part is designed to enable a N-acetylglucosamine <br />
based chemotaxis in <i>Bacillus subtilis</i>.<br />
<p class="title4">Design</p><br />
<p class="texte">The encoded protein is a chimera of two proteins:<br><br />
- the methyl accepting Chemotaxis protein (McpA from <i>Bacillus subtilis</i> which is required for taxis towards glucose.<br><br />
- the N-acetylglucosamine regulated methyl-accepting chemotaxis protein from <i>Vibrio cholerae</i> (VCD).<br><br />
<br><br />
The chimeric protein contains the intracellular domains of McpA from <i>Bacillus subtilis</i> to enable the transduction of the signal: <br />
these domains correspond to the amino acids 1-37 and 282-610. <br />
The extracellular domain of VCD is inserted between the intracellular regions of McpA <br />
to sense N-acetylglucosamine and corresponds to the amino acids 31-310.<br></p><br />
<img src="https://static.igem.org/mediawiki/2014/a/a2/BBa_K1364000.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Translation unit</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part is not tested yet.</p><br />
<p class="title4">References</p><br />
<p class="texte">Uniprot:<br><br />
- <a href="http://www.uniprot.org/uniprot/P39214">McpA</a><br><br />
- <a href="http://www.uniprot.org/uniprot/C3NYT2">N-acetylglucosamine regulated methyl-accepting chemotaxis protein</a><br></p><br />
<!--à compléter--><br />
</p><br />
<br><br />
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<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364004">BBa_K1364004</a>: P<sub>veg</sub> + N-acetylatedglucosamine based chemotaxis for <i>B. subtilis</i></div><br />
<div class="thelanguage"><br />
<br />
<br><br />
<p class="texte">This part is designed to enable a N-acetylglucosamine based chemotaxis in <i>Bacillus subtilis</i>.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This expression cassette is designed for the expression of an antifungal peptide, <br />
D4E1 and for its secretion in <i>Bacillus subtilis</i>.<br />
It is composed of the strong, constitutive promoter of <i>B. subtilis</i> <br />
P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">K823003</a>),<br />
strong RBS for <i>B. subtilis</i> (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>),<br />
the open reading frame of D4E1<br />
and a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>)</p><br />
<img src="https://static.igem.org/mediawiki/2014/3/35/BBa_K1364004.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was not tested yet.</p><br />
<p class="title4">References</p><br />
<p class="texte">Uniprot:<br><br />
- <a href="http://www.uniprot.org/uniprot/P39214">McpA</a><br><br />
- <a href="http://www.uniprot.org/uniprot/C3NYT2">N-acetylglucosamine regulated methyl-accepting chemotaxis protein</a></p><br />
<br />
<br />
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<br></br><br />
<br />
<p class="title2">II. Binding</p><br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364005">BBa_K1364005</a>: P<sub>veg</sub> + Chitin Binding Cell Wall protein</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte">This part is designed to enable the binding of <i>Bacillus subtilis</i> to the fungi wall made of chitin.<p/><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a Cell Wall Binding (CWB) sequence <br />
and a Chitin Binding Domain (CBD) linked with a 6 amino acids linker.<br><br />
<br><br />
The CWB domain has been extracted from LytC (<a href="http://parts.igem.org/Part:BBa_K316030">BBa_K316030</a>) of the iGEM 2010 Imperial College team.<br><br />
The CBD is composed of the Domain 4 of the GbpA protein of <i>Vibrio cholerae</i>,<br />
a protein reported to mediate bacterial attachment to chitin. <br />
It has been shown that the Domain 4 was essential to bind chitin.</p><br />
<img src="https://static.igem.org/mediawiki/2014/f/f5/BBa_K1364005.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested with chitin beads. (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Binding module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)<br><br />
- The Vibrio cholerae Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373<br><br />
</p><br />
<br />
<br><br />
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<br></br><br />
<br />
<p class="title2"> III. Fungicides</p><br />
<p class="title3">GAFP-1</p><br />
<br />
<br><br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364002">BBa_K1364002</a>: RBS - Antifungal GAFP-1</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte">The <i>Gastrodia</i> anti-fungal protein(GAFP-1), also known as gastrodianin, is a mannose and chitin binding lectin<br />
originating from the Asiatic orchid Gastrodia elata, a traditional Chinese medicinal herb cultured for thousands of years.<br />
GAFP-1 is composed of 15 amino acids LDSLSFSYNNFEEDD and is able to inhibit the growth of multiples species of plant pathogenic fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a Strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>) and<br />
the open reading frame of the <i>Gastrodia</i> anti-fungal protein 1 (GAFP-1)<br />
optimized for its expression and its secretion in <i>Bacillus subtilis</i>.<br />
The codon optimization was made thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/5/5f/BBa_K1364002.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Translational unit</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)<br><br />
- The Vibrio cholerae Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373<br></p><br />
<br><br />
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<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364007">BBa_K1364007</a>: RBS + Antifungal GAFP-1 + Double terminator</div><br />
<div class="thelanguage"><br />
<br />
<br />
<p class="texte">The <i>Gastrodia</i> anti-fungal protein(GAFP-1), also known as gastrodianin, is a mannose and chitin binding lectin<br />
originating from the Asiatic orchid Gastrodia elata, a traditional Chinese medicinal herb cultured for thousands of years.<br />
GAFP-1 is composed of 15 amino acids LDSLSFSYNNFEEDD and is able to inhibit the growth of multiples species of plant pathogenic fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a Strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>),<br />
the open reading frame of the <i>Gastrodia</i> anti-fungal protein 1 (GAFP-1) <br />
and a double terminator <a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>.<br />
optimized for its expression and its secretion in <i>Bacillus subtilis</i>.<br />
The codon optimization was made thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/4/42/BBa_K1364007.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Composite part</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)<br><br />
- The Vibrio cholerae Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373<br></p><br />
<br><br />
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<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364008">BBa_K1364008</a>: P<sub>veg</sub> - strong RBS - Antifungal GAFP-1 - Double terminator</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte">This expression cassette is designed for the expression and secretion of the <i>Gastrodia</i> anti-fungal protein(GAFP-1), also known as gastrodianin. GAFP-1 is a mannose and chitin binding lectin<br />
originating from the Asiatic orchid Gastrodia elata, a traditional Chinese medicinal herb cultured for thousands of years.<br />
GAFP-1 is composed of 15 amino acids LDSLSFSYNNFEEDD and is able to inhibit the growth of multiples species of plant pathogenic fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of the strong, constitutive promoter of <i>Bacillus subtilis</i> P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">K823003</a>),<br />
strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>),<br />
the open reading frame of the <i>Gastrodia</i> anti-fungal protein 1 (GAFP-1) <br />
and a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>).<br />
optimized for its expression and its secretion in <i>Bacillus subtilis</i>.<br />
The codon optimization was made thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/6/67/BBa_K1364008.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)<br><br />
- The Vibrio cholerae Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373<br><br />
</p><br />
<br />
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<br></br><br />
<br />
<p class="title3">D4E1</p><br />
<br />
<br><br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364003">BBa_K1364003</a>:RBS - Antifungal D4E1 - Double terminator</div><br />
<div class="thelanguage"><br />
<br />
<br />
<p class="texte">D4E1 is a linear synthetic peptide of 17 amino acids <br />
which has shown to have antifungal activities <br />
by complexing with a sterol present in conodial wall of a varety of fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a Strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>),<br />
the open reading frame of D4E1 and <br />
a Double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>)<br />
optimized for its expression and its secretion in <i>Bacillus subtilis</i>.<br />
This part was optimized for the expression and its secretion in <i>Bacillus subtilis </i><br />
thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/b/bb/BBa_K1364003.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Translational unit</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- De Lucca AJ, Bland JM, Grimm C, Jacks TJ, Cary JW, Jaynes JM, Cleveland TE, Walsh TJ. Fungicidal properties, sterol binding, and proteolytic resistance of the synthetic peptide D4E1.<br><br />
- Can J Microbiol. 1998 Jun;44(6):514-20.</p><br />
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<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364009">BBa_K1364009</a>:P<sub>veg</sub> - RBS - Antifungal D4E1 - Double Terminator</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte"><br />
D4E1 is a linear synthetic peptide of 17 amino acids <br />
which has shown to have antifungal activities <br />
by complexing with a sterol present in conodial wall of a varety of fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of the strong, constitutive promoter of <i>B. subtilis</i> <br />
P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">K823003</a>),<br />
a strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>),<br />
the open reading frame of D4E1 and <br />
a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>).<br />
This part was optimized for the expression and its secretion in <i>Bacillus subtilis </i><br />
thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/4/47/BBa_K1364009.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Translational unit</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- De Lucca AJ, Bland JM, Grimm C, Jacks TJ, Cary JW, Jaynes JM, Cleveland TE, Walsh TJ. Fungicidal properties, sterol binding, and proteolytic resistance of the synthetic peptide D4E1.<br><br />
- Can J Microbiol. 1998 Jun;44(6):514-20.</p><br />
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<p class="title3">EcAMP-1</p><br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364010">BBa_K1364010</a>: P<sub>veg</sub> - SpoVG - Antifungal EcAMP-1</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte"><br />
EcAMP-1 is an antimicrobial peptide of 37 amino acids <br />
originated from the specie <i>Echinochloa crus-galli</i>, <br />
a type of wild grass. <br />
This peptide has a particular structure : it is helical because of two disulfide bonds.<br><br />
EcAMP-1 has shown to have antifungal activities. Its mode of action may be the prevention of hyphae elongation.<br><br />
This part was added to the Registry by the iGEM Utah State team in 2013.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of the constitutive promoter P<sub>veg</sub> and<br />
strong RBS for <i>B. subtilis</i><br />
(<a href="http://parts.igem.org/Part:BBa_K733013">K733013</a>) and<br />
the open reading frame of EcAMP-1.<br><br />
The EcAMP-1 part was codon optimized for <i>E. coli</i> by the iGEM Utah State team<br />
and thanks to the Life Technologies GeneArt software program. </p><br />
<img src="https://static.igem.org/mediawiki/2014/5/5f/BBa_K1364010.png"><br />
<p class="title4">Type</p><br />
<p class="texte"> Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part is not tested yet.</p><br />
<p class="title4">References</p><br />
<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 (Echinochloa crus-galli)</b>. The journal of Biological Chemistry. 2011, Vol. 286, 25145–25153</p><br />
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<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364011">BBa_K1364011</a>: P<sub>veg</sub> - SpoVG - Antifungal EcAMP-1 - Double terminator</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte" id="select10"><br />
EcAMP-1 is an antimicrobial peptide of 37 amino acids <br />
originated from the specie <i>Echinochloa crus-galli</i>, <br />
a type of wild grass. <br />
This peptide has a particular structure : it is helical because of two disulfide bonds.<br><br />
EcAMP-1 has shown to have antifungal activities. Its mode of action may be the prevention of hyphae elongation.<br><br />
This part was added to the Registry by the iGEM Utah State team in 2013.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of the constitutive promoter P<sub>veg</sub> and<br />
strong RBS for <i>B. subtilis</i><br />
(<a href="http://parts.igem.org/Part:BBa_K733013">K733013</a>),<br />
the open reading frame of EcAMP-1 and <br />
a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>).<br><br />
The EcAMP-1 part was codon optimized for <i>E. coli</i> by the iGEM Utah State team<br />
and thanks to the Life Technologies GeneArt software program. </p><br />
<img src="https://static.igem.org/mediawiki/2014/7/74/BBa_K1364011.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part is not tested yet.</p><br />
<p class="title4">References</p><br />
<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 (Echinochloa crus-galli)</b>. The journal of Biological Chemistry. 2011, Vol. 286, 25145–25153</p><br />
<br />
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</div><br />
<br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364019">BBa_K1364019</a>: P<sub>veg</sub> - RBS - Antifungal EcAMP (revised with a stop codon) - Double terminator</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte">EcAMP-1 is an antimicrobial peptide of 37 amino acids <br />
originated from the specie <i>Echinochloa crus-galli</i>, <br />
a type of wild grass. <br />
This peptide has a particular structure : it is helical because of two disulfide bonds.<br><br />
EcAMP-1 has shown to have antifungal activities. Its mode of action may be the prevention of hyphae elongation.<br><br />
This part was added to the Registry by the iGEM Utah State team in 2013.</br><br />
We observed the presence of the stop codon in the suffix. When we have digested this Biobrick, it disappeared. So we added this codon in upstream of the suffix and now, we can reuse this Biobrick.<br />
</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of the constitutive promoter P<sub>veg</sub> and<br />
strong RBS for <i>B. subtilis</i><br />
(<a href="http://parts.igem.org/Part:BBa_K733013">K733013</a>),<br />
<b>the revised open reading frame of EcAMP-1 (with a stop codon) </b> and <br />
a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>).<br><br />
The EcAMP-1 part was codon optimized for <i>E. coli</i> by the iGEM Utah State team<br />
and thanks to the Life Technologies GeneArt software program. </p><br />
<img src="https://static.igem.org/mediawiki/2014/7/74/BBa_K1364011.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Composite</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was not tested yet</p><br />
<p class="title4">References</p><br />
<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 (Echinochloa crus-galli)</b>. The journal of Biological Chemistry. 2011, Vol. 286, 25145–25153</p><br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 13); return false">Collapse</a></p><br />
</div><br />
<br />
<br></br><br />
<br />
<p class="title3">Fungicide operons</p><br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364013">BBa_K1364013</a>: P<sub>veg</sub> - SpoVG - Antifungals GAFP-1 and D4E1 - Double terminator</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte"><br />
<p class="title4">Design</p><br />
<p class="texte">This part is designed for the co-expression of two different peptides <br />
with anti-fungal activities : D4E1 and GAFP-1. <br />
It is composed of the strong, constitutive promoter of <i>B. subtilis</i> <br />
P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">K823003</a>) <br />
and the translation unit with GAFP-1 and D4E1 (BBa_K1364012).</p><br />
<img src="https://static.igem.org/mediawiki/2014/4/40/BBa_K1364013.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
See <a href="http://parts.igem.org/Part:BBa_K1364002">BBa_K1364002</a> and <a href="http://parts.igem.org/Part:BBa_K1364003">BBa_K1364003</a>.</p><br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 10); return false">Collapse</a></p><br />
</div><br />
<br />
<br></br><br />
<br />
<p class="title2">Basic tools</p><br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364015">BBa_K1364015</a>: P<sub>veg</sub> + RFP</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte"><!--Compléter--><br />
This part is designed to enable the expression of a Red Fluorescent Protein in Bacillus subtilis under the control of a constitutive promoter Pveg. This construction has been checked by sequencing and has shown to work also in E. coli<br />
</p><br />
<img src="https://static.igem.org/mediawiki/parts/6/69/Pveg%2BRFP.jpg"><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a constitutive promoter Pveg (K823003), spoVG RBS (K143021), the coding sequence of the RFP (E1010) and a double terminator (B0015) </p><br />
<p class="title4">Type</p><br />
<p class="texte">Reporter</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested in both E.coli and B. subtilis. The sequences was verified by sequencing. </p><br />
<br />
<br />
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</div><br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364016">BBa_K1364016</a>: P<sub>lepA</sub> + RFP</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte"><!--Compléter--><br />
This part is designed to enable the expression of a Red Fluorescent Protein in Bacillus subtilis under the control of a constitutive promoter PlepA. This construction has been checked by sequencing and has shown to work also in E. coli<br />
</p><br />
<img src="https://static.igem.org/mediawiki/parts/1/12/PlepA%2BRFP.jpg"><br />
<br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a constitutive promoter PlepA (K823002), spoVG RBS (K143021), the coding sequence of the RFP (E1010) and a double terminator (B0015) </p><br />
<p class="title4">Type</p><br />
<p class="texte">Reporter</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested in both E.coli and B. subtilis. The sequences was verified by sequencing.</p><br />
<br />
<br />
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</div><br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364017">BBa_K1364017</a>: P<sub>lepA</sub> + RBS SpoVG</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte"><!--Compléter--><br />
PlepA is a constitutive promoter in Bacillus subtilis (BBa_K823002) coupled with a RBS spoVG (BBa_K143021). To get the highest level of translation from this Promoter-RBS combination it must be connected to a coding region preceded by a coding region prefix. A standard prefix will increase the distance between the RBS and the start codon, reducing translational efficiency. <br />
This construction is also working with E. coli and has been verified by sequencing.<br />
</p><br />
<img src="https://static.igem.org/mediawiki/parts/d/da/PlepA%2BSpoVG.jpg"><br />
<p class="title4">Type</p><br />
<p class="texte">Composite</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was checked by sequencing</p><br />
<br />
<br />
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</div><br />
<br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364021">BBa_K1364021</a>: Integrative plasmid for <i>Bacillus subtilis</i> (pSBbs4E)</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte">This part is an empty backbone vector for the usage in <i>Bacillus subtilis</i>. It integrates in the thrC locus and can be selected with Erythromycin . It has an Ampicillin resistance for cloning in E.coli. The backbones contains an RFP in the BioBrick site (J04450) to facilitate the cloning in coli. <br />
</p><br />
<p class="title4">Design</p><br />
<p class="texte">We digested pKL190 plasmid and PCR products of BBa_J04450 with BamHI and EcoRI. We proceded to ligation.</p><br />
<p class="title4">Type</p><br />
<p class="texte">Backbone</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested by transforming <i>B. subtilis</i>. Clones obtained were Auxotrophic of Threonine and resistant to Erythromycin.</p><br />
<p class="title4">References</p><br />
<p class="texte"><br />
R. Bernard, K.A. Marquis, and D.Z. Rudner. <b>Nucleoid occlusion prevents cell division during replication fork arrest in Bacillus subtilis</b>Mol Microbiol. Nov 2010; 78(4): 866–882.</p><br />
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{{:Team:Toulouse/template/footer}}</div>Jourdanhttp://2014.igem.org/Team:Toulouse/Result/parts/Submitted_partsTeam:Toulouse/Result/parts/Submitted parts2014-10-17T09:48:31Z<p>Jourdan: </p>
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<h2>Parts</h2><br />
<p>What did we send to the Registry?</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Results&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Parts</p> <br />
</div><br />
<br />
<br />
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<div class="centering" style="padding-top: 20px; padding-bottom:40px;"><br />
<br />
<br />
<p class="title1">Submitted parts</p><br />
<br />
<p class="texte"><br />
We have deposed 16 new BioBrick parts to the Registry. <br />
All of them were cloned into the standard plasmid pSB1C3, tested and sequenced.<br><br />
<!--à vérifier et confirmer--><br />
</p><br />
<br />
<p class="title2">I. Chemotaxis</p><br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</a>: N-acetylglucosamine based chemotaxis for <i>Bacillus subtilis</i></div><br />
<div class="thelanguage"><br />
<br />
<p class="texte">This part is designed to enable a N-acetylglucosamine <br />
based chemotaxis in <i>Bacillus subtilis</i>.<br />
<p class="title4">Design</p><br />
<p class="texte">The encoded protein is a chimera of two proteins:<br><br />
- the methyl accepting Chemotaxis protein (McpA from <i>Bacillus subtilis</i> which is required for taxis towards glucose.<br><br />
- the N-acetylglucosamine regulated methyl-accepting chemotaxis protein from <i>Vibrio cholerae</i> (VCD).<br><br />
<br><br />
The chimeric protein contains the intracellular domains of McpA from <i>Bacillus subtilis</i> to enable the transduction of the signal: <br />
these domains correspond to the amino acids 1-37 and 282-610. <br />
The extracellular domain of VCD is inserted between the intracellular regions of McpA <br />
to sense N-acetylglucosamine and corresponds to the amino acids 31-310.<br></p><br />
<img src="https://static.igem.org/mediawiki/2014/a/a2/BBa_K1364000.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Translation unit</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part is not tested yet.</p><br />
<p class="title4">References</p><br />
<p class="texte">Uniprot:<br><br />
- <a href="http://www.uniprot.org/uniprot/P39214">McpA</a><br><br />
- <a href="http://www.uniprot.org/uniprot/C3NYT2">N-acetylglucosamine regulated methyl-accepting chemotaxis protein</a><br></p><br />
<!--à compléter--><br />
</p><br />
<br><br />
<br />
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</div><br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364004">BBa_K1364004</a>: P<sub>veg</sub> + N-acetylatedglucosamine based chemotaxis for <i>B. subtilis</i></div><br />
<div class="thelanguage"><br />
<br />
<br><br />
<p class="texte">This part is designed to enable a N-acetylglucosamine based chemotaxis in <i>Bacillus subtilis</i>.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This expression cassette is designed for the expression of an antifungal peptide, <br />
D4E1 and for its secretion in <i>Bacillus subtilis</i>.<br />
It is composed of the strong, constitutive promoter of <i>B. subtilis</i> <br />
P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">K823003</a>),<br />
strong RBS for <i>B. subtilis</i> (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>),<br />
the open reading frame of D4E1<br />
and a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>)</p><br />
<img src="https://static.igem.org/mediawiki/2014/3/35/BBa_K1364004.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was not tested yet.</p><br />
<p class="title4">References</p><br />
<p class="texte">Uniprot:<br><br />
- <a href="http://www.uniprot.org/uniprot/P39214">McpA</a><br><br />
- <a href="http://www.uniprot.org/uniprot/C3NYT2">N-acetylglucosamine regulated methyl-accepting chemotaxis protein</a></p><br />
<br />
<br />
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<br></br><br />
<br />
<p class="title2">II. Binding</p><br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364005">BBa_K1364005</a>: P<sub>veg</sub> + Chitin Binding Cell Wall protein</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte">This part is designed to enable the binding of <i>Bacillus subtilis</i> to the fungi wall made of chitin.<p/><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a Cell Wall Binding (CWB) sequence <br />
and a Chitin Binding Domain (CBD) linked with a 6 amino acids linker.<br><br />
<br><br />
The CWB domain has been extracted from LytC (<a href="http://parts.igem.org/Part:BBa_K316030">BBa_K316030</a>) of the iGEM 2010 Imperial College team.<br><br />
The CBD is composed of the Domain 4 of the GbpA protein of <i>Vibrio cholerae</i>,<br />
a protein reported to mediate bacterial attachment to chitin. <br />
It has been shown that the Domain 4 was essential to bind chitin.</p><br />
<img src="https://static.igem.org/mediawiki/2014/f/f5/BBa_K1364005.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested with chitin beads. (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Binding module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)<br><br />
- The Vibrio cholerae Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373<br><br />
</p><br />
<br />
<br><br />
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</div><br />
<br />
<br></br><br />
<br />
<p class="title2"> III. Fungicides</p><br />
<p class="title3">GAFP-1</p><br />
<br />
<br><br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364002">BBa_K1364002</a>: RBS - Antifungal GAFP-1</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte">The <i>Gastrodia</i> anti-fungal protein(GAFP-1), also known as gastrodianin, is a mannose and chitin binding lectin<br />
originating from the Asiatic orchid Gastrodia elata, a traditional Chinese medicinal herb cultured for thousands of years.<br />
GAFP-1 is composed of 15 amino acids LDSLSFSYNNFEEDD and is able to inhibit the growth of multiples species of plant pathogenic fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a Strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>) and<br />
the open reading frame of the <i>Gastrodia</i> anti-fungal protein 1 (GAFP-1)<br />
optimized for its expression and its secretion in <i>Bacillus subtilis</i>.<br />
The codon optimization was made thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/5/5f/BBa_K1364002.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Translational unit</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)<br><br />
- The Vibrio cholerae Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373<br></p><br />
<br><br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 3); return false">Collapse</a></p><br />
</div><br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364007">BBa_K1364007</a>: RBS + Antifungal GAFP-1 + Double terminator</div><br />
<div class="thelanguage"><br />
<br />
<br />
<p class="texte">The <i>Gastrodia</i> anti-fungal protein(GAFP-1), also known as gastrodianin, is a mannose and chitin binding lectin<br />
originating from the Asiatic orchid Gastrodia elata, a traditional Chinese medicinal herb cultured for thousands of years.<br />
GAFP-1 is composed of 15 amino acids LDSLSFSYNNFEEDD and is able to inhibit the growth of multiples species of plant pathogenic fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a Strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>),<br />
the open reading frame of the <i>Gastrodia</i> anti-fungal protein 1 (GAFP-1) <br />
and a double terminator <a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>.<br />
optimized for its expression and its secretion in <i>Bacillus subtilis</i>.<br />
The codon optimization was made thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/4/42/BBa_K1364007.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Composite part</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)<br><br />
- The Vibrio cholerae Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373<br></p><br />
<br><br />
<br />
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</div><br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364008">BBa_K1364008</a>: P<sub>veg</sub> - strong RBS - Antifungal GAFP-1 - Double terminator</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte">This expression cassette is designed for the expression and secretion of the <i>Gastrodia</i> anti-fungal protein(GAFP-1), also known as gastrodianin. GAFP-1 is a mannose and chitin binding lectin<br />
originating from the Asiatic orchid Gastrodia elata, a traditional Chinese medicinal herb cultured for thousands of years.<br />
GAFP-1 is composed of 15 amino acids LDSLSFSYNNFEEDD and is able to inhibit the growth of multiples species of plant pathogenic fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of the strong, constitutive promoter of <i>Bacillus subtilis</i> P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">K823003</a>),<br />
strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>),<br />
the open reading frame of the <i>Gastrodia</i> anti-fungal protein 1 (GAFP-1) <br />
and a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>).<br />
optimized for its expression and its secretion in <i>Bacillus subtilis</i>.<br />
The codon optimization was made thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/6/67/BBa_K1364008.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)<br><br />
- The Vibrio cholerae Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373<br><br />
</p><br />
<br />
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</div><br />
<br />
<br></br><br />
<br />
<p class="title3">D4E1</p><br />
<br />
<br><br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364003">BBa_K1364003</a>:RBS - Antifungal D4E1 - Double terminator</div><br />
<div class="thelanguage"><br />
<br />
<br />
<p class="texte">D4E1 is a linear synthetic peptide of 17 amino acids <br />
which has shown to have antifungal activities <br />
by complexing with a sterol present in conodial wall of a varety of fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a Strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>),<br />
the open reading frame of D4E1 and <br />
a Double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>)<br />
optimized for its expression and its secretion in <i>Bacillus subtilis</i>.<br />
This part was optimized for the expression and its secretion in <i>Bacillus subtilis </i><br />
thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/b/bb/BBa_K1364003.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Translational unit</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- De Lucca AJ, Bland JM, Grimm C, Jacks TJ, Cary JW, Jaynes JM, Cleveland TE, Walsh TJ. Fungicidal properties, sterol binding, and proteolytic resistance of the synthetic peptide D4E1.<br><br />
- Can J Microbiol. 1998 Jun;44(6):514-20.</p><br />
<br />
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</div><br />
<br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364009">BBa_K1364009</a>:P<sub>veg</sub> - RBS - Antifungal D4E1 - Double Terminator</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte"><br />
D4E1 is a linear synthetic peptide of 17 amino acids <br />
which has shown to have antifungal activities <br />
by complexing with a sterol present in conodial wall of a varety of fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of the strong, constitutive promoter of <i>B. subtilis</i> <br />
P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">K823003</a>),<br />
a strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>),<br />
the open reading frame of D4E1 and <br />
a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>).<br />
This part was optimized for the expression and its secretion in <i>Bacillus subtilis </i><br />
thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/4/47/BBa_K1364009.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Translational unit</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- De Lucca AJ, Bland JM, Grimm C, Jacks TJ, Cary JW, Jaynes JM, Cleveland TE, Walsh TJ. Fungicidal properties, sterol binding, and proteolytic resistance of the synthetic peptide D4E1.<br><br />
- Can J Microbiol. 1998 Jun;44(6):514-20.</p><br />
<br />
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</div><br />
<br />
<br></br><br />
<br />
<p class="title3">EcAMP-1</p><br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364010">BBa_K1364010</a>: P<sub>veg</sub> - SpoVG - Antifungal EcAMP-1</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte"><br />
EcAMP-1 is an antimicrobial peptide of 37 amino acids <br />
originated from the specie <i>Echinochloa crus-galli</i>, <br />
a type of wild grass. <br />
This peptide has a particular structure : it is helical because of two disulfide bonds.<br><br />
EcAMP-1 has shown to have antifungal activities. Its mode of action may be the prevention of hyphae elongation.<br><br />
This part was added to the Registry by the iGEM Utah State team in 2013.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of the constitutive promoter P<sub>veg</sub> and<br />
strong RBS for <i>B. subtilis</i><br />
(<a href="http://parts.igem.org/Part:BBa_K733013">K733013</a>) and<br />
the open reading frame of EcAMP-1.<br><br />
The EcAMP-1 part was codon optimized for <i>E. coli</i> by the iGEM Utah State team<br />
and thanks to the Life Technologies GeneArt software program. </p><br />
<img src="https://static.igem.org/mediawiki/2014/5/5f/BBa_K1364010.png"><br />
<p class="title4">Type</p><br />
<p class="texte"> Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part is not tested yet.</p><br />
<p class="title4">References</p><br />
<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 (Echinochloa crus-galli)</b>. The journal of Biological Chemistry. 2011, Vol. 286, 25145–25153</p><br />
<br />
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</div><br />
<br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364011">BBa_K1364011</a>: P<sub>veg</sub> - SpoVG - Antifungal EcAMP-1 - Double terminator</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte" id="select10"><br />
EcAMP-1 is an antimicrobial peptide of 37 amino acids <br />
originated from the specie <i>Echinochloa crus-galli</i>, <br />
a type of wild grass. <br />
This peptide has a particular structure : it is helical because of two disulfide bonds.<br><br />
EcAMP-1 has shown to have antifungal activities. Its mode of action may be the prevention of hyphae elongation.<br><br />
This part was added to the Registry by the iGEM Utah State team in 2013.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of the constitutive promoter P<sub>veg</sub> and<br />
strong RBS for <i>B. subtilis</i><br />
(<a href="http://parts.igem.org/Part:BBa_K733013">K733013</a>),<br />
the open reading frame of EcAMP-1 and <br />
a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>).<br><br />
The EcAMP-1 part was codon optimized for <i>E. coli</i> by the iGEM Utah State team<br />
and thanks to the Life Technologies GeneArt software program. </p><br />
<img src="https://static.igem.org/mediawiki/2014/7/74/BBa_K1364011.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part is not tested yet.</p><br />
<p class="title4">References</p><br />
<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 (Echinochloa crus-galli)</b>. The journal of Biological Chemistry. 2011, Vol. 286, 25145–25153</p><br />
<br />
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</div><br />
<br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364019">BBa_K1364019</a>: P<sub>veg</sub> - RBS - Antifungal EcAMP (revised with a stop codon) - Double terminator</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte">EcAMP-1 is an antimicrobial peptide of 37 amino acids <br />
originated from the specie <i>Echinochloa crus-galli</i>, <br />
a type of wild grass. <br />
This peptide has a particular structure : it is helical because of two disulfide bonds.<br><br />
EcAMP-1 has shown to have antifungal activities. Its mode of action may be the prevention of hyphae elongation.<br><br />
This part was added to the Registry by the iGEM Utah State team in 2013.</br><br />
We observed the presence of the stop codon in the suffix. When we have digested this Biobrick, it disappeared. So we added this codon in upstream of the suffix and now, we can reuse this Biobrick.<br />
</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of the constitutive promoter P<sub>veg</sub> and<br />
strong RBS for <i>B. subtilis</i><br />
(<a href="http://parts.igem.org/Part:BBa_K733013">K733013</a>),<br />
<b>the revised open reading frame of EcAMP-1 (with a stop codon) </b> and <br />
a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>).<br><br />
The EcAMP-1 part was codon optimized for <i>E. coli</i> by the iGEM Utah State team<br />
and thanks to the Life Technologies GeneArt software program. </p><br />
<img src="https://static.igem.org/mediawiki/2014/7/74/BBa_K1364011.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Composite</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was not tested yet</p><br />
<p class="title4">References</p><br />
<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 (Echinochloa crus-galli)</b>. The journal of Biological Chemistry. 2011, Vol. 286, 25145–25153</p><br />
<br />
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</div><br />
<br />
<br></br><br />
<br />
<p class="title3">Fungicide operons</p><br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364013">BBa_K1364013</a>: P<sub>veg</sub> - SpoVG - Antifungals GAFP-1 and D4E1 - Double terminator</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte"><br />
<p class="title4">Design</p><br />
<p class="texte">This part is designed for the co-expression of two different peptides <br />
with anti-fungal activities : D4E1 and GAFP-1. <br />
It is composed of the strong, constitutive promoter of <i>B. subtilis</i> <br />
P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">K823003</a>) <br />
and the translation unit with GAFP-1 and D4E1 (BBa_K1364012).</p><br />
<img src="https://static.igem.org/mediawiki/2014/4/40/BBa_K1364013.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
See <a href="http://parts.igem.org/Part:BBa_K1364002">BBa_K1364002</a> and <a href="http://parts.igem.org/Part:BBa_K1364003">BBa_K1364003</a>.</p><br />
<br />
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</div><br />
<br />
<br></br><br />
<br />
<p class="title2">Basic tools</p><br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364015">BBa_K1364015</a>: P<sub>veg</sub> + RFP</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte"><!--Compléter--><br />
This part is designed to enable the expression of a Red Fluorescent Protein in Bacillus subtilis under the control of a constitutive promoter Pveg. This construction has been checked by sequencing and has shown to work also in E. coli<br />
</p><br />
<img src="https://static.igem.org/mediawiki/parts/6/69/Pveg%2BRFP.jpg"><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a constitutive promoter Pveg (K823003), spoVG RBS (K143021), the coding sequence of the RFP (E1010) and a double terminator (B0015) </p><br />
<p class="title4">Type</p><br />
<p class="texte">Reporter</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested in both E.coli and B. subtilis. The sequences was verified by sequencing. </p><br />
<br />
<br />
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</div><br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364016">BBa_K1364016</a>: P<sub>lepA</sub> + RFP</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte"><!--Compléter--><br />
This part is designed to enable the expression of a Red Fluorescent Protein in Bacillus subtilis under the control of a constitutive promoter PlepA. This construction has been checked by sequencing and has shown to work also in E. coli<br />
</p><br />
<img src="https://static.igem.org/mediawiki/parts/1/12/PlepA%2BRFP.jpg"><br />
<br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a constitutive promoter PlepA (K823002), spoVG RBS (K143021), the coding sequence of the RFP (E1010) and a double terminator (B0015) </p><br />
<p class="title4">Type</p><br />
<p class="texte">Reporter</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested in both E.coli and B. subtilis. The sequences was verified by sequencing.</p><br />
<br />
<br />
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</div><br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364017">BBa_K1364017</a>: P<sub>lepA</sub> + RBS SpoVG</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte"><!--Compléter--><br />
PlepA is a constitutive promoter in Bacillus subtilis (BBa_K823002) coupled with a RBS spoVG (BBa_K143021). To get the highest level of translation from this Promoter-RBS combination it must be connected to a coding region preceded by a coding region prefix. A standard prefix will increase the distance between the RBS and the start codon, reducing translational efficiency. <br />
This construction is also working with E. coli and has been verified by sequencing.<br />
</p><br />
<img src="https://static.igem.org/mediawiki/parts/d/da/PlepA%2BSpoVG.jpg"><br />
<p class="title4">Type</p><br />
<p class="texte">Composite</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was checked by sequencing</p><br />
<br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 12); return false">Collapse</a></p><br />
</div><br />
<br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364021">BBa_K1364021</a>: Integrative plasmid for <i>Bacillus subtilis</i> (pSBbs4E)</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte">This part is an empty backbone vector for the usage in Bacillus subtilis. It integrates in the thrC locus and can be selected with Erythromycin cassette. It has a Ampicillin resistance for cloning in E.coli. <br />
</p><br />
<p class="title4">Design</p><br />
<p class="texte">We digested pKL190 plasmid and PCR products of BBa_J04450 with BamHI and EcoRI. We proceded to ligation.</p><br />
<p class="title4">Type</p><br />
<p class="texte">Backbone</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested by transforming <i>B. subtilis</i>. Clones obtained were Auxotrophic of Threonine and resistant to Erythromycin.</p><br />
<p class="title4">References</p><br />
<p class="texte"><br />
R. Bernard, K.A. Marquis, and D.Z. Rudner. <b>Nucleoid occlusion prevents cell division during replication fork arrest in Bacillus subtilis</b>Mol Microbiol. Nov 2010; 78(4): 866–882.</p><br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 13); return false">Collapse</a></p><br />
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color:#666; font-size:18px;">Achievement</br><br />
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{{:Team:Toulouse/template/footer}}</div>Jourdanhttp://2014.igem.org/Team:Toulouse/Team/Fun_factsTeam:Toulouse/Team/Fun facts2014-10-17T09:40:37Z<p>Jourdan: </p>
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<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Team&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Fun Facts</p> <br />
<br />
</div> <br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<div style="float:left; width:525px;"><br />
<img src="https://static.igem.org/mediawiki/2014/3/30/Kilometers.jpg" style="margin-top:5px; width:470px" /><br />
</div><br />
<br />
<p class="title2">Have you ever tried to estimate the kilometers traveled by your team during this summer?</p><br />
<p class="texte"><br />
According to our calculations, one member walks approximately 4 kilometers per day all around the <br />
laboratory. This represents 5,544 kilometers for the whole team during the 126 days of this epic <br />
adventure. <br />
What does that mean exactly? Simply that we could walk, cycle, swim or whatever you want until <br />
Kazakhstan, Russia, Kenya… We could even have reached the USA but we decided to stay in the lab <br />
and take the plane to go to Boston!</p><br />
<br />
<br> </br><br />
<br />
<div style="float:right; width:500px;"><br />
<img src="https://static.igem.org/mediawiki/2014/5/52/Interview.jpg" style="margin-top:5px; margin-left: 62px; width:450px" /><br />
</div> <br />
<br />
<p class="title2">What do trees lining the “Canal du Midi” think about SubtiTree?</p><br />
<p class="texte"><br />
According to our homemade impartial survey, 94% of the questioned plane trees approve our project and are interested in <br />
serving as guinea pigs for our new bacterial medicine. This percentage represents 41,580 trees which <br />
are also gathered in the association called: “Happy tree friends“.</p><br />
<br />
<br />
<div style="float:left; width:500px; margin-top:50px;"><br />
<img src="https://static.igem.org/mediawiki/2014/a/ab/Multidisciplinary_yes_we_are.jpg" style="margin-top:5px; width:450px" /><br />
</div> <br />
<br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br> <br />
<p class="title2">Multidisciplinary… Yes we are!</p><br />
<p class="texte">Housework in our laboratory became necessary when most of people were in vacations except us. <br />
But do you know the novelty this year in our team? Times are changing because now men are <br />
cleaning! ;-)</p><br />
<br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br> <br> <br><br />
<br />
<div style="float:right; width:500px; margin-top:50px;"><br />
<img src="https://static.igem.org/mediawiki/2014/e/e2/World_cup.jpg" style="margin-top:5px; width:450px; margin-left:62px;" /><br />
</div> <br />
<br> <br> <br> <br> <br> <br><br />
<p class="title2">The “can’t miss it” event of the summer: the 2014 Football (also called Soccer in some third zone countries!!) World Cup!</p><br />
<p class="texte">From 06/12/14 to 07/13/14, the Toulouse iGEM Team was cheering on the French team. During the <br />
games of the French team, our group was juggling with wet lab and large screen projections! Despite <br />
our support, the French team did not win the World Cup. However, we have not said our last world <br />
yet: let’s see what will happen in 2018… ;-)</p><br />
<br />
<div class="clear"></div><br />
<br />
<div style="margin-top:50px; text-align:center;"><br />
<p class="title2">Have you ever forgotten a culture tube or a petri dish?</p><br />
<p class="texte" style="text-align:center;">Never? Let us show you what happens in that case!</p><br />
</div><br />
<center><br />
<table><br />
<tr><td><img src="https://static.igem.org/mediawiki/2014/f/fa/Old_tube1.png" width="160px"></td><br />
<td><img src="https://static.igem.org/mediawiki/2014/1/1b/Old_tube2.JPG" width="400px"></td></tr><br />
<tr><td><img src="https://static.igem.org/mediawiki/2014/d/d4/Old_petri1.png" width="370px"></td><br />
<td><img src="https://static.igem.org/mediawiki/2014/d/d7/Old_petri2.JPG" width="400px"></td></tr><br />
</table><br />
</center><br />
<br />
<br><br />
<br><br />
<br> <br />
<br />
<div style="text-align:center; width:760px; margin:0 auto; margin-top:15px; border-top:1px solid #555; padding-top:60px;"><br />
<p class="title2" style="padding-bottom:15px;">To finish this part, let’s do the official Awards Ceremony of the 2014 Toulouse iGEM Team!</p><br />
<ul><br />
<li class="tree"><p class="texte">The Geek Award goes to... <b>Florie</b> <br />
who spent the longest time in front of her laptop for the modeling part!</p></li><br />
<li class="tree"><p class="texte">The latest-survivor-of-weekly-meetings goes to... <b>Diane</b> who stayed up until 3am because she was skyping from South Korea!<br />
</p></li><br />
<li class="tree"><p class="texte">The worst singer award goes to ...<b>Abdel</b> who <br />
spent the whole day singing badly in the lab!</p></li><br />
<li class="tree"><p class="texte">The dancer award goes to ... <b>Camille</b><br />
who did the famous Plasmid Dance!</p></li><br />
<li class="tree"><p class="texte">The “hello you” award goes to... <b>Pierre</b> who was always saying <br />
“Hello you” each time he meets someone (approximatively 256 times per day)!</p></li><br />
<li class="tree"><p class="texte">The most tired award goes to...<b>Manon</b> but we still do not know why!</p></li><br />
<li class="tree"><p class="texte">The misplaced ideas award goes to... <b>Mathieu</b> but you do not want to know why!</p></li><br />
<li class="tree"><p class="texte">The perseverance award goes to... <b>Emeline</b> who succeeded a cloning after twelve trials!</p></li><br />
<li class="tree"><p class="texte">The drawing award goes to... <b>Fanny</b> <br />
who drew our first SubtiTree logo!</p></li><br />
<li class="tree"><p class="texte">The best phone-caller award goes to...<b>Laureen</b><br />
who was our perfect lab secretary!</p></li><br />
<li class="tree"><p class="texte">The biggest blunder in the lab award goes to ... <b>Aurélie</b> who poured an agarose gel without gel tray!</p></li><br />
<li class="tree"><p class="texte">And last but not least ... The Best Nervous breakdown Award goes to ... <b>Our deep freezer</b>! The whole team is grateful for its hard work during a hot summer! Three successive breakdowns during the hot days of summer: thank you freezer, so long chap, we’ll unplug you after iGEM is finished and you’ll retire, hopefully not in the Canal du Midi…</li></p><br />
</ul><br />
</div><br />
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<!-------------------------------- FOOTER ---------------------------------><br />
{{:Team:Toulouse/template/footer}}</div>Jourdanhttp://2014.igem.org/Team:Toulouse/Team/Fun_factsTeam:Toulouse/Team/Fun facts2014-10-16T21:01:55Z<p>Jourdan: </p>
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<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed; margin-top:30px;"><br />
<div style="margin:0 auto; width:960px;"><br />
<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Team&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Fun Facts</p> <br />
<br />
</div> <br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<div style="float:left; width:525px;"><br />
<img src="https://static.igem.org/mediawiki/2014/3/30/Kilometers.jpg" style="margin-top:5px; width:470px" /><br />
</div><br />
<br />
<p class="title2">Have you ever tried to estimate the kilometers traveled by your team during this summer?</p><br />
<p class="texte"><br />
According to our calculations, one member walks approximately 4 kilometers per day all around the <br />
laboratory. This represents 5,544 kilometers for the whole team during the 126 days of this epic <br />
adventure. <br />
What does that mean exactly? Simply that we could walk, cycle, swim or whatever you want until <br />
Kazakhstan, Russia, Kenya… We could even have reached the USA but we decided to stay in the lab <br />
and take the plane to go to Boston!</p><br />
<br />
<br> </br><br />
<br />
<div style="float:right; width:500px;"><br />
<img src="https://static.igem.org/mediawiki/2014/5/52/Interview.jpg" style="margin-top:5px; margin-left: 62px; width:450px" /><br />
</div> <br />
<br />
<p class="title2">What do trees lining the “Canal du Midi” think about SubtiTree?</p><br />
<p class="texte"><br />
According to our homemade impartial survey, 94% of the questioned plane trees approve our project and are interested in <br />
serving as guinea pigs for our new bacterial medicine. This percentage represents 41,580 trees which <br />
are also gathered in the association called: “Happy tree friends“.</p><br />
<br />
<br />
<div style="float:left; width:500px; margin-top:50px;"><br />
<img src="https://static.igem.org/mediawiki/2014/a/ab/Multidisciplinary_yes_we_are.jpg" style="margin-top:5px; width:450px" /><br />
</div> <br />
<br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br> <br />
<p class="title2">Multidisciplinary… Yes we are!</p><br />
<p class="texte">Housework in our laboratory became necessary when most of people were in vacations except us. <br />
But do you know the novelty this year in our team? Times are changing because now men are <br />
cleaning! ;-)</p><br />
<br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br> <br> <br><br />
<br />
<div style="float:right; width:500px; margin-top:50px;"><br />
<img src="https://static.igem.org/mediawiki/2014/e/e2/World_cup.jpg" style="margin-top:5px; width:450px; margin-left:62px;" /><br />
</div> <br />
<br> <br> <br> <br> <br> <br><br />
<p class="title2">The “can’t miss it” event of the summer: the 2014 Football (also called Soccer in some third zone countries!!) World Cup!</p><br />
<p class="texte">From 06/12/14 to 07/13/14, the Toulouse iGEM Team was cheering on the French team. During the <br />
games of the French team, our group was juggling with wet lab and large screen projections! Despite <br />
our support, the French team did not win the World Cup. However, we have not said our last world <br />
yet: let’s see what will happen in 2018… ;-)</p><br />
<br />
<div class="clear"></div><br />
<br />
<div style="margin-top:50px; text-align:center;"><br />
<p class="title2">Have you ever forgotten a culture tube or a petri dish?</p><br />
<p class="texte" style="text-align:center;">Never? Let us show you what happens in that case!</p><br />
</div><br />
<center><br />
<table><br />
<tr><td><img src="https://static.igem.org/mediawiki/2014/f/fa/Old_tube1.png" width="160px"></td><br />
<td><img src="https://static.igem.org/mediawiki/2014/1/1b/Old_tube2.JPG" width="400px"></td></tr><br />
<tr><td><img src="https://static.igem.org/mediawiki/2014/d/d4/Old_petri1.png" width="370px"></td><br />
<td><img src="https://static.igem.org/mediawiki/2014/d/d7/Old_petri2.JPG" width="400px"></td></tr><br />
</table><br />
</center><br />
<br />
<br><br />
<br><br />
<br> <br />
<br />
<div style="text-align:center; width:760px; margin:0 auto; margin-top:15px; border-top:1px solid #555; padding-top:60px;"><br />
<p class="title2" style="padding-bottom:15px;">To finish this part, let’s do the official Awards Ceremony of the 2014 Toulouse iGEM Team!</p><br />
<ul><br />
<li class="tree"><p class="texte">The Geek Awardgoes to <b>Florie</b> <br />
who spent the longest time in front of her laptop for the modeling part!</p></li><br />
<li class="tree"><p class="texte">The latest-survivor-of-weekly-meetings goes to... <b>Diane</b> who stayed up until 3am because she was skyping from South Korea!<br />
</p></li><br />
<li class="tree"><p class="texte">The worst singer award goes to ...<b>Abdel</b> who <br />
spent the whole day singing badly in the lab!</p></li><br />
<li class="tree"><p class="texte">The dancer award goes to ... <b>Camille</b><br />
who did the famous Plasmid Dance!</p></li><br />
<li class="tree"><p class="texte">The “hello you” award goes to... <b>Pierre</b> who was always saying <br />
“Hello you” each time he meets someone (approximatively 256 times per day)!</p></li><br />
<li class="tree"><p class="texte">The most tired award goes to...<b>Manon</b> but we still do not know why!</p></li><br />
<li class="tree"><p class="texte">The misplaced ideas award goes to... <b>Mathieu</b> but you do not want to know why!</p></li><br />
<li class="tree"><p class="texte">The perseverance award goes to... <b>Emeline</b> who succeeded a cloning after twelve trials!</p></li><br />
<li class="tree"><p class="texte">The drawing award goes to... <b>Fanny</b> <br />
who drew our first SubtiTree logo!</p></li><br />
<li class="tree"><p class="texte">The best phone-caller award goes to...<b>Laureen</b><br />
who was our perfect lab secretary!</p></li><br />
<li class="tree"><p class="texte">The biggest blunder in the lab award goes to ... <b>Aurélie</b> who poured an agarose gel without gel tray!</p></li><br />
<li class="tree"><p class="texte">And last but not least ... The Best Nervous breakdown Award goes to ... <b>Our deep freezer</b>! The whole team is grateful for its hard work during a hot summer! Three successive breakdowns during the hot days of summer: thank you freezer, so long chap, we’ll unplug you after iGEM is finished and you’ll retire, hopefully not in the Canal du Midi…</li></p><br />
</ul><br />
</div><br />
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{{:Team:Toulouse/template/footer}}</div>Jourdanhttp://2014.igem.org/Team:Toulouse/Team/Fun_factsTeam:Toulouse/Team/Fun facts2014-10-16T21:00:41Z<p>Jourdan: </p>
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<div class="fils-ariane" style="width:100%; height:60px; background:#ededed; margin-top:30px;"><br />
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<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Team&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Fun Facts</p> <br />
<br />
</div> <br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<div style="float:left; width:525px;"><br />
<img src="https://static.igem.org/mediawiki/2014/3/30/Kilometers.jpg" style="margin-top:5px; width:470px" /><br />
</div><br />
<br />
<p class="title2">Have you ever tried to estimate the kilometers traveled by your team during this summer?</p><br />
<p class="texte"><br />
According to our calculations, one member walks approximately 4 kilometers per day all around the <br />
laboratory. This represents 5,544 kilometers for the whole team during the 126 days of this epic <br />
adventure. <br />
What does that mean exactly? Simply that we could walk, cycle, swim or whatever you want until <br />
Kazakhstan, Russia, Kenya… We could even have reached the USA but we decided to stay in the lab <br />
and take the plane to go to Boston!</p><br />
<br />
<br> </br><br />
<br />
<div style="float:right; width:500px;"><br />
<img src="https://static.igem.org/mediawiki/2014/5/52/Interview.jpg" style="margin-top:5px; margin-left: 62px; width:450px" /><br />
</div> <br />
<br />
<p class="title2">What do trees lining the “Canal du Midi” think about SubtiTree?</p><br />
<p class="texte"><br />
According to our homemade impartial survey, 94% of the questioned plane trees approve our project and are interested in <br />
serving as guinea pigs for our new bacterial medicine. This percentage represents 41,580 trees which <br />
are also gathered in the association called: “Happy tree friends“.</p><br />
<br />
<br />
<div style="float:left; width:500px; margin-top:50px;"><br />
<img src="https://static.igem.org/mediawiki/2014/a/ab/Multidisciplinary_yes_we_are.jpg" style="margin-top:5px; width:450px" /><br />
</div> <br />
<br />
<br><br />
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<br> <br />
<p class="title2">Multidisciplinary… Yes we are!</p><br />
<p class="texte">Housework in our laboratory became necessary when most of people were in vacations except us. <br />
But do you know the novelty this year in our team? Times are changing because now men are <br />
cleaning! ;-)</p><br />
<br />
<br><br />
<br><br />
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<br> <br> <br><br />
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<div style="float:right; width:500px; margin-top:50px;"><br />
<img src="https://static.igem.org/mediawiki/2014/e/e2/World_cup.jpg" style="margin-top:5px; width:450px; margin-left:62px;" /><br />
</div> <br />
<br> <br> <br> <br> <br> <br><br />
<p class="title2">The “can’t miss it” event of the summer: the 2014 Football (also called Soccer in some third zone countries!!) World Cup!</p><br />
<p class="texte">From 06/12/14 to 07/13/14, the Toulouse iGEM Team was cheering on the French team. During the <br />
games of the French team, our group was juggling with wet lab and large screen projections! Despite <br />
our support, the French team did not win the World Cup. However, we have not said our last world <br />
yet: let’s see what will happen in 2018… ;-)</p><br />
<br />
<div class="clear"></div><br />
<br />
<div style="margin-top:50px; text-align:center;"><br />
<p class="title2">Have you ever forgotten a culture tube or a petri dish?</p><br />
<p class="texte" style="text-align:center;">Never? Let us show you what happens in that case!</p><br />
</div><br />
<center><br />
<table><br />
<tr><td><img src="https://static.igem.org/mediawiki/2014/f/fa/Old_tube1.png" width="160px"></td><br />
<td><img src="https://static.igem.org/mediawiki/2014/1/1b/Old_tube2.JPG" width="400px"></td></tr><br />
<tr><td><img src="https://static.igem.org/mediawiki/2014/d/d4/Old_petri1.png" width="370px"></td><br />
<td><img src="https://static.igem.org/mediawiki/2014/d/d7/Old_petri2.JPG" width="400px"></td></tr><br />
</table><br />
</center><br />
<br />
<br><br />
<br><br />
<br> <br />
<br />
<div style="text-align:center; width:760px; margin:0 auto; margin-top:15px; border-top:1px solid #555; padding-top:60px;"><br />
<p class="title2" style="padding-bottom:15px;">To finish this part, let’s do the official Awards Ceremony of the 2014 Toulouse iGEM Team!</p><br />
<ul><br />
<li class="tree"><p class="texte">The Geek Awardgoes to <b>Florie</b> <br />
who spent the longest time in front of her laptop for the modeling part!</p></li><br />
<li class="tree"><p class="texte">The latest-survivor-of-weekly-meetings goes to... <b>Diane</b> who stayed up until 3am because she was skyping from South Korea!<br />
</p></li><br />
<li class="tree"><p class="texte">The worst singer award goes to ...<b>Abdel</b> who <br />
spent the whole day singing badly in the lab!</p></li><br />
<li class="tree"><p class="texte">The dancer award goes to ... <b>Camille</b><br />
who did the famous Plasmid Dance!</p></li><br />
<li class="tree"><p class="texte">The “hello you” award goes to... <b>Pierre</b> who was always saying <br />
“Hello you” each time he meets someone (approximatively 256 times per day)!</p></li><br />
<li class="tree"><p class="texte">The most tired award: The three nominees are Laureen, Manon, Aurélie. And the winner is... <br />
<b>Manon</b> but we still do not know why!</p></li><br />
<li class="tree"><p class="texte">The misplaced ideas award goes to... <b>Mathieu</b> but you do not want to know why!</p></li><br />
<li class="tree"><p class="texte">The perseverance award: The three nominees are Emeline, Diane, Abdel. And the winner is... <br />
<b>Emeline</b> who succeeded a cloning after twelve trials!</p></li><br />
<li class="tree"><p class="texte">The drawing award goes to... <b>Fanny</b> <br />
who drew our first SubtiTree logo!</p></li><br />
<li class="tree"><p class="texte">The best phone-caller award goes to...<b>Laureen</b><br />
who was our perfect lab secretary!</p></li><br />
<li class="tree"><p class="texte">The biggest blunder in the lab award goes to ... <b>Aurélie</b> who poured an agarose gel without gel tray!</p></li><br />
<li class="tree"><p class="texte">And last but not least ... The Best Nervous breakdown Award goes to ... <b>Our deep freezer</b>! The whole team is grateful for its hard work during a hot summer! Three successive breakdowns during the hot days of summer: thank you freezer, so long chap, we’ll unplug you after iGEM is finished and you’ll retire, hopefully not in the Canal du Midi…</li></p><br />
</ul><br />
</div><br />
<br />
</div><br />
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<!-------------------------------- FOOTER ---------------------------------><br />
{{:Team:Toulouse/template/footer}}</div>Jourdanhttp://2014.igem.org/Team:Toulouse/Result/parts/Submitted_partsTeam:Toulouse/Result/parts/Submitted parts2014-10-16T20:48:00Z<p>Jourdan: </p>
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<h2>Parts</h2><br />
<p>What did we send to the Registry?</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Results&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Parts</p> <br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 20px; padding-bottom:40px;"><br />
<br />
<br />
<p class="title1">Submitted parts</p><br />
<br />
<p class="texte"><br />
We have deposed 16 new BioBrick parts to the Registry. <br />
All of them were cloned into the standard plasmid pSB1C3, tested and sequenced.<br><br />
<!--à vérifier et confirmer--><br />
</p><br />
<br />
<p class="title2">I. Chemotaxis</p><br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</a>: N-acetylglucosamine based chemotaxis for <i>Bacillus subtilis</i></div><br />
<div class="thelanguage"><br />
<br />
<p class="texte">This part is designed to enable a N-acetylglucosamine <br />
based chemotaxis in <i>Bacillus subtilis</i>.<br />
<p class="title4">Design</p><br />
<p class="texte">The encoded protein is a chimera of two proteins:<br><br />
- the methyl accepting Chemotaxis protein (McpA from <i>Bacillus subtilis</i> which is required for taxis towards glucose.<br><br />
- the N-acetylglucosamine regulated methyl-accepting chemotaxis protein from <i>Vibrio cholerae</i> (VCD).<br><br />
<br><br />
The chimeric protein contains the intracellular domains of McpA from <i>Bacillus subtilis</i> to enable the transduction of the signal: <br />
these domains correspond to the amino acids 1-37 and 282-610. <br />
The extracellular domain of VCD is inserted between the intracellular regions of McpA <br />
to sense N-acetylglucosamine and corresponds to the amino acids 31-310.<br></p><br />
<img src="https://static.igem.org/mediawiki/2014/a/a2/BBa_K1364000.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Translation unit</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part is not tested yet.</p><br />
<p class="title4">References</p><br />
<p class="texte">Uniprot:<br><br />
- <a href="http://www.uniprot.org/uniprot/P39214">McpA</a><br><br />
- <a href="http://www.uniprot.org/uniprot/C3NYT2">N-acetylglucosamine regulated methyl-accepting chemotaxis protein</a><br></p><br />
<!--à compléter--><br />
</p><br />
<br><br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 0); return false">Collapse</a></p><br />
</div><br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364004">BBa_K1364004</a>: P<sub>veg</sub> + N-acetylatedglucosamine based chemotaxis for <i>B. subtilis</i></div><br />
<div class="thelanguage"><br />
<br />
<br><br />
<p class="texte">This part is designed to enable a N-acetylglucosamine based chemotaxis in <i>Bacillus subtilis</i>.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This expression cassette is designed for the expression of an antifungal peptide, <br />
D4E1 and for its secretion in <i>Bacillus subtilis</i>.<br />
It is composed of the strong, constitutive promotor of <i>B. subtilis</i> <br />
P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">K823003</a>),<br />
strong RBS for <i>B. subtilis</i> (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>),<br />
the open reading frame of D4E1<br />
and a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>)</p><br />
<img src="https://static.igem.org/mediawiki/2014/3/35/BBa_K1364004.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was not tested yet.</p><br />
<p class="title4">References</p><br />
<p class="texte">Uniprot:<br><br />
- <a href="http://www.uniprot.org/uniprot/P39214">McpA</a><br><br />
- <a href="http://www.uniprot.org/uniprot/C3NYT2">N-acetylglucosamine regulated methyl-accepting chemotaxis protein</a></p><br />
<br />
<br />
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 0); return false">Collapse</a></p><br />
</div><br />
<br />
<br></br><br />
<br />
<p class="title2">II. Binding</p><br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364005">BBa_K1364005</a>: P<sub>veg</sub> + Chitin Binding Cell Wall protein</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte">This part is designed to enable the binding of <i>Bacillus subtilis</i> to the fungi wall made of chitin.<p/><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a Cell Wall Binding (CWB) sequence <br />
and a Chitin Binding Domain (CBD) linked with a 6 amino acids linker.<br><br />
<br><br />
The CWB domain has been extracted from LytC (<a href="http://parts.igem.org/Part:BBa_K316030">BBa_K316030</a>) of the iGEM 2010 Imperial College team.<br><br />
The CBD is composed of the Domain 4 of the GbpA protein of <i>Vibrio cholerae</i>,<br />
a protein reported to mediate bacterial attachment to chitin. <br />
It has been shown that the Domain 4 was essential to bind chitin.</p><br />
<img src="https://static.igem.org/mediawiki/2014/f/f5/BBa_K1364005.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested with chitin beads. (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Binding module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)<br><br />
- The Vibrio cholerae Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373<br><br />
</p><br />
<br />
<br><br />
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</div><br />
<br />
<br></br><br />
<br />
<p class="title2"> III. Fungicides</p><br />
<p class="title3">GAFP-1</p><br />
<br />
<br><br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364002">BBa_K1364002</a>: RBS - Antifungal GAFP-1</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte">The <i>Gastrodia</i> anti-fungal protein(GAFP-1), also known as gastrodianin, is a mannose and chitin binding lectin<br />
originating from the Asiatic orchid Gastrodia elata, a traditional Chinese medicinal herb cultured for thousands of years.<br />
GAFP-1 is composed of 15 amino acids LDSLSFSYNNFEEDD and is able to inhibit the growth of multiples species of plant pathogenic fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a Strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>) and<br />
the open reading frame of the <i>Gastrodia</i> anti-fungal protein 1 (GAFP-1)<br />
optimized for its expression and its secretion in <i>Bacillus subtilis</i>.<br />
The codon optimization was made thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/5/5f/BBa_K1364002.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Translational unit</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)<br><br />
- The Vibrio cholerae Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373<br></p><br />
<br><br />
<br />
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</div><br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364007">BBa_K1364007</a>: RBS + Antifungal GAFP-1 + Double terminator</div><br />
<div class="thelanguage"><br />
<br />
<br />
<p class="texte">The <i>Gastrodia</i> anti-fungal protein(GAFP-1), also known as gastrodianin, is a mannose and chitin binding lectin<br />
originating from the Asiatic orchid Gastrodia elata, a traditional Chinese medicinal herb cultured for thousands of years.<br />
GAFP-1 is composed of 15 amino acids LDSLSFSYNNFEEDD and is able to inhibit the growth of multiples species of plant pathogenic fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a Strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>),<br />
the open reading frame of the <i>Gastrodia</i> anti-fungal protein 1 (GAFP-1) <br />
and a double terminator <a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>.<br />
optimized for its expression and its secretion in <i>Bacillus subtilis</i>.<br />
The codon optimization was made thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/4/42/BBa_K1364007.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Composite part</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)<br><br />
- The Vibrio cholerae Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373<br></p><br />
<br><br />
<br />
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</div><br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364008">BBa_K1364008</a>: P<sub>veg</sub> - strong RBS - Antifungal GAFP-1 - Double terminator</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte">This expression cassette is designed for the expression and secretion of the <i>Gastrodia</i> anti-fungal protein(GAFP-1), also known as gastrodianin. GAFP-1 is a mannose and chitin binding lectin<br />
originating from the Asiatic orchid Gastrodia elata, a traditional Chinese medicinal herb cultured for thousands of years.<br />
GAFP-1 is composed of 15 amino acids LDSLSFSYNNFEEDD and is able to inhibit the growth of multiples species of plant pathogenic fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of the strong, constitutive promoter of <i>Bacillus subtilis</i> P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">K823003</a>),<br />
strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>),<br />
the open reading frame of the <i>Gastrodia</i> anti-fungal protein 1 (GAFP-1) <br />
and a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>).<br />
optimized for its expression and its secretion in <i>Bacillus subtilis</i>.<br />
The codon optimization was made thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/6/67/BBa_K1364008.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)<br><br />
- The Vibrio cholerae Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373<br><br />
</p><br />
<br />
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</div><br />
<br />
<br></br><br />
<br />
<p class="title3">D4E1</p><br />
<br />
<br><br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364003">BBa_K1364003</a>:RBS - Antifungal D4E1 - Double terminator</div><br />
<div class="thelanguage"><br />
<br />
<br />
<p class="texte">D4E1 is a linear synthetic peptide of 17 amino acids <br />
which has shown to have antifungal activities <br />
by complexing with a sterol present in conodial wall of a varety of fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a Strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>),<br />
the open reading frame of D4E1 and <br />
a Double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>)<br />
optimized for its expression and its secretion in <i>Bacillus subtilis</i>.<br />
This part was optimized for the expression and its secretion in <i>Bacillus subtilis </i><br />
thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/b/bb/BBa_K1364003.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Translational unit</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- De Lucca AJ, Bland JM, Grimm C, Jacks TJ, Cary JW, Jaynes JM, Cleveland TE, Walsh TJ. Fungicidal properties, sterol binding, and proteolytic resistance of the synthetic peptide D4E1.<br><br />
- Can J Microbiol. 1998 Jun;44(6):514-20.</p><br />
<br />
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</div><br />
<br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364009">BBa_K1364009</a>:P<sub>veg</sub> - RBS - Antifungal D4E1 - Double Terminator</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte"><br />
D4E1 is a linear synthetic peptide of 17 amino acids <br />
which has shown to have antifungal activities <br />
by complexing with a sterol present in conodial wall of a varety of fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of the strong, constitutive promotor of <i>B. subtilis</i> <br />
P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">K823003</a>),<br />
a strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>),<br />
the open reading frame of D4E1 and <br />
a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>).<br />
This part was optimized for the expression and its secretion in <i>Bacillus subtilis </i><br />
thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/4/47/BBa_K1364009.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Translational unit</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- De Lucca AJ, Bland JM, Grimm C, Jacks TJ, Cary JW, Jaynes JM, Cleveland TE, Walsh TJ. Fungicidal properties, sterol binding, and proteolytic resistance of the synthetic peptide D4E1.<br><br />
- Can J Microbiol. 1998 Jun;44(6):514-20.</p><br />
<br />
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</div><br />
<br />
<br></br><br />
<br />
<p class="title3">EcAMP-1</p><br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364010">BBa_K1364010</a>: P<sub>veg</sub> - SpoVG - Antifungal EcAMP-1</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte"><br />
EcAMP-1 is an antimicrobial peptide of 37 amino acids <br />
originated from the specie <i>Echinochloa crus-galli</i>, <br />
a type of wild grass. <br />
This peptide has a particular structure : it is helical because of two disulfide bonds.<br><br />
EcAMP-1 has shown to have antifungal activities. Its mode of action may be the prevention of hyphae elongation.<br><br />
This part was added to the Registry by the iGEM Utah State team in 2013.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of the constitutive promoter P<sub>veg</sub> and<br />
strong RBS for <i>B. subtilis</i><br />
(<a href="http://parts.igem.org/Part:BBa_K733013">K733013</a>) and<br />
the open reading frame of EcAMP-1.<br><br />
The EcAMP-1 part was codon optimized for <i>E. coli</i> by the iGEM Utah State team<br />
and thanks to the Life Technologies GeneArt software program. </p><br />
<img src="https://static.igem.org/mediawiki/2014/5/5f/BBa_K1364010.png"><br />
<p class="title4">Type</p><br />
<p class="texte"> Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part is not tested yet.</p><br />
<p class="title4">References</p><br />
<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 (Echinochloa crus-galli)</b>. The journal of Biological Chemistry. 2011, Vol. 286, 25145–25153</p><br />
<br />
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</div><br />
<br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364011">BBa_K1364011</a>: P<sub>veg</sub> - SpoVG - Antifungal EcAMP-1 - Double terminator</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte" id="select10"><br />
EcAMP-1 is an antimicrobial peptide of 37 amino acids <br />
originated from the specie <i>Echinochloa crus-galli</i>, <br />
a type of wild grass. <br />
This peptide has a particular structure : it is helical because of two disulfide bonds.<br><br />
EcAMP-1 has shown to have antifungal activities. Its mode of action may be the prevention of hyphae elongation.<br><br />
This part was added to the Registry by the iGEM Utah State team in 2013.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of the constitutive promoter P<sub>veg</sub> and<br />
strong RBS for <i>B. subtilis</i><br />
(<a href="http://parts.igem.org/Part:BBa_K733013">K733013</a>),<br />
the open reading frame of EcAMP-1 and <br />
a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>).<br><br />
The EcAMP-1 part was codon optimized for <i>E. coli</i> by the iGEM Utah State team<br />
and thanks to the Life Technologies GeneArt software program. </p><br />
<img src="https://static.igem.org/mediawiki/2014/7/74/BBa_K1364011.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part is not tested yet.</p><br />
<p class="title4">References</p><br />
<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 (Echinochloa crus-galli)</b>. The journal of Biological Chemistry. 2011, Vol. 286, 25145–25153</p><br />
<br />
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</div><br />
<br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364019">BBa_K1364019</a>: P<sub>veg</sub> - RBS - Antifungal EcAMP (revised with a stop codon) - Double terminator</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte">EcAMP-1 is an antimicrobial peptide of 37 amino acids <br />
originated from the specie <i>Echinochloa crus-galli</i>, <br />
a type of wild grass. <br />
This peptide has a particular structure : it is helical because of two disulfide bonds.<br><br />
EcAMP-1 has shown to have antifungal activities. Its mode of action may be the prevention of hyphae elongation.<br><br />
This part was added to the Registry by the iGEM Utah State team in 2013.</br><br />
We observed the presence of the stop codon in the suffix. When we have digested this Biobrick, it disappeared. So we added this codon in upstream of the suffix and now, we can reuse this Biobrick.<br />
</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of the constitutive promoter P<sub>veg</sub> and<br />
strong RBS for <i>B. subtilis</i><br />
(<a href="http://parts.igem.org/Part:BBa_K733013">K733013</a>),<br />
<b>the revised open reading frame of EcAMP-1 (with a stop codon) </b> and <br />
a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>).<br><br />
The EcAMP-1 part was codon optimized for <i>E. coli</i> by the iGEM Utah State team<br />
and thanks to the Life Technologies GeneArt software program. </p><br />
<img src="https://static.igem.org/mediawiki/2014/7/74/BBa_K1364011.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Composite</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was not tested yet</p><br />
<p class="title4">References</p><br />
<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 (Echinochloa crus-galli)</b>. The journal of Biological Chemistry. 2011, Vol. 286, 25145–25153</p><br />
<br />
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</div><br />
<br />
<br></br><br />
<br />
<p class="title3">Fungicide operons</p><br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364013">BBa_K1364013</a>: P<sub>veg</sub> - SpoVG - Antifungals GAFP-1 and D4E1 - Double terminator</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte"><br />
<p class="title4">Design</p><br />
<p class="texte">This part is designed for the co-expression of two different peptides <br />
with anti-fungal activities : D4E1 and GAFP-1. <br />
It is composed of the strong, constitutive promotor of <i>B. subtilis</i> <br />
P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">K823003</a>) <br />
and the translation unit with GAFP-1 and D4E1 (BBa_K1364012).</p><br />
<img src="https://static.igem.org/mediawiki/2014/4/40/BBa_K1364013.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
See <a href="http://parts.igem.org/Part:BBa_K1364002">BBa_K1364002</a> and <a href="http://parts.igem.org/Part:BBa_K1364003">BBa_K1364003</a>.</p><br />
<br />
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</div><br />
<br />
<br></br><br />
<br />
<p class="title2">Basic tools</p><br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364015">BBa_K1364015</a>: P<sub>veg</sub> + RFP</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte"><!--Compléter--><br />
This part is designed to enable the expression of a Red Fluorescent Protein in Bacillus subtilis under the control of a constitutive promotor Pveg. This construction has been checked by sequencing and has shown to work also in E. coli<br />
</p><br />
<img src="https://static.igem.org/mediawiki/parts/6/69/Pveg%2BRFP.jpg"><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a constitutive promotor Pveg (K823003), spoVG RBS (K143021), the coding sequence of the RFP (E1010) and a double terminator (B0015) </p><br />
<p class="title4">Type</p><br />
<p class="texte">Reporter</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested in both E.coli and B. subtilis. The sequences was verified by sequencing. </p><br />
<br />
<br />
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</div><br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364016">BBa_K1364016</a>: P<sub>lepA</sub> + RFP</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte"><!--Compléter--><br />
This part is designed to enable the expression of a Red Fluorescent Protein in Bacillus subtilis under the control of a constitutive promotor PlepA. This construction has been checked by sequencing and has shown to work also in E. coli<br />
</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a constitutive promotor PlepA (K823002), spoVG RBS (K143021), the coding sequence of the RFP (E1010) and a double terminator (B0015) </p><br />
<p class="title4">Type</p><br />
<p class="texte">Reporter</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested in both E.coli and B. subtilis. The sequences was verified by sequencing.</p><br />
<br />
<br />
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</div><br />
<br />
<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364017">BBa_K1364017</a>: P<sub>lepA</sub> + RBS SpoVG</div><br />
<div class="thelanguage"><br />
<br />
<p class="texte"><!--Compléter--><br />
PlepA is a constitutive promotor in Bacillus subtilis (BBa_K823002) coupled with a RBS spoVG (BBa_K143021). To get the highest level of translation from this Promoter-RBS combination it must be connected to a coding region preceded by a coding region prefix. A standard prefix will increase the distance between the RBS and the start codon, reducing translational efficiency. <br />
This construction is also working with E. coli and has been verified by sequencing.<br />
</p><br />
<br />
<p class="title4">Type</p><br />
<p class="texte">Composite</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was checked by sequencing</p><br />
<br />
<br />
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<div class="technology"><a href="http://parts.igem.org/Part:BBa_K1364021">BBa_K1364021</a>: Integrative plasmid for <i>Bacillus subtilis</i></div><br />
<div class="thelanguage"><br />
<br />
<p class="texte"><!--Compléter--><br />
</p><br />
<p class="title4">Design</p><br />
<p class="texte">Coming soon! </p><br />
<p class="title4">Type</p><br />
<p class="texte">Composite</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was not tested yet</p><br />
<p class="title4">References</p><br />
<p class="texte">Coming soon!</p><br />
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{{:Team:Toulouse/template/footer}}</div>Jourdanhttp://2014.igem.org/Team:Toulouse/Project/FungicidesTeam:Toulouse/Project/Fungicides2014-10-16T20:30:09Z<p>Jourdan: </p>
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<h2>Fungicides</h2><br />
<p>To eradicate fungal diseases</p><br />
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<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Project&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Fungicides</p> <br />
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<!--Short description : à changer!!!--><br />
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<center><img style="width:700px; " src="https://static.igem.org/mediawiki/2014/0/0c/Recap_fungicides.jpg"><br />
<br><br />
<p class="legend">Figure 1: Scheme of the fungicide module</p></center><br />
<br />
<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. This triple therapy minimizes the risk of resistance.</p> <br><br />
<br />
<p class="textesimple">Originally from plants, these peptides have different targets to maximize the lethality on <i>C. platani</i>.</p><br />
<br></br> <br />
<ul><br />
<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 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><br />
<br />
<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 originating from the Asiatic orchid <i>Gastrodia elata</i>, a traditional Chinese medicinal herb cultured for thousands of years. 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 ascomycete and basidiomycete fungal plant pathogens.</p></li><br />
<br />
<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. 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. EcAMP-1 exhibits high activity against fungi of the genus <i>Fusarium</i>.</p></li><br />
</ul><br />
</p><br />
<br><br />
<p class="title1" style="margin-top:30px;">More information about this module </p><br />
<p class="texte"><br />
We built different genetic constructions to test each fungicide separately and to test them all together on the same operon where the three genes coding for the antifungal peptides are placed under the control of the constitutive promoter P<sub>veg</sub> in <i>Bacillus subtilis</i>.</p><br />
<br />
<center><img style="width:930px; float:left; margin: 30px 0 45px;" src="https://static.igem.org/mediawiki/parts/d/d0/Fungicideprod.jpg"><br />
<p class="legend">Figure 2: Fungicide operon</p></center><br />
<br />
<p class="title2">Added parts</p><br />
<p class="title3">EcAMP-1</p><br />
<p class="texte">EcAMP-1 was already present in the Registry, added by the Utah State 2013 iGEM team (<a href="http://parts.igem.org/Part:BBa_K1162001"_blank">BBa_K1162001</a>). This part has been modified and improved by our team (<a href="http://parts.igem.org/Part:BBa_K1364019"_blank">BBa_K1364019</a>). </p><br />
<p class="title3">D4E1 and GAFP-1</p><br />
<p class="texte">We added D4E1 and GAFP-1 to the Registry of Standard Biological Parts (See <a href="https://2014.igem.org/Team:Toulouse/Result/parts/Submitted_parts"_blank">Submitted parts</a>). <br>We ordered the genes to a synthesis company and did cloning. These new BioBricks were designed in order to be expressed and secreted with <i>Bacillus subtilis</i>. </p><br />
<br><br />
<p class="title2">Secretion</p><br />
<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 a signal peptide (amyE signal peptide) followed by a pro peptide, cleaved during the secretion process.</p><br><br />
<br />
<br />
<center><img style="width:400px; " src="https://static.igem.org/mediawiki/2014/2/2e/Secretion.jpg"><br />
<img style="width:400px; " src="https://static.igem.org/mediawiki/2014/d/d7/Fongpep.jpg"><br />
<br><p class="legend">Figure 3: Design of GAFP-1 and D4E1</p></center><br />
<br><br />
<br />
<br />
<p class="title1">References</p><br />
<br />
<ul><br />
<li class="tree"><p class="texte">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. </p></li><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><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><br />
<li class="tree"><p class="texte">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</p></li><br />
<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><br />
<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 (Echinochloa crus-galli)</b>. The journal of Biological Chemistry. 2011, Vol. 286, 25145–25153</p></li><br />
</ul><br />
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{{:Team:Toulouse/template/footer}}</div>Jourdanhttp://2014.igem.org/Team:Toulouse/Result/parts/Used_partsTeam:Toulouse/Result/parts/Used parts2014-10-16T20:10:40Z<p>Jourdan: </p>
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<h2>Parts</h2><br />
<p>What did we send to the Registry?</p><br />
</div><br />
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</div><br />
<br />
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<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Results&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Parts</p> <br />
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<br />
<!--SOUS-TITRE--><br />
<p class="title1">Used parts</p><br />
<br />
<br><br />
<p class="texte"> <br />
Here are listed all the bricks we used from the iGEM kits to make our project ! </p><br />
<br />
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<!--1ere ligne--><br />
<tr><td><p class="texte"><b>BioBrick</b></p></td><br />
<td><p class="texte"><b>Description</b></p></td><br />
<td><p class="texte"><b>Origine</b></p></td><br />
<td><p class="texte"><b>Module(s)</b></p></td></tr><br />
<!--2eme ligne--><br />
<tr><td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K733013">K733013</a></p></td><br />
<td><p class="texte">P<sub>veg</sub> (<a href="http://parts.igem.org/Part:BBa_K316001">K316001</a>) + RBS-SpoVG (<a href="http://parts.igem.org/Part:BBa_K143021">K143021</a>) : Constitutive promotor P<sub>veg</sub> and strong RBS for <i>B. subtilis</i></p></td><br />
<td><p class="texte">2014 Kit plate 1, 7H</p></td><br />
<td><p class="texte">Fungicides (EcAMP)</p></td><br />
</tr><br />
<!--3eme ligne--><br />
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<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K823003">K823003</a></p></td><br />
<td><p class="texte">P<sub>veg</sub> : strong, constitutive promotor of <i>B. subtilis</i></p></td><br />
<td><p class="texte">2014 Kit plate 1, 20G</p></td><br />
<td><p class="texte">Fungicides (D4E1 GAFP-1)<br>Chemotaxis<br>Binding</p></td><br />
</tr><br />
<!--4eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K823002">K823002</a></p></td><br />
<td><p class="texte">P<sub>lepA</sub> : constitutive promotor of <i>B. subtilis</i>.<br />
<br>It is the promotor of the lepA gene of <i>B. subtilis</i></p></td><br />
<td><p class="texte">2014 Kit plate 1, 20E</p></td><br />
<td><p class="texte">Fungicides (D4E1)<br>Chemotaxis<br>Binding</p></td><br />
</tr><br />
<!--5eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K606061">K606061</a></p></td><br />
<td><p class="texte">SpoVG RBS for <i>B. subtilis</i><br />
<br>Strong ribosome binding site from subtilis. Works fine in <i>E. coli</i></p></td><br />
<td><p class="texte">2014 Kit plate 1, 15A</p></td><br />
<td><p class="texte">Optional</p></td><br />
</tr><br />
<!--6eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K316027">K316027</a></p></td><br />
<td><p class="texte"><i>B. subtilis</i> transformation vector with LacI, targets amyE locus.</p></td><br />
<td><p class="texte">2014 Kit plate 1, 2N</p></td><br />
<td><p class="texte">Fungicides<br>Chemotaxis<br>Binding</p></td><br />
</tr><br />
<!--7eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_B0015">B0015</a></p></td><br />
<td><p class="texte">Double terminator (<a href="http://parts.igem.org/Part:BBa_B0010">BBa_B0010</a> + <a href="http://parts.igem.org/Part:BBa_B0012">BBa_B0012</a>)</p></td><br />
<td><p class="texte">2014 Kit plate 3, 3F</p></td><br />
<td><p class="texte">Fungicides (GAFP-1, EcAMP-1)</p></td><br />
</tr><br />
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<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K780000">K780000</a></p></td><br />
<td><p class="texte">Terminator for <i>B. subtilis</i></p></td><br />
<td><p class="texte">Sent by the Warsaw iGEM team</p></td><br />
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<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K780001">K780001</a></p></td><br />
<td><p class="texte">Strong RBS for <i>B. subtilis</i></p></td><br />
<td><p class="texte">Sent by the Warsaw iGEM team</p></td><br />
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<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K780002">K780002</a></p></td><br />
<td><p class="texte">Strong RBS for <i>B. subtilis</i></p></td><br />
<td><p class="texte">Sent by the Warsaw iGEM team</p></td><br />
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<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K780003">K780003</a></p></td><br />
<td><p class="texte">Strong constitutive promotor for <i>B. subtilis</i></p></td><br />
<td><p class="texte">Sent by the Warsaw iGEM team</p></td><br />
<td><p class="texte">Fungicides<br>Chemotaxis<br>Binding</p></td><br />
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<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K1162001">K1162001</a></p></td><br />
<td><p class="texte">EcAMP-1</p></td><br />
<td><p class="texte">Sent by the Utah State iGEM team</p></td><br />
<td><p class="texte">Fungicides (EcAMP-1)</p></td><br />
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<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K823023">K823023</a></p></td><br />
<td><p class="texte">Empty backbone vector pSB<sub>BS</sub>1C for integration into <i>B. subtilis</i><br />
<br>It integrates in the amyE locus</p></td><br />
<td><p class="texte">Sent by the Munich iGEM team</p></td><br />
<td><p class="texte">Fungicides<br>Chemotaxis<br>Binding</p></td></tr><br />
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<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K823022">K823022</a></p></td><br />
<td><p class="texte">Empty backbone vector pSB<sub>BS</sub>4S for integration into<i>B. subtilis</i><br />
<br>It integrates in the thrC locus</p></td><br />
<td><p class="texte">Sent by the Munich iGEM team</p></td><br />
<td><p class="texte">Fungicides<br>Chemotaxis<br>Binding</p></td><br />
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<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K823021">K823021</a></p></td><br />
<td><p class="texte">Reporter vector pSB<sub>BS</sub>1C-lacZ for <i>B. subtilis</i></p></td><br />
<td><p class="texte">Sent by the Munich iGEM team</p></td><br />
<td><p class="texte">Fungicides<br>Chemotaxis<br>Binding</p></td></tr><br />
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color:#666; font-size:18px;">Achievement</br><br />
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{{:Team:Toulouse/template/footer}}</div>Jourdanhttp://2014.igem.org/Team:Toulouse/Result/achievementTeam:Toulouse/Result/achievement2014-10-16T15:32:08Z<p>Jourdan: </p>
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<h2>Achievement</h2><br />
<p>Bronze, Silver or Gold Medal? That is the question!</p><br />
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<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Results&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Achievement</p> <br />
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<h1 class="title1" style="text-align:center;">Let's sum up what we did during this summer!</h1><br />
<br></br><br />
<p class="texte"> <br />
After a summer of hard work, our team is proud of the achieved results. The first step of our project was to have a <b>proof of concept</b> of the 3 functions requiered to fight against Canker. Once these results obtained, we decided to move on to next step : In order to get closer to our final objective, the 3 genetic modules have to be assembled and to be tested in <b>model plants</b>. In the short time we had, these tests were only performed with the antifungal module, but the results were very encouraging. So encouraging that we moved on to the third step of our project and think about the <b>real injection of SubtiTree in diseased trees</b>. We then considered the <b>spreading</b> problems and proposed different strategies to avoid this major issue. <b>Modeling</b> enables us to predict the growth and the lifespan of SubtiTree in Plane Trees. <br><br />
Well aware that we are part of the <b>iGEM Community</b>, our team also worked for this community by improving the iGEMer's Bible: the Registry of Standard Biological Parts. We improved the EcAMP BioBrick by adding a promotor, a RBS, but mainly by the addition of a STOP codon. As <i>Bacillus subtilis</i> appears to be for us an ideal chassis, we also developed new genetic tools to facilitate its utilization. Thanks to numerous interactions with other iGEM teams, we didn't see the time flying by during this <b>amazing summer</b> !<br />
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<h1 class="title1" style="text-align:center;">Medals Fulfillment</h1> <br />
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<td style="border-bottom:4px solid #e5e6e6; border-top-left-radius:9px;"><center><br>Bronze Medal <img src="https://static.igem.org/mediawiki/2013/8/86/Medal_bronze.png" class="imgcontenttable"/></center><br></td><br />
<td style="border-bottom:4px solid #e5e6e6;"><center><br>Silver Medal <img src="https://static.igem.org/mediawiki/2013/d/dd/Medal_silver.png" class="imgcontenttable"/></center><br></td><br />
<td style="border-bottom:4px solid #e5e6e6; border-top-right-radius:9px;"><center><br>Gold Medal<img src="https://static.igem.org/mediawiki/2013/b/bd/Medal_gold_3.png" class="imgcontenttable"/></center><br></td><br />
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<td style="border-right:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/>Register the team, have a great summer, and plan to have fun at the Jamboree</td><br />
<td style="border-right:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/>Experimentally validate that at least three new BioBrick Part or Device of your own design and construction work as expected.</td><br />
<td><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Improve the function of an existing BioBrick Part or Device and improve the <i>Bacillus subtilis</i> genetic tools, enter this information in the Registry : <a href="http://parts.igem.org/Part:BBa_K1364019"target="_blank">BBa_K1364019</a>, <a href="http://parts.igem.org/Part:BBa_K1364016"target="_blank">BBa_K1364016</a>, <a href="http://parts.igem.org/Part:BBa_K1364017"target="_blank">BBa_K1364017</a>, <a href="http://parts.igem.org/Part:BBa_K1364021"target="_blank">BBa_K1364021</a>, </td><br />
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<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Successfully complete and submit this iGEM Judging form </td><br />
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/>Document the characterization of these three parts in the 'Main Page' of that Part's/Device's Registry entry<br />
Submit this new part to the iGEM Parts Registry:<br> <a href="http://parts.igem.org/Part:BBa_K1364005"target="_blank">BBa_K1364005</a>, <a href="http://parts.igem.org/Part:BBa_K1364009"target="_blank">BBa_K1364009</a>, <a href="http://parts.igem.org/Part:BBa_K1364013"target="_blank">BBa_K1364013.</a></td><br />
<td style="border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Your project may have implications for the environment, security, safety and ethics and/or ownership and sharing. Describe a novel approach that your team has used to help you and others consider these aspects of the design and outcomes of synthetic biology efforts. → <a href="https://2014.igem.org/Team:Toulouse/ethics" target="_blank">Ethic Part</a> </td><br />
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<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Create and share a Description of the team’s project using the iGEM wiki and the team’s parts using the Registry Biological Parts </td><br />
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Submit these new part to the iGEM Parts Registry (submissions must adhere to the iGEM Registry guidelines)</td><br />
<td style="border-top:1px solid #e5e6e6;"> </td><br />
</tr><br />
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<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Plan to present a Poster and Talk at the iGEM Jamboree </td><br />
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Your project may have implications for the environment, security, safety and ethics and/or ownership and sharing. Describe one or more ways in which these or other broader implications have been taken into consideration in the design and execution of your project. → <a href="https://2014.igem.org/Team:Toulouse/ethics" target="_blank"> Ethical Page</a></td><br />
<td style="border-top:1px solid #e5e6e6;"></td><br />
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<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;"><img style="width:20px;" src="https://static.igem.org/mediawiki/2014/3/34/Career-check.jpg" class="imgcontenttableleft"/> Document at least one new standard BioBrick Part or Device used in your project/central to your project and submit this part to the iGEM Registry : <a href="http://parts.igem.org/Part:BBa_K1364000" target="_blank">BBa_K1364000</a>, <a href="http://parts.igem.org/Part:BBa_K1364002"target="_blank">BBa_K1364002</a>, <a href="http://parts.igem.org/Part:BBa_K1364003"target="_blank">BBa_K1364003</a>, <a href="http://parts.igem.org/Part:BBa_K1364005"target="_blank">BBa_K1364005</a>. </td><br />
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{{:Team:Toulouse/template/footer}}</div>Jourdanhttp://2014.igem.org/Team:Toulouse/Notebook/ProtocolsTeam:Toulouse/Notebook/Protocols2014-10-16T15:02:26Z<p>Jourdan: </p>
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<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Notebook&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Protocols</p> <br />
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<h3 class="title2" style="margin-top:10px; color:#333;">Summary :</h3><br />
<ul class="menuleft"><br />
<li style="margin-top:25px;"><a href="#select1"><i>E. coli</i> competent cells</a></li><br />
<li><a href="#select2"><i>E. coli</i> transformation protocol</a></li><br />
<li><a href="#select3">Miniprep and alcaline lysis</a></li><br />
<li><a href="#select4">Cloning</a></li><br />
<li><a href="#select5">Checking of the genetic constructions</a></li><br />
<li><a href="#select6"><i>B. subtilis</i> transformation</a></li><br />
<li><a href="#select7">Checking of the genetic constructions after plasmid integration in <i>Bacillus subtilis</i> </a></li><br />
<li><a href="#select8">Final Tests</a></li><br />
</ul><br />
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<p class="title1" id="select1"><I>E. coli</I> competent cells</p><br />
<p class="texte"><I> <CENTER> MANIPULATION IN ICE </CENTER> </I></p><br />
<p class="texte"><B> Day 0 </B><br />
<br>- Make an Escherichia coli cell culture in LB medium overnight<br />
<p><br />
<p class="texte"><B> Day 1 </B><br />
<br/> <br />
- Freeze 0.1 M CaCl2 and 4 Falcon tubes of 50mL at 4°C<br />
<br/> <br />
- Streak 2% culture in LB to get an OD of 0.3 to 0.4 (it takes approximately 1h30)<br />
<br/> <br />
- Centrifuge 10 minutes at 4500 RPM<br />
<br/><br />
- Remove the supernatant<br />
<br/><br />
- Resuspend the pellet in 7.5 mL of 0.1 M frozen CaCl2 <br />
<br/><br />
- Centrifuge 10 minutes at 4 500 RPM<br />
<br/><br />
- Resuspend the pellet in 500µL of 0.1 M CaCl2<br />
<br/><br />
- Add glycerol for a final concentration of 15%<br />
<br/><br />
- Keep the tubes at -80°C<br />
</p><br />
<br />
<p class="title1" id="select2"> <I>E. coli</I> transformation protocol </p><br />
<p class="texte"><br />
- Let the LB agar medium plates dry in a sterile area<br />
<br/><br />
- Thaw out the competent cell aliquotes for about 10 to 20 minutes<br />
<br/><br />
- Add 20 to 100 ng of plasmid or 3µL of kit plate DNA <br />
<br/><br />
NB: for kit plate, resuspend the well in 10µL of sterile water<br />
- Put the tubes 20minutes in the ice<br />
<br/><br />
- Put the tubes 2 minutes at 42°C in the water bath<br />
<br/><br />
- Put the tubes back in ice immediately to create the thermic shock<br />
<br/><br />
- Add 1mL of LB medium<br />
<br/><br />
- Put the tube 2 hours in the 37°C water bath (1hour if it concerns an ampicillin resistant strain) to allow the phenotypic expression<br />
<br/><br />
- Centrifuge for 1 minute at 13 000 RPM<br />
<br/><br />
- Remove the supernatant <br />
<br/><br />
- Resuspend in 250 µL of LB medium<br />
<br/><br />
- Streak the final mix on LB agar selective medium: 200 µL on one plate, 50µL on the second plate.<br />
<br/><br />
</p><br />
<br />
<p class="title1" id="select3"> Miniprep and alcaline lysis </p><br />
<p class="texte"><B> Day 0 </B></p><br />
<p class="texte"><br />
<br/>- Resuspend 4 to 12 colonies from the plate and name each colony taken on the tubes and on the plate (A, B, C…)<br />
<br/><br />
- Resuspend one colony/culture tube in 5mL of LB medium with antibiotic<br />
<br/><br />
- Leave the culture shakes overnight at 37°C <br />
<p class="texte"><B> Day 1 </B></p><br />
<p class="texte"><br />
<br>- Use the QIAprep spin Miniprep Kit for each culture tube. The last step consisting in the elution of the DNA is made with a hot elution buffer or water at 55°C.<br />
<br/><br />
- Keep the tubes at -20°C <br />
<br><br />
<br />
<br/><I>NB: It is possible to purify the plasmid with an alcaline lysis without any purification column. For 2 mL of culture, 200µL of buffer 1 is added to resuspend the pellet, 400µL of buffer 2 to allow the lysis of the cells and the denaturation of the protein and 300µL of buffer 3 to precipitate the DNA and the proteins. The solution is then centrifuged 10 minutes at 13 000 RPM.<br />
60µL of isopropanol is added to the supernatant and the solution is centrifuged again. The pellet is then resuspended in 100µL of pH 7.4 TE buffer. A part of the contamination by the RNA can avoid by the addition of pH 7.4 TE buffer + 0.2 µL of RNAse. <br />
<br> Buffer 1: Tris 10 mM pH 8 + EDTA 1mM<br />
<br> Buffer 2: NaOH 2mM + SDS 1%<br />
<br> Bufer 3: A COOK 3M + A COOH 15% <br />
</I><br />
<br />
<p class="title1" id="select4"> Cloning </p><br />
<p class="texte"><br />
<br>After taking the competent cells, transforming the Biobricks and making the miniprep, make the digestion mix.<br />
<br><br />
<p class="texte"><B> First Step </B><br />
<br> <B> BOTH PARTS HAVE THE SAME ANTIBIOTIC RESISTANCE </B><br />
<br> 1) Digestion mix<br />
<br> For the vector :<br />
<br>- 5 µL of miniprep plasmid <br />
<br><br />
- 2 µL of each restriction enzymes<br />
<br><br />
- 2 µL of Green Buffer<br />
<br><br />
- 9 µL of Milli-Q water <br />
<br><br />
<br> For the insert :<br />
<br>- 10 µL of miniprep plasmid <br />
<br><br />
- 2 µL of each restriction enzymes<br />
<br><br />
- 2 µL of Green Buffer<br />
<br><br />
- 4 µL of Milli-Q water <br />
<br><br />
- Incubate 15 minutes at 37°C <br />
<br />
<p class="texte">2) Gel extraction<br />
<br><br />
- Prepare a 1% of 2% electrophoresis agarose gel with 0.5x TAE buffer<br />
<br><br />
- Put 20µL of sample + 6µL of marker (1kb for 1% gel and 100pb for 2%) into the well<br />
<br><br />
- Migration for 30 min at 100V or 1hour at 50V.<br />
<br><br />
- The revelation is made in BET (10minutes) and then 5minutes in water.<br />
<br><br />
- The gel extraction is realized thanks to the THERMO SCIENTIFIC GeneJET Gel Extraction and DNA Clean Up Microkit.<br />
<br />
<p class="texte">3) Inactivation of the enzymes for the vector<br />
<br>There are two ways to inactivate the enzymes :<br />
<br>- Use of DNA Clean up kit for the DNA fragment above 200 pb<br />
<br>- Heat inactivation at 95°C for 10 minutes.<br />
<br />
<p class="texte"><br />
<B> THE TWO PARTS HAVE A DIFFERENT ANTIBIOTIC RESISTANCE </B><br />
<br>1) Digestion mix <br />
<br>For each part, add : <br />
<br>- 5 µL of miniprep plasmid <br />
<br>- 1 µL of each restriction enzymes<br />
<br>- 2 µL of Green Buffer<br />
<br>- 9 µL of Milli-Q water <br />
<br>- Incubate 15 minutes at 37°C <br />
<br />
<p class="texte"><br />
2) Inactivation of the enzymes for the vector<br />
<br>There are two ways to inactivate the enzymes :<br />
<br>- Use of DNA Clean up kit for the DNA fragment above 200 pb<br />
<br>- Heat inactivation at 95°C for 10 minutes.<br />
<br />
<p class="texte"><br />
<B> Second step </B><br />
<br><B> Ligation </B><br />
<br/><br />
- Mix 10 µL of insert + 4µL of vector + 2µL of 10x T4 buffer + 0.5µL of T4 ligase + 3.5µL of Milli-Q water<br />
<br/><br />
A control without insert must be made<br />
<br/><br />
- Incubate the ligation mix 15 minutes at room temperature (22°C) and keep the tubes in ice or at -20°C to prepare the transformation<br />
<br/><br />
</p><br />
<br />
<p class="texte"><br />
2) Transformation<br />
<br/><br />
- Take 5µL of the ligation mix for 50µL of competent cells and use the Toulouse iGEM Team 2014 transformation protocol.<br />
<br/><br />
- Plate the solution on selective medium overnight at 37°C.<br />
<br/><br />
</p><br />
<br />
<p class="title1 " id="select5">Checking of the genetic constructions </p><br />
<p class="texte"><br />
1) Colony PCR<br />
<br/><br />
- Add 0.5µL of plasmid + 25µL of DreamTAQ MasterMix + 2µL of each 10µM primer (VR and VF2) + H20 qsp 25µL and take a colony.<br />
<br/><br />
- Look for the number of necessary cycles and the proper temperature thanks to AmplifX or Serial Cloner 2.1 softwares.<br />
<br/>The following cycles have been used : <br />
<br/>- 94°C - 5 min<br />
<br/>- (94°C 45sec ; 55°C 45 sec ; 72°C 1min/kb ) * 25 cycles <br />
<br/>- 72°C 5min<br />
<br/>- Then 4°C<br />
<br/><br />
<br />
<p class="texte"><br />
2) Analytic digestion<br />
<br/><br />
- Put a colony in 5mL of LB selective medium and wait for 6 hours<br />
<br/><br />
- Make a purification thanks to the Miniprep kit<br />
<br/><br />
- Mix 2µL of plasmid + 2µL of Fast Digest Green Buffer + 1µL of each enzyme + Milli-Q water qsp 20µL<br />
<br/><br />
- Wait 15 minutes at 37°C and put the mix on a 1% or 2% gel for 30 minutes at 100V.<br />
<br/><br />
<br />
<p class="texte"><br />
<br/>3) Sequencing<br />
<br/>The sequencing of the genetic constructions was performed by Eurofins Genomics Company by mixing 15µL of pure plasmid solution with 2µL of one primer.<br />
<br />
<p class="title1" id="select6"> <I>B. subtilis</I> transformation</p><br />
<p class="texte"><br />
<br/>Strain: <I>Bacillus subtilis</I> 168. <br />
<br/>Plasmid : pSBBS4S given by the Munich University iGEM Team. l’équipe iGEM de l’université de Munich. This plasmid is replicative in <I>E. coli</I> and integrative in <I>Bacillus subtilis</I>.<br />
<br />
<p class="texte"><br />
<B> Day 0 </B><br />
<br/>- Streak out the Bacillus strain and plate this on an LB agar plate overnight at 37°C<br />
<br />
<p class="texte"><B> Day 1 </B><br />
<br />
<br/>- Pick up a nice big colony of <I>B. Subtilis </I> strain and drop it in 2 ml of completed 1x MC<br />
<br/><br />
- Grow at 37°C for 5 hours<br />
<br/><br />
- Mix 400 µl of culture in a fresh tube ( tubes loosely closed for the aeration) and put 5µL of Miniprep DNA.<br />
<br/><br />
- Grow the cells at 37°C for an additional 2 hours<br />
<br/><br />
- Spread the complete 400 µl reaction mix on selective antibiotic plates, and incubate at 37°C overnight <br />
<br/><br />
<br />
<p class="texte"><br />
<b> Preparation of solutions </b><br />
<I> <br> 300 mM Tri-Na Citrate:</I><br />
<br>- 0.88 g Tri-Na Citrate<br />
<br>- 10mL MQ water<br />
<p class="texte"><br />
<I> <br> Ferric NH4 citrate:</I><br />
<br>- 0.22g Ferric NH4<br />
<br>- 10mL MQ water<br />
<p class="texte"><br />
<I> <br> 10x Competence Medium </I><br />
<br> For 10mL:<br />
<br>- 1.40g K2HPO4<br />
<br>- 0.52g KH2PO4<br />
<br>- 2g glucose<br />
<br>- 1 mL 300 mM Tri-Na citrate <br />
<br>- 0.1 mL Ferric NH4 citrate<br />
<br>- 0.1g Casein Hydrolysate<br />
<br>- 0.2 g Potassium glutamate<br />
<br>The complete mixture should be dissolved in 10 ml. First add 5 ml milliQ water and mix. When everything is dissolved add MQ water till 10 ml. Filter sterilize the complete mixture and store at -20°C.<br />
<br />
<p class="texte"><br />
<I> <br> 1x Competence Medium </I><br />
<br>- 1.8 mL MQ water<br />
<br>- 200 µL 10x Competence Medium solution (previously filter sterilized)<br />
<br>- 6.7 µL 1M MgSO4 (previously autoclaved)<br />
<br>- 10 µL 1% tryptophan (previously filter sterilized and stored in aluminium foil)<br />
<br>The complete mixture should be dissolved in 100 ml. First add 50 ml milliQ water and mix. When everything is dissolved add MQ water till 100 ml. Filter sterilize the complete mixture and store at -20°C.<br />
<br />
<p class="title1" id="select7">Checking of the genetic constructions after plasmid integration in <I>Bacillus subtilis</I></p><br />
<p class="texte"><br />
<br>Test of the pSBBS4S plasmid integration in Bacillus subtilis genome on the threonine site:<br />
<br>- Plate the transformed Bacillus strain on a selective medium (LB + spectinomycin) overnight <br />
<br>- The obtained clones are then plated on different media: Medium Competence (Thr+), Medium Competence (Thr-) and LB + spectinomycine. <br />
<br>When the plasmid is integrated, the clone can grow on minimum medium without threonine but can not grow on the other media.<br />
Moreover, a colony PCR can be performed with the same protocol as previously presented. The used primers are VFBS and VRBS.<br />
<br />
<p class="title1" id="select8">Final Tests</p><br />
<p class="title2">Chemotaxis test</p><br />
<p class="texte"><br />
Many chemotaxis tests exist such as plate tests or capillary tests. We tried all of them and we decided to optimize the « Capillary essay » from Imperial College 2011 iGEM team.<br />
<br>- Prepare the bacteria in LB medium so they reach an OD between 0.5 and 0.6 (exponential growth phase). This step takes about 5 hours.<br />
<br>- Prepare the multichannel pipette: improve the cohesion between the tips and the pipette with Blu-Tack to avoid air and the possibility of leakage.<br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2014/b/b1/Installation_1.gif"></center><br />
<p class="texte"><br />
<br>- Put 200µl of the different chemoattractants in the wells of the ELISA plate and pipette 15µL of each with the multichannel pipette: galactose which represents our negative control, glucose which represents our positive control and N-acetylglucosamine (NAG). <br />
NB: The NAG is the most important test because it is the monosaccharide which composes the chitin on Ceratocystis platani wall. The volume in the tips must be marked.<br />
<br>- Put the tips with chemoattractants in 300µL of the bacterial solution in exponential growth phase in the ELISA plate.<br />
<br>- Let the installation settle for 1 hour at room temperature.<br />
<br>- After an hour, put the volume of the tips on parafilm. <br />
<br>- Each solution is diluted 1/10,000 and 100µL is spread on LA medium.<br />
<br>- The plates are then incubated overnight at 37°C.</p><br />
<br />
<p class="title2">Binding test</p><br />
<p class="texte"><I>CBB (Chitin Binding Buffer):</I><br />
<br>- 500 mM NaCl<br />
<br>- 20 mM Tris-HCl<br />
<br>- 1 mM EDTA<br />
<br>- 0,05% Triton X-100, 25°C, pH=8<br />
</p><br />
<br />
<p class="texte"><br />
<I>Column activation:</I> <br />
<br>- Vortex the beads <br />
<br>- Put 50 µL of beads in a 1.5mL centrifuge tube<br />
<br>- Wash with 500 µL of CBB<br />
<br>- Put the centrifuge tube on a magnetic rack<br />
<br>- Wait 30 seconds<br />
<br>- Remove supernatant<br />
<br>- Repeat the wash<br />
</p><br />
<br />
<br />
<p class="texte"><I>Bacterial fixation on the chitin beads:</I><br />
<br>- Add 200 µL of bacteria solution (105 bactéria/mL)to the washed beads <br />
<br>- Shake during 1h at 4°C<br />
<br>- Add 500 µL of CBB (washing A)<br />
<br>- Put the centrifuge tube on a magnetic rack<br />
<br>- Wait 30 seconds<br />
<br>- Remove supernatant<br />
<br>- Add 500 µL of CBB (washing B)<br />
<br>- Put the centrifuge tube on a magnetic rack<br />
<br>- Wait 30 seconds<br />
<br>- Remove supernatant<br />
<br>- Add 500 µL of CBB to recover the beads directly<br />
</p><br />
<br />
<p class="texte"><I>Bacteria count:</I><br />
<br>- Make different dilutions : 10-1, 10-3, 10-5 of the first bacterial culture and spread on LA plates<br />
<br>- Make different dilutions : 1, 10-2, 10-4 of washings (A and B) and of the beads in CBB medium and spread on LA plates<br />
<br>- Place the plates at 37°C overnight<br />
<br>- Count colonies on different plates<br />
</p><br />
<br />
<p class="title2">Fungicide test: anti-fungal activities</p><br />
<p class="texte"><br />
CAUTION : all the lab equipment must be desinfected before the manipulations with the fungi. <br />
<br>Three different funguss strains were used : <I>Aspergillus brasiliensis, Aspergillus nidulans, Trichoderma reesei</I><br />
<br>- The conidia can be taken by adding one drop of Tween 80 on the fungus plate.<br />
<br>- Then the drop is mixed with 1mL of sterile water in an Eppedorf.<br />
<br>- A microscopy count can be performed thanks to Thoma cell to determine the conidia concentration.<br />
<br>NB : The conidia solutions are then diluted and spread on sap medium to get 10,000 conidia/plate.<br />
<br>- After 72hours of liquid culture of the different clones of <i>B. subtilis</i> with the fungicides module, the culture can be centrifugated.<br />
<br>- 130µL of the supernatant is used to soak a pad placed on the conidia plate. The bacterial pellet is resuspended in 130µL of LB medium and also put on a pad. <br />
<br>- The plates containing 10,000 conidia and the soaked pads are then put at room temperature for a few days according to the growth speed of the fungi. Controls are also realized with wild type strains or copper sulfate at 10 and 20mg/mL.<br />
</p><br />
<br />
<p class="title2">Fungicide test: <i>in planta</i> assay</p><br />
<p class="texte"><br />
The first step is related to the inoculation of SubtiTree in plants through stomata (opened in wet condition). We diluted our bacterial samples to get two concentrations: 5.10^6 and 10^8 bacteria per mL. The WT and trasnformed bacteria are introduced into plants (a control test without bacteria was performed). Thanks to a 1 ml syringe (without needle), the plant was injected with bacteria by pressure. Five leaves of each plant were used, marked with a marker point. It is necessary to repeat the operation on both sides of the leaf and the excess is wipe off. The plants are placed in a growth chamber (phytotron) to control light, high humidity, temperature and bacterial non-proliferation. Bacterial growth in the plant is left for 24 h.<br />
</br><br />
<br />
The next step begins with the preparation of the fungal samples.Fungal culture is crushed and mixed with PDB (Potato Dextrose Broth). Then the mix passes through a 100 µm filter (to remove large aggregates) and through a 40 µm filter. The caught hyphae are mixed with PDB for 24 to 48 hours until OD(600nm) 2.5 isobtained. The previously seeded leaves are taken from the plant using a scalpel and placed in boxes above wet absorbent paper (leaves are kept alive for a week). Above each leaf, 5µl of the fungal suspension is deposited (using beveled tips because it is too viscous). As control, we kept inoculated leaves without fungus and leaves with only fungus. Pictures are taken at different times. All the plants are destroyed by autoclaving.<br />
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{{:Team:Toulouse/template/footer}}</div>Jourdanhttp://2014.igem.org/Team:Toulouse/Notebook/ProtocolsTeam:Toulouse/Notebook/Protocols2014-10-16T15:00:49Z<p>Jourdan: </p>
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<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Notebook&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Protocols</p> <br />
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<h3 class="title2" style="margin-top:10px; color:#333;">Summary :</h3><br />
<ul class="menuleft"><br />
<li style="margin-top:25px;"><a href="#select1"><i>E. coli</i> competent cells</a></li><br />
<li><a href="#select2"><i>E. coli</i> transformation protocol</a></li><br />
<li><a href="#select3">Miniprep and alcaline lysis</a></li><br />
<li><a href="#select4">Cloning</a></li><br />
<li><a href="#select5">Checking of the genetic constructions</a></li><br />
<li><a href="#select6"><i>B. subtilis</i> transformation</a></li><br />
<li><a href="#select7">Checking of the genetic constructions after plasmid integration in <i>Bacillus subtilis</i> </a></li><br />
<li><a href="#select8">Final Tests</a></li><br />
</ul><br />
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<p class="title1" id="select1"><I>E. coli</I> competent cells</p><br />
<p class="texte"><I> <CENTER> MANIPULATION IN ICE </CENTER> </I></p><br />
<p class="texte"><B> Day 0 </B><br />
<br>- Make an Escherichia coli cell culture in LB medium overnight<br />
<p><br />
<p class="texte"><B> Day 1 </B><br />
<br/> <br />
- Freeze 0.1 M CaCl2 and 4 Falcon tubes of 50mL at 4°C<br />
<br/> <br />
- Streak 2% culture in LB to get an OD of 0.3 to 0.4 (it takes approximately 1h30)<br />
<br/> <br />
- Centrifuge 10 minutes at 4500 RPM<br />
<br/><br />
- Remove the supernatant<br />
<br/><br />
- Resuspend the pellet in 7.5 mL of 0.1 M frozen CaCl2 <br />
<br/><br />
- Centrifuge 10 minutes at 4 500 RPM<br />
<br/><br />
- Resuspend the pellet in 500µL of 0.1 M CaCl2<br />
<br/><br />
- Add glycerol for a final concentration of 15%<br />
<br/><br />
- Keep the tubes at -80°C<br />
</p><br />
<br />
<p class="title1" id="select2"> <I>E. coli</I> transformation protocol </p><br />
<p class="texte"><br />
- Let the LB agar medium plates dry in a sterile area<br />
<br/><br />
- Thaw out the competent cell aliquotes for about 10 to 20 minutes<br />
<br/><br />
- Add 20 to 100 ng of plasmid or 3µL of kit plate DNA <br />
<br/><br />
NB: for kit plate, resuspend the well in 10µL of sterile water<br />
- Put the tubes 20minutes in the ice<br />
<br/><br />
- Put the tubes 2 minutes at 42°C in the water bath<br />
<br/><br />
- Put the tubes back in ice immediately to create the thermic shock<br />
<br/><br />
- Add 1mL of LB medium<br />
<br/><br />
- Put the tube 2 hours in the 37°C water bath (1hour if it concerns an ampicillin resistant strain) to allow the phenotypic expression<br />
<br/><br />
- Centrifuge for 1 minute at 13 000 RPM<br />
<br/><br />
- Remove the supernatant <br />
<br/><br />
- Resuspend in 250 µL of LB medium<br />
<br/><br />
- Streak the final mix on LB agar selective medium: 200 µL on one plate, 50µL on the second plate.<br />
<br/><br />
</p><br />
<br />
<p class="title1" id="select3"> Miniprep and alcaline lysis </p><br />
<p class="texte"><B> Day 0 </B></p><br />
<p class="texte"><br />
<br/>- Resuspend 4 to 12 colonies from the plate and name each colony taken on the tubes and on the plate (A, B, C…)<br />
<br/><br />
- Resuspend one colony/culture tube in 5mL of LB medium with antibiotic<br />
<br/><br />
- Leave the culture shakes overnight at 37°C <br />
<p class="texte"><B> Day 1 </B></p><br />
<p class="texte"><br />
<br>- Use the QIAprep spin Miniprep Kit for each culture tube. The last step consisting in the elution of the DNA is made with a hot elution buffer or water at 55°C.<br />
<br/><br />
- Keep the tubes at -20°C <br />
<br><br />
<br />
<br/><I>NB: It is possible to purify the plasmid with an alcaline lysis without any purification column. For 2 mL of culture, 200µL of buffer 1 is added to resuspend the pellet, 400µL of buffer 2 to allow the lysis of the cells and the denaturation of the protein and 300µL of buffer 3 to precipitate the DNA and the proteins. The solution is then centrifuged 10 minutes at 13 000 RPM.<br />
60µL of isopropanol is added to the supernatant and the solution is centrifuged again. The pellet is then resuspended in 100µL of pH 7.4 TE buffer. A part of the contamination by the RNA can avoid by the addition of pH 7.4 TE buffer + 0.2 µL of RNAse. <br />
<br> Buffer 1: Tris 10 mM pH 8 + EDTA 1mM<br />
<br> Buffer 2: NaOH 2mM + SDS 1%<br />
<br> Bufer 3: A COOK 3M + A COOH 15% <br />
</I><br />
<br />
<p class="title1" id="select4"> Cloning </p><br />
<p class="texte"><br />
<br>After taking the competent cells, transforming the Biobricks and making the miniprep, make the digestion mix.<br />
<br><br />
<p class="texte"><B> First Step </B><br />
<br> <B> BOTH PARTS HAVE THE SAME ANTIBIOTIC RESISTANCE </B><br />
<br> 1) Digestion mix<br />
<br> For the vector :<br />
<br>- 5 µL of miniprep plasmid <br />
<br><br />
- 2 µL of each restriction enzymes<br />
<br><br />
- 2 µL of Green Buffer<br />
<br><br />
- 9 µL of Milli-Q water <br />
<br><br />
<br> For the insert :<br />
<br>- 10 µL of miniprep plasmid <br />
<br><br />
- 2 µL of each restriction enzymes<br />
<br><br />
- 2 µL of Green Buffer<br />
<br><br />
- 4 µL of Milli-Q water <br />
<br><br />
- Incubate 15 minutes at 37°C <br />
<br />
<p class="texte">2) Gel extraction<br />
<br><br />
- Prepare a 1% of 2% electrophoresis agarose gel with 0.5x TAE buffer<br />
<br><br />
- Put 20µL of sample + 6µL of marker (1kb for 1% gel and 100pb for 2%) into the well<br />
<br><br />
- Migration for 30 min at 100V or 1hour at 50V.<br />
<br><br />
- The revelation is made in BET (10minutes) and then 5minutes in water.<br />
<br><br />
- The gel extraction is realized thanks to the THERMO SCIENTIFIC GeneJET Gel Extraction and DNA Clean Up Microkit.<br />
<br />
<p class="texte">3) Inactivation of the enzymes for the vector<br />
<br>There are two ways to inactivate the enzymes :<br />
<br>- Use of DNA Clean up kit for the DNA fragment above 200 pb<br />
<br>- Heat inactivation at 95°C for 10 minutes.<br />
<br />
<p class="texte"><br />
<B> THE TWO PARTS HAVE A DIFFERENT ANTIBIOTIC RESISTANCE </B><br />
<br>1) Digestion mix <br />
<br>For each part, add : <br />
<br>- 5 µL of miniprep plasmid <br />
<br>- 1 µL of each restriction enzymes<br />
<br>- 2 µL of Green Buffer<br />
<br>- 9 µL of Milli-Q water <br />
<br>- Incubate 15 minutes at 37°C <br />
<br />
<p class="texte"><br />
2) Inactivation of the enzymes for the vector<br />
<br>There are two ways to inactivate the enzymes :<br />
<br>- Use of DNA Clean up kit for the DNA fragment above 200 pb<br />
<br>- Heat inactivation at 95°C for 10 minutes.<br />
<br />
<p class="texte"><br />
<B> Second step </B><br />
<br><B> Ligation </B><br />
<br/><br />
- Mix 10 µL of insert + 4µL of vector + 2µL of 10x T4 buffer + 0.5µL of T4 ligase + 3.5µL of Milli-Q water<br />
<br/><br />
A control without insert must be made<br />
<br/><br />
- Incubate the ligation mix 15 minutes at room temperature (22°C) and keep the tubes in ice or at -20°C to prepare the transformation<br />
<br/><br />
</p><br />
<br />
<p class="texte"><br />
2) Transformation<br />
<br/><br />
- Take 5µL of the ligation mix for 50µL of competent cells and use the Toulouse iGEM Team 2014 transformation protocol.<br />
<br/><br />
- Plate the solution on selective medium overnight at 37°C.<br />
<br/><br />
</p><br />
<br />
<p class="title1 " id="select5">Checking of the genetic constructions </p><br />
<p class="texte"><br />
1) Colony PCR<br />
<br/><br />
- Add 0.5µL of plasmid + 25µL of DreamTAQ MasterMix + 2µL of each 10µM primer (VR and VF2) + H20 qsp 25µL and take a colony.<br />
<br/><br />
- Look for the number of necessary cycles and the proper temperature thanks to AmplifX or Serial Cloner 2.1 softwares.<br />
<br/>The following cycles have been used : <br />
<br/>- 94°C - 5 min<br />
<br/>- (94°C 45sec ; 55°C 45 sec ; 72°C 1min/kb ) * 25 cycles <br />
<br/>- 72°C 5min<br />
<br/>- Then 4°C<br />
<br/><br />
<br />
<p class="texte"><br />
2) Analytic digestion<br />
<br/><br />
- Put a colony in 5mL of LB selective medium and wait for 6 hours<br />
<br/><br />
- Make a purification thanks to the Miniprep kit<br />
<br/><br />
- Mix 2µL of plasmid + 2µL of Fast Digest Green Buffer + 1µL of each enzyme + Milli-Q water qsp 20µL<br />
<br/><br />
- Wait 15 minutes at 37°C and put the mix on a 1% or 2% gel for 30 minutes at 100V.<br />
<br/><br />
<br />
<p class="texte"><br />
<br/>3) Sequencing<br />
<br/>The sequencing of the genetic constructions was performed by Eurofins Genomics Company by mixing 15µL of pure plasmid solution with 2µL of one primer.<br />
<br />
<p class="title1" id="select6"> <I>B. subtilis</I> transformation</p><br />
<p class="texte"><br />
<br/>Strain: <I>Bacillus subtilis</I> 168. <br />
<br/>Plasmid : pSBBS4S given by the Munich University iGEM Team. l’équipe iGEM de l’université de Munich. This plasmid is replicative in <I>E. coli</I> and integrative in <I>Bacillus subtilis</I>.<br />
<br />
<p class="texte"><br />
<B> Day 0 </B><br />
<br/>- Streak out the Bacillus strain and plate this on an LB agar plate overnight at 37°C<br />
<br />
<p class="texte"><B> Day 1 </B><br />
<br />
<br/>- Pick up a nice big colony of <I>B. Subtilis </I> strain and drop it in 2 ml of completed 1x MC<br />
<br/><br />
- Grow at 37°C for 5 hours<br />
<br/><br />
- Mix 400 µl of culture in a fresh tube ( tubes loosely closed for the aeration) and put 5µL of Miniprep DNA.<br />
<br/><br />
- Grow the cells at 37°C for an additional 2 hours<br />
<br/><br />
- Spread the complete 400 µl reaction mix on selective antibiotic plates, and incubate at 37°C overnight <br />
<br/><br />
<br />
<p class="texte"><br />
<b> Preparation of solutions </b><br />
<I> <br> 300 mM Tri-Na Citrate:</I><br />
<br>- 0.88 g Tri-Na Citrate<br />
<br>- 10mL MQ water<br />
<p class="texte"><br />
<I> <br> Ferric NH4 citrate:</I><br />
<br>- 0.22g Ferric NH4<br />
<br>- 10mL MQ water<br />
<p class="texte"><br />
<I> <br> 10x Competence Medium </I><br />
<br> For 10mL:<br />
<br>- 1.40g K2HPO4<br />
<br>- 0.52g KH2PO4<br />
<br>- 2g glucose<br />
<br>- 1 mL 300 mM Tri-Na citrate <br />
<br>- 0.1 mL Ferric NH4 citrate<br />
<br>- 0.1g Casein Hydrolysate<br />
<br>- 0.2 g Potassium glutamate<br />
<br>The complete mixture should be dissolved in 10 ml. First add 5 ml milliQ water and mix. When everything is dissolved add MQ water till 10 ml. Filter sterilize the complete mixture and store at -20°C.<br />
<br />
<p class="texte"><br />
<I> <br> 1x Competence Medium </I><br />
<br>- 1.8 mL MQ water<br />
<br>- 200 µL 10x Competence Medium solution (previously filter sterilized)<br />
<br>- 6.7 µL 1M MgSO4 (previously autoclaved)<br />
<br>- 10 µL 1% tryptophan (previously filter sterilized and stored in aluminium foil)<br />
<br>The complete mixture should be dissolved in 100 ml. First add 50 ml milliQ water and mix. When everything is dissolved add MQ water till 100 ml. Filter sterilize the complete mixture and store at -20°C.<br />
<br />
<p class="title1" id="select7">Checking of the genetic constructions after plasmid integration in <I>Bacillus subtilis</I></p><br />
<p class="texte"><br />
<br>Test of the pSBBS4S plasmid integration in Bacillus subtilis genome on the threonine site:<br />
<br>- Plate the transformed Bacillus strain on a selective medium (LB + spectinomycin) overnight <br />
<br>- The obtained clones are then plated on different media: Medium Competence (Thr+), Medium Competence (Thr-) and LB + spectinomycine. <br />
<br>When the plasmid is integrated, the clone can grow on minimum medium without threonine but can not grow on the other media.<br />
Moreover, a colony PCR can be performed with the same protocol as previously presented. The used primers are VFBS and VRBS.<br />
<br />
<p class="title1" id="select8">Final Tests</p><br />
<p class="title2">Chemotaxis test</p><br />
<p class="texte"><br />
Many chemotaxis tests exist such as plate tests or capillary tests. We tried all of them and we decided to optimize the « Capillary essay » from Imperial College 2011 iGEM team.<br />
<br>- Prepare the bacteria in LB medium so they reach an OD between 0.5 and 0.6 (exponential growth phase). This step takes about 5 hours.<br />
<br>- Prepare the multichannel pipette: improve the cohesion between the tips and the pipette with Blu-Tack to avoid air and the possibility of leakage.<br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2014/b/b1/Installation_1.gif"></center><br />
<p class="texte"><br />
<br>- Put 200µl of the different chemoattractants in the wells of the ELISA plate and pipette 15µL of each with the multichannel pipette: galactose which represents our negative control, glucose which represents our positive control and N-acetylglucosamine (NAG). <br />
NB: The NAG is the most important test because it is the monosaccharide which composes the chitin on Ceratocystis platani wall. The volume in the tips must be marked.<br />
<br>- Put the tips with chemoattractants in 300µL of the bacterial solution in exponential growth phase in the ELISA plate.<br />
<br>- Let the installation settle for 1 hour at room temperature.<br />
<br>- After an hour, put the volume of the tips on parafilm. <br />
<br>- Each solution is diluted 1/10,000 and 100µL is spread on LA medium.<br />
<br>- The plates are then incubated overnight at 37°C.</p><br />
<br />
<p class="title2">Binding test</p><br />
<p class="texte"><I>CBB (Chitin Binding Buffer):</I><br />
<br>- 500 mM NaCl<br />
<br>- 20 mM Tris-HCl<br />
<br>- 1 mM EDTA<br />
<br>- 0,05% Triton X-100, 25°C, pH=8<br />
</p><br />
<br />
<p class="texte"><br />
<I>Column activation:</I> <br />
<br>- Vortex the beads <br />
<br>- Put 50 µL of beads in a 1.5mL centrifuge tube<br />
<br>- Wash with 500 µL of CBB<br />
<br>- Put the centrifuge tube on a magnetic rack<br />
<br>- Wait 30 seconds<br />
<br>- Remove supernatant<br />
<br>- Repeat the wash<br />
</p><br />
<br />
<br />
<p class="texte"><I>Bacterial fixation on the chitin beads:</I><br />
<br>- Add 200 µL of bacteria solution (105 bactéria/mL)to the washed beads <br />
<br>- Shake during 1h at 4°C<br />
<br>- Add 500 µL of CBB (washing A)<br />
<br>- Put the centrifuge tube on a magnetic rack<br />
<br>- Wait 30 seconds<br />
<br>- Remove supernatant<br />
<br>- Add 500 µL of CBB (washing B)<br />
<br>- Put the centrifuge tube on a magnetic rack<br />
<br>- Wait 30 seconds<br />
<br>- Remove supernatant<br />
<br>- Add 500 µL of CBB to recover the beads directly<br />
</p><br />
<br />
<p class="texte"><I>Bacteria count:</I><br />
<br>- Make different dilutions : 10-1, 10-3, 10-5 of the first bacterial culture and spread on LA plates<br />
<br>- Make different dilutions : 1, 10-2, 10-4 of washings (A and B) and of the beads in CBB medium and spread on LA plates<br />
<br>- Place the plates at 37°C overnight<br />
<br>- Count colonies on different plates<br />
</p><br />
<br />
<p class="title2">Fungicide test: anti-fungal activities</p><br />
<p class="texte"><br />
CAUTION : all the lab equipment must be desinfected before the manipulations with the fungi. <br />
<br>Three different funguss strains were used : <I>Aspergillus brasiliensis, Aspergillus nidulans, Trichoderma reesei</I><br />
<br>- The conidia can be taken by adding one drop of Tween 80 on the fungus plate.<br />
<br>- Then the drop is mixed with 1mL of sterile water in an Eppedorf.<br />
<br>- A microscopy count can be performed thanks to Thoma cell to determine the conidia concentration.<br />
<br>NB : The conidia solutions are then diluted and spread on sap medium to get 10,000 conidia/plate.<br />
<br>- After 72hours of liquid culture of the different clones of <i>B. subtilis</i> with the fungicides module, the culture can be centrifugated.<br />
<br>- 130µL of the supernatant is used to soak a pad placed on the conidia plate. The bacterial pellet is resuspended in 130µL of LB medium and also put on a pad. <br />
<br>- The plates containing 10,000 conidia and the soaked pads are then put at room temperature for a few days according to the growth speed of the fungi. Controls are also realized with wild type strains or copper sulfate at 10 and 20mg/mL.<br />
</p><br />
<br />
<p class="title2">Fungicide test: <i>in planta</i> assay</p><br />
<p class="texte"><br />
The first step is related to the inoculation of SubtiTree in plants through stomata (opened in wet condition). We diluted our bacterial samples to get two concentrations: 5.10^6 and 10^8 bacteria per mL. The WT and trasnformed bacteria are introduced into plants (a control test without bacteria was performed). Thanks to a 1 ml syringe (without needle), the plant was injected with bacteria by pressure. Five leaves of each plant were used, marked with a marker point. It is necessary to repeat the operation on both sides of the leaf and the excess is wipe off. The plants are placed in a growth chamber (phytotron) to control light, high humidity, temperature and bacterial non-proliferation. Bacterial growth in the plant is left for 24 h.<br />
</br><br />
<br />
The next step begins with the preparation of the fungal samples.Fungal culture is crushed and mixed with PDB (Potato Dextrose Broth). Then the mix passes through a 100 µm filter (to remove large aggregates) and through a 40 µm filter. The caught hyphae are mixed with PDB for 24 to 48 hours until OD(600nm) 2.5 isobtained. The previously seeded leaves are taken from the plant using a scalpel and placed in boxes above wet absorbent paper (leaves are kept alive for a week). Above each leaf 5µl of the fungal suspension is placed (using beveled tips because it is too viscous. As control, we kept inoculated leaves without fungus and leaves with only fungus. Pictures are taken at different times. All the plants are destroyed by autoclaving.<br />
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{{:Team:Toulouse/template/footer}}</div>Jourdanhttp://2014.igem.org/Team:Toulouse/Notebook/ProtocolsTeam:Toulouse/Notebook/Protocols2014-10-16T14:59:51Z<p>Jourdan: </p>
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<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Notebook&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Protocols</p> <br />
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<h3 class="title2" style="margin-top:10px; color:#333;">Summary :</h3><br />
<ul class="menuleft"><br />
<li style="margin-top:25px;"><a href="#select1"><i>E. coli</i> competent cells</a></li><br />
<li><a href="#select2"><i>E. coli</i> transformation protocol</a></li><br />
<li><a href="#select3">Miniprep and alcaline lysis</a></li><br />
<li><a href="#select4">Cloning</a></li><br />
<li><a href="#select5">Checking of the genetic constructions</a></li><br />
<li><a href="#select6"><i>B. subtilis</i> transformation</a></li><br />
<li><a href="#select7">Checking of the genetic constructions after plasmid integration in <i>Bacillus subtilis</i> </a></li><br />
<li><a href="#select8">Final Tests</a></li><br />
</ul><br />
</div><br />
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<br />
<p class="title1" id="select1"><I>E. coli</I> competent cells</p><br />
<p class="texte"><I> <CENTER> MANIPULATION IN ICE </CENTER> </I></p><br />
<p class="texte"><B> Day 0 </B><br />
<br>- Make an Escherichia coli cell culture in LB medium overnight<br />
<p><br />
<p class="texte"><B> Day 1 </B><br />
<br/> <br />
- Freeze 0.1 M CaCl2 and 4 Falcon tubes of 50mL at 4°C<br />
<br/> <br />
- Streak 2% culture in LB to get an OD of 0.3 to 0.4 (it takes approximately 1h30)<br />
<br/> <br />
- Centrifuge 10 minutes at 4500 RPM<br />
<br/><br />
- Remove the supernatant<br />
<br/><br />
- Resuspend the pellet in 7.5 mL of 0.1 M frozen CaCl2 <br />
<br/><br />
- Centrifuge 10 minutes at 4 500 RPM<br />
<br/><br />
- Resuspend the pellet in 500µL of 0.1 M CaCl2<br />
<br/><br />
- Add glycerol for a final concentration of 15%<br />
<br/><br />
- Keep the tubes at -80°C<br />
</p><br />
<br />
<p class="title1" id="select2"> <I>E. coli</I> transformation protocol </p><br />
<p class="texte"><br />
- Let the LB agar medium plates dry in a sterile area<br />
<br/><br />
- Thaw out the competent cell aliquotes for about 10 to 20 minutes<br />
<br/><br />
- Add 20 to 100 ng of plasmid or 3µL of kit plate DNA <br />
<br/><br />
NB: for kit plate, resuspend the well in 10µL of sterile water<br />
- Put the tubes 20minutes in the ice<br />
<br/><br />
- Put the tubes 2 minutes at 42°C in the water bath<br />
<br/><br />
- Put the tubes back in ice immediately to create the thermic shock<br />
<br/><br />
- Add 1mL of LB medium<br />
<br/><br />
- Put the tube 2 hours in the 37°C water bath (1hour if it concerns an ampicillin resistant strain) to allow the phenotypic expression<br />
<br/><br />
- Centrifuge for 1 minute at 13 000 RPM<br />
<br/><br />
- Remove the supernatant <br />
<br/><br />
- Resuspend in 250 µL of LB medium<br />
<br/><br />
- Streak the final mix on LB agar selective medium: 200 µL on one plate, 50µL on the second plate.<br />
<br/><br />
</p><br />
<br />
<p class="title1" id="select3"> Miniprep and alcaline lysis </p><br />
<p class="texte"><B> Day 0 </B></p><br />
<p class="texte"><br />
<br/>- Resuspend 4 to 12 colonies from the plate and name each colony taken on the tubes and on the plate (A, B, C…)<br />
<br/><br />
- Resuspend one colony/culture tube in 5mL of LB medium with antibiotic<br />
<br/><br />
- Leave the culture shakes overnight at 37°C <br />
<p class="texte"><B> Day 1 </B></p><br />
<p class="texte"><br />
<br>- Use the QIAprep spin Miniprep Kit for each culture tube. The last step consisting in the elution of the DNA is made with a hot elution buffer or water at 55°C.<br />
<br/><br />
- Keep the tubes at -20°C <br />
<br><br />
<br />
<br/><I>NB: It is possible to purify the plasmid with an alcaline lysis without any purification column. For 2 mL of culture, 200µL of buffer 1 is added to resuspend the pellet, 400µL of buffer 2 to allow the lysis of the cells and the denaturation of the protein and 300µL of buffer 3 to precipitate the DNA and the proteins. The solution is then centrifuged 10 minutes at 13 000 RPM.<br />
60µL of isopropanol is added to the supernatant and the solution is centrifuged again. The pellet is then resuspended in 100µL of pH 7.4 TE buffer. A part of the contamination by the RNA can avoid by the addition of pH 7.4 TE buffer + 0.2 µL of RNAse. <br />
<br> Buffer 1: Tris 10 mM pH 8 + EDTA 1mM<br />
<br> Buffer 2: NaOH 2mM + SDS 1%<br />
<br> Bufer 3: A COOK 3M + A COOH 15% <br />
</I><br />
<br />
<p class="title1" id="select4"> Cloning </p><br />
<p class="texte"><br />
<br>After taking the competent cells, transforming the Biobricks and making the miniprep, make the digestion mix.<br />
<br><br />
<p class="texte"><B> First Step </B><br />
<br> <B> BOTH PARTS HAVE THE SAME ANTIBIOTIC RESISTANCE </B><br />
<br> 1) Digestion mix<br />
<br> For the vector :<br />
<br>- 5 µL of miniprep plasmid <br />
<br><br />
- 2 µL of each restriction enzymes<br />
<br><br />
- 2 µL of Green Buffer<br />
<br><br />
- 9 µL of Milli-Q water <br />
<br><br />
<br> For the insert :<br />
<br>- 10 µL of miniprep plasmid <br />
<br><br />
- 2 µL of each restriction enzymes<br />
<br><br />
- 2 µL of Green Buffer<br />
<br><br />
- 4 µL of Milli-Q water <br />
<br><br />
- Incubate 15 minutes at 37°C <br />
<br />
<p class="texte">2) Gel extraction<br />
<br><br />
- Prepare a 1% of 2% electrophoresis agarose gel with 0.5x TAE buffer<br />
<br><br />
- Put 20µL of sample + 6µL of marker (1kb for 1% gel and 100pb for 2%) into the well<br />
<br><br />
- Migration for 30 min at 100V or 1hour at 50V.<br />
<br><br />
- The revelation is made in BET (10minutes) and then 5minutes in water.<br />
<br><br />
- The gel extraction is realized thanks to the THERMO SCIENTIFIC GeneJET Gel Extraction and DNA Clean Up Microkit.<br />
<br />
<p class="texte">3) Inactivation of the enzymes for the vector<br />
<br>There are two ways to inactivate the enzymes :<br />
<br>- Use of DNA Clean up kit for the DNA fragment above 200 pb<br />
<br>- Heat inactivation at 95°C for 10 minutes.<br />
<br />
<p class="texte"><br />
<B> THE TWO PARTS HAVE A DIFFERENT ANTIBIOTIC RESISTANCE </B><br />
<br>1) Digestion mix <br />
<br>For each part, add : <br />
<br>- 5 µL of miniprep plasmid <br />
<br>- 1 µL of each restriction enzymes<br />
<br>- 2 µL of Green Buffer<br />
<br>- 9 µL of Milli-Q water <br />
<br>- Incubate 15 minutes at 37°C <br />
<br />
<p class="texte"><br />
2) Inactivation of the enzymes for the vector<br />
<br>There are two ways to inactivate the enzymes :<br />
<br>- Use of DNA Clean up kit for the DNA fragment above 200 pb<br />
<br>- Heat inactivation at 95°C for 10 minutes.<br />
<br />
<p class="texte"><br />
<B> Second step </B><br />
<br><B> Ligation </B><br />
<br/><br />
- Mix 10 µL of insert + 4µL of vector + 2µL of 10x T4 buffer + 0.5µL of T4 ligase + 3.5µL of Milli-Q water<br />
<br/><br />
A control without insert must be made<br />
<br/><br />
- Incubate the ligation mix 15 minutes at room temperature (22°C) and keep the tubes in ice or at -20°C to prepare the transformation<br />
<br/><br />
</p><br />
<br />
<p class="texte"><br />
2) Transformation<br />
<br/><br />
- Take 5µL of the ligation mix for 50µL of competent cells and use the Toulouse iGEM Team 2014 transformation protocol.<br />
<br/><br />
- Plate the solution on selective medium overnight at 37°C.<br />
<br/><br />
</p><br />
<br />
<p class="title1 " id="select5">Checking of the genetic constructions </p><br />
<p class="texte"><br />
1) Colony PCR<br />
<br/><br />
- Add 0.5µL of plasmid + 25µL of DreamTAQ MasterMix + 2µL of each 10µM primer (VR and VF2) + H20 qsp 25µL and take a colony.<br />
<br/><br />
- Look for the number of necessary cycles and the proper temperature thanks to AmplifX or Serial Cloner 2.1 softwares.<br />
<br/>The following cycles have been used : <br />
<br/>- 94°C - 5 min<br />
<br/>- (94°C 45sec ; 55°C 45 sec ; 72°C 1min/kb ) * 25 cycles <br />
<br/>- 72°C 5min<br />
<br/>- Then 4°C<br />
<br/><br />
<br />
<p class="texte"><br />
2) Analytic digestion<br />
<br/><br />
- Put a colony in 5mL of LB selective medium and wait for 6 hours<br />
<br/><br />
- Make a purification thanks to the Miniprep kit<br />
<br/><br />
- Mix 2µL of plasmid + 2µL of Fast Digest Green Buffer + 1µL of each enzyme + Milli-Q water qsp 20µL<br />
<br/><br />
- Wait 15 minutes at 37°C and put the mix on a 1% or 2% gel for 30 minutes at 100V.<br />
<br/><br />
<br />
<p class="texte"><br />
<br/>3) Sequencing<br />
<br/>The sequencing of the genetic constructions was performed by Eurofins Genomics Company by mixing 15µL of pure plasmid solution with 2µL of one primer.<br />
<br />
<p class="title1" id="select6"> <I>B. subtilis</I> transformation</p><br />
<p class="texte"><br />
<br/>Strain: <I>Bacillus subtilis</I> 168. <br />
<br/>Plasmid : pSBBS4S given by the Munich University iGEM Team. l’équipe iGEM de l’université de Munich. This plasmid is replicative in <I>E. coli</I> and integrative in <I>Bacillus subtilis</I>.<br />
<br />
<p class="texte"><br />
<B> Day 0 </B><br />
<br/>- Streak out the Bacillus strain and plate this on an LB agar plate overnight at 37°C<br />
<br />
<p class="texte"><B> Day 1 </B><br />
<br />
<br/>- Pick up a nice big colony of <I>B. Subtilis </I> strain and drop it in 2 ml of completed 1x MC<br />
<br/><br />
- Grow at 37°C for 5 hours<br />
<br/><br />
- Mix 400 µl of culture in a fresh tube ( tubes loosely closed for the aeration) and put 5µL of Miniprep DNA.<br />
<br/><br />
- Grow the cells at 37°C for an additional 2 hours<br />
<br/><br />
- Spread the complete 400 µl reaction mix on selective antibiotic plates, and incubate at 37°C overnight <br />
<br/><br />
<br />
<p class="texte"><br />
<b> Preparation of solutions </b><br />
<I> <br> 300 mM Tri-Na Citrate:</I><br />
<br>- 0.88 g Tri-Na Citrate<br />
<br>- 10mL MQ water<br />
<p class="texte"><br />
<I> <br> Ferric NH4 citrate:</I><br />
<br>- 0.22g Ferric NH4<br />
<br>- 10mL MQ water<br />
<p class="texte"><br />
<I> <br> 10x Competence Medium </I><br />
<br> For 10mL:<br />
<br>- 1.40g K2HPO4<br />
<br>- 0.52g KH2PO4<br />
<br>- 2g glucose<br />
<br>- 1 mL 300 mM Tri-Na citrate <br />
<br>- 0.1 mL Ferric NH4 citrate<br />
<br>- 0.1g Casein Hydrolysate<br />
<br>- 0.2 g Potassium glutamate<br />
<br>The complete mixture should be dissolved in 10 ml. First add 5 ml milliQ water and mix. When everything is dissolved add MQ water till 10 ml. Filter sterilize the complete mixture and store at -20°C.<br />
<br />
<p class="texte"><br />
<I> <br> 1x Competence Medium </I><br />
<br>- 1.8 mL MQ water<br />
<br>- 200 µL 10x Competence Medium solution (previously filter sterilized)<br />
<br>- 6.7 µL 1M MgSO4 (previously autoclaved)<br />
<br>- 10 µL 1% tryptophan (previously filter sterilized and stored in aluminium foil)<br />
<br>The complete mixture should be dissolved in 100 ml. First add 50 ml milliQ water and mix. When everything is dissolved add MQ water till 100 ml. Filter sterilize the complete mixture and store at -20°C.<br />
<br />
<p class="title1" id="select7">Checking of the genetic constructions after plasmid integration in <I>Bacillus subtilis</I></p><br />
<p class="texte"><br />
<br>Test of the pSBBS4S plasmid integration in Bacillus subtilis genome on the threonine site:<br />
<br>- Plate the transformed Bacillus strain on a selective medium (LB + spectinomycin) overnight <br />
<br>- The obtained clones are then plated on different media: Medium Competence (Thr+), Medium Competence (Thr-) and LB + spectinomycine. <br />
<br>When the plasmid is integrated, the clone can grow on minimum medium without threonine but can not grow on the other media.<br />
Moreover, a colony PCR can be performed with the same protocol as previously presented. The used primers are VFBS and VRBS.<br />
<br />
<p class="title1" id="select8">Final Tests</p><br />
<p class="title2">Chemotaxis test</p><br />
<p class="texte"><br />
Many chemotaxis tests exist such as plate tests or capillary tests. We tried all of them and we decided to optimize the « Capillary essay » from Imperial College 2011 iGEM team.<br />
<br>- Prepare the bacteria in LB medium so they reach an OD between 0.5 and 0.6 (exponential growth phase). This step takes about 5 hours.<br />
<br>- Prepare the multichannel pipette: improve the cohesion between the tips and the pipette with Blu-Tack to avoid air and the possibility of leakage.<br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2014/b/b1/Installation_1.gif"></center><br />
<p class="texte"><br />
<br>- Put 200µl of the different chemoattractants in the wells of the ELISA plate and pipette 15µL of each with the multichannel pipette: galactose which represents our negative control, glucose which represents our positive control and N-acetylglucosamine (NAG). <br />
NB: The NAG is the most important test because it is the monosaccharide which composes the chitin on Ceratocystis platani wall. The volume in the tips must be marked.<br />
<br>- Put the tips with chemoattractants in 300µL of the bacterial solution in exponential growth phase in the ELISA plate.<br />
<br>- Let the installation settle for 1 hour at room temperature.<br />
<br>- After an hour, put the volume of the tips on parafilm. <br />
<br>- Each solution is diluted 1/10,000 and 100µL is spread on LA medium.<br />
<br>- The plates are then incubated overnight at 37°C.</p><br />
<br />
<p class="title2">Binding test</p><br />
<p class="texte"><I>CBB (Chitin Binding Buffer):</I><br />
<br>- 500 mM NaCl<br />
<br>- 20 mM Tris-HCl<br />
<br>- 1 mM EDTA<br />
<br>- 0,05% Triton X-100, 25°C, pH=8<br />
</p><br />
<br />
<p class="texte"><br />
<I>Column activation:</I> <br />
<br>- Vortex the beads <br />
<br>- Put 50 µL of beads in a 1.5mL centrifuge tube<br />
<br>- Wash with 500 µL of CBB<br />
<br>- Put the centrifuge tube on a magnetic rack<br />
<br>- Wait 30 seconds<br />
<br>- Remove supernatant<br />
<br>- Repeat the wash<br />
</p><br />
<br />
<br />
<p class="texte"><I>Bacterial fixation on the chitin beads:</I><br />
<br>- Add 200 µL of bacteria solution (105 bactéria/mL)to the washed beads <br />
<br>- Shake during 1h at 4°C<br />
<br>- Add 500 µL of CBB (washing A)<br />
<br>- Put the centrifuge tube on a magnetic rack<br />
<br>- Wait 30 seconds<br />
<br>- Remove supernatant<br />
<br>- Add 500 µL of CBB (washing B)<br />
<br>- Put the centrifuge tube on a magnetic rack<br />
<br>- Wait 30 seconds<br />
<br>- Remove supernatant<br />
<br>- Add 500 µL of CBB to recover the beads directly<br />
</p><br />
<br />
<p class="texte"><I>Bacteria count:</I><br />
<br>- Make different dilutions : 10-1, 10-3, 10-5 of the first bacterial culture and spread on LA plates<br />
<br>- Make different dilutions : 1, 10-2, 10-4 of washings (A and B) and of the beads in CBB medium and spread on LA plates<br />
<br>- Place the plates at 37°C overnight<br />
<br>- Count colonies on different plates<br />
</p><br />
<br />
<p class="title2">Fungicide test: anti-fungal activities</p><br />
<p class="texte"><br />
CAUTION : all the lab equipment must be desinfected before the manipulations with the fungi. <br />
<br>Three different funguss strains were used : <I>Aspergillus brasiliensis, Aspergillus nidulans, Trichoderma reesei</I><br />
<br>- The conidia can be taken by adding one drop of Tween 80 on the fungus plate.<br />
<br>- Then the drop is mixed with 1mL of sterile water in an Eppedorf.<br />
<br>- A microscopy count can be performed thanks to Thoma cell to determine the conidia concentration.<br />
<br>NB : The conidia solutions are then diluted and spread on sap medium to get 10,000 conidia/plate.<br />
<br>- After 72hours of liquid culture of the different clones of <i>B. subtilis>/i> with the fungicides module, the culture can be centrifugated.<br />
<br>- 130µL of the supernatant is used to soak a pad placed on the conidia plate. The bacterial pellet is resuspended in 130µL of LB medium and also put on a pad. <br />
<br>- The plates containing 10,000 conidia and the soaked pads are then put at room temperature for a few days according to the growth speed of the fungi. Controls are also realized with wild type strains or copper sulfate at 10 and 20mg/mL.<br />
</p><br />
<br />
<p class="title2">Fungicide test: <i>in planta</i> assay</p><br />
<p class="texte"><br />
The first step is related to the inoculation of SubtiTree in plants through stomata (opened in wet condition). We diluted our bacterial samples to get two concentrations: 5.10^6 and 10^8 bacteria per mL. The WT and trasnformed bacteria are introduced into plants (a control test without bacteria was performed). Thanks to a 1 ml syringe (without needle), the plant was injected with bacteria by pressure. Five leaves of each plant were used, marked with a marker point. It is necessary to repeat the operation on both sides of the leaf and the excess is wipe off. The plants are placed in a growth chamber (phytotron) to control light, high humidity, temperature and bacterial non-proliferation. Bacterial growth in the plant is left for 24 h.<br />
</br><br />
<br />
The next step begins with the preparation of the fungal samples.Fungal culture is crushed and mixed with PDB (Potato Dextrose Broth). Then the mix passes through a 100 µm filter (to remove large aggregates) and through a 40 µm filter. The caught hyphae are mixed with PDB for 24 to 48 hours until OD(600nm) 2.5 isobtained. The previously seeded leaves are taken from the plant using a scalpel and placed in boxes above wet absorbent paper (leaves are kept alive for a week). Above each leaf 5µl of the fungal suspension is placed (using beveled tips because it is too viscous. As control, we kept inoculated leaves without fungus and leaves with only fungus. Pictures are taken at different times. All the plants are destroyed by autoclaving.<br />
</p><br />
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