Team:INSA-Lyon/Protocol

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<h4>PROTOCOLS</h4>
<h4>PROTOCOLS</h4>
<div id="icones">
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<ul style="margin:auto;">
<a href="https://2014.igem.org/Team:INSA-Lyon/Biology" class="hu-icon"><li class="iconmulti"  style="line-height:20px;  ">WETLAB SUMMARY</li></a>
<a href="https://2014.igem.org/Team:INSA-Lyon/Biology" class="hu-icon"><li class="iconmulti"  style="line-height:20px;  ">WETLAB SUMMARY</li></a>
<a href="https://2014.igem.org/Team:INSA-Lyon/Results" class="hu-icon"><li class="iconmulti"  style="line-height:40px;  ">RESULTS</li></a>
<a href="https://2014.igem.org/Team:INSA-Lyon/Results" class="hu-icon"><li class="iconmulti"  style="line-height:40px;  ">RESULTS</li></a>
<a href="https://2014.igem.org/Team:INSA-Lyon/Notebook" class="hu-icon"><li class="iconmulti"  style="line-height:40px;  ">NOTEBOOK</li></a>
<a href="https://2014.igem.org/Team:INSA-Lyon/Notebook" class="hu-icon"><li class="iconmulti"  style="line-height:40px;  ">NOTEBOOK</li></a>
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<a href="https://2014.igem.org/Team:INSA-Lyon/Parts" class="hu-icon"><li class="iconmulti"  style="line-height:40px;  ">PARTS</li></a>
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<a href="https://2014.igem.org/Team:INSA-Lyon/Protocol" class="hu-icon"><li class="iconmulti"  style="line-height:40px;  ">PROTOCOLS</li></a>
<a href="https://2014.igem.org/Team:INSA-Lyon/DataPage" class="hu-icon"><li class="iconmulti"  style="line-height:40px;  ">DATA PAGE</li></a>
<a href="https://2014.igem.org/Team:INSA-Lyon/DataPage" class="hu-icon"><li class="iconmulti"  style="line-height:40px;  ">DATA PAGE</li></a>
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<div id="presentation">
 
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<div class="protocolsContainer">
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<div class="protocolsInnerContainer">
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<div class="protocolsButtonCell" onclick="redirectWindow('https://static.igem.org/mediawiki/2014/0/0e/ABC_miniprep.pdf')">
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<div class="protocolsButtonImage">
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<img src="https://static.igem.org/mediawiki/2014/b/b8/ABC.png" alt="ABC Minipreping" class="pImgOver">
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<img src="https://static.igem.org/mediawiki/2014/a/ad/Fond_picto_protocols.png" alt="Fond" class="pImgUnder">
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</div>
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<div class="protocolsButtonText">
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<span>ABC Minipreping</span>
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<div class="protocolsButtonCell" onclick="redirectWindow('https://static.igem.org/mediawiki/2014/8/80/Adhesion_test_protocole.pdf')">
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<div class="protocolsButtonImage">
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<img src="https://static.igem.org/mediawiki/2014/1/16/Adhesion_picto.png" alt="Adhesion test" class="pImgOver">
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<img src="https://static.igem.org/mediawiki/2014/a/ad/Fond_picto_protocols.png" alt="Fond" class="pImgUnder">
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<div class="protocolsButtonText">
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<span>Adhesion test</span>
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<div class="protocolsButtonCell" onclick="redirectWindow('https://static.igem.org/mediawiki/2014/e/ef/Crystal_Violet_protocole.pdf')">
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<div class="protocolsButtonImage">
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<img src="https://static.igem.org/mediawiki/2014/b/b4/Insainsa_crystal.png" alt="Crystal violet" class="pImgOver">
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<img src="https://static.igem.org/mediawiki/2014/a/ad/Fond_picto_protocols.png" alt="Fond" class="pImgUnder">
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</div>
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<div class="protocolsButtonText">
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<span>Crystal Violet</span>
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</div>
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<div class="protocolsButtonCell" onclick="redirectWindow('https://static.igem.org/mediawiki/2014/7/7e/Culture_confocal_analyse.pdf')">
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<div class="protocolsButtonImage">
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<img src="https://static.igem.org/mediawiki/2014/d/d0/New_microscope.png" alt="Confocal analyses" class="pImgOver">
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<img src="https://static.igem.org/mediawiki/2014/a/ad/Fond_picto_protocols.png" alt="Fond" class="pImgUnder">
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</div>
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<div class="protocolsButtonText">
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<span>Confocal analyses </span>
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<div class="protocolsButtonCell" onclick="redirectWindow('https://static.igem.org/mediawiki/2014/1/13/Digestion.pdf')">
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    <li><a href="#contenu1" onclick="$('#contenu1').slideToggle('slow')"><h1><img src="https://static.igem.org/mediawiki/2014/d/d5/Insa_fleche_titre.png" width="20px" />Nickel chelation</h1></a><hr/></li>
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          <ul id="contenu1" style="list-style-type: none !important;display:none;">
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<img src="https://static.igem.org/mediawiki/2014/3/38/Digestion.png" alt="Digestion" class="pImgOver">
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              <li><p>
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<img src="https://static.igem.org/mediawiki/2014/a/ad/Fond_picto_protocols.png" alt="Fond" class="pImgUnder">
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<p>What about chelation ? <br/>
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</div>
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Nickel(II) chelated for each of the constructions (WT, HIS1,  HIS2) is evaluated by using dimethylglyoxime (DMG) as the precipitating reagent. This is achieved by using absorbing properties of DMG-Ni(II) pink-colored complex (peak absorption at 554nm). <br/>
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<div class="protocolsButtonText">
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Firstly, a calibration set of Nickel and DMG was done. <br/>
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<span>Digestion</span>
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</div>
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</div>
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Then, strains 265, 266 and 267 were assayed for biofilm nickel absorption on liquid cultures using the calibration set, after measuring the OD of the complex formed for each strain at 554nm.<br/>
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<div class="protocolsButtonCell" onclick="redirectWindow('https://static.igem.org/mediawiki/2014/9/9d/Ligation.pdf')">
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This technique is more qualitative than quantitative due to the spectrometer precision. <br/>
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Here are the outcomes. </p><br/>
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<img src="https://static.igem.org/mediawiki/2014/2/2a/New_ligation.png" alt="Ligation" class="pImgOver">
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<img src="https://static.igem.org/mediawiki/2013/e/ea/Wikiphotogamme.png" alt="photo de la gamme"  
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<img src="https://static.igem.org/mediawiki/2014/a/ad/Fond_picto_protocols.png" alt="Fond" class="pImgUnder">
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width="600px"/> <br/>
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<img src="https://static.igem.org/mediawiki/2014/7/77/DMGgammeet.png" alt="gamme graphe"  
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<div class="protocolsButtonText">
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width="600px"/>  <br/> <br/>
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<span>Ligation</span>
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<br/>
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</div>
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</div>
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A second method has been used, more quantitative and more precise:  mass spectrometry assays have been realized. The quantity of chelated nickel for each strain has been compared to the quantity of curlis formed by each strain.<br/>
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Here are the results : <br/>
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<img src="https://static.igem.org/mediawiki/2014/1/10/ICP.png" alt="ICP"  
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width="600px"/> <br/>
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As confirmed by this second method, strain 267 chelates twice more than strain 266. That means that two Histags are twice more effective than one. Moreover, one histag allows a better chelation than none, but not a really significative one.
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</p></li>
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<div class="protocolsButtonCell" onclick="redirectWindow('https://static.igem.org/mediawiki/2014/f/f7/Ni_chelation.pdf')">
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<div class="protocolsButtonImage">
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<img src="https://static.igem.org/mediawiki/2014/c/ca/Insa_ICP.png" alt="ICP-MS" class="pImgOver">
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<img src="https://static.igem.org/mediawiki/2014/a/ad/Fond_picto_protocols.png" alt="Fond" class="pImgUnder">
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</div>
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<span>Ni Chelation (ICP-MS)</span>
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<div class="protocolsButtonCell" onclick="redirectWindow('https://static.igem.org/mediawiki/2014/0/01/Ni_chelation_DMG_n.pdf')">
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<div class="protocolsButtonImage">
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<img src="https://static.igem.org/mediawiki/2014/d/d9/DMG.png" alt="DMG" class="pImgOver">
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<img src="https://static.igem.org/mediawiki/2014/a/ad/Fond_picto_protocols.png" alt="Fond" class="pImgUnder">
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</div>
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<div class="protocolsButtonText">
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<span>Ni Chelation (DMG)</span>
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</div>
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</div>
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<div class="protocolsButtonCell" onclick="redirectWindow('https://static.igem.org/mediawiki/2014/1/1b/TSS_transformation.pdf')">
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<div class="protocolsButtonImage">
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<img src="https://static.igem.org/mediawiki/2014/6/6f/New_TSS.png" alt="Transformation" class="pImgOver">
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<img src="https://static.igem.org/mediawiki/2014/a/ad/Fond_picto_protocols.png" alt="Fond" class="pImgUnder">
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</div>
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<div class="protocolsButtonText">
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<span>TSS Transformation</span>
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</div>
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</div>
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    <li><a href="#contenu2" onclick="$('#contenu2').slideToggle('slow')"><h1><img src="https://static.igem.org/mediawiki/2014/d/d5/Insa_fleche_titre.png" width="20px" />Effectiveness of His-Tag motif</h1></a><hr/></li>
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<div class="protocolsButtonCell" onclick="redirectWindow('https://static.igem.org/mediawiki/2014/1/1e/UV_temperature.pdf')">
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          <ul id="contenu2" style="list-style-type: none !important;display:none;">
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              <li><p>
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<img src="https://static.igem.org/mediawiki/2014/b/b2/UV.png" alt="UV" class="pImgOver">
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TEXT
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<img src="https://static.igem.org/mediawiki/2014/a/ad/Fond_picto_protocols.png" alt="Fond" class="pImgUnder">
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</p></li>
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</div>
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          </ul>
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<div class="protocolsButtonText">
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<span>UV & Temperature </span>
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    <li><a href="#contenu3" onclick="$('#contenu3').slideToggle('slow')"><h1><img src="https://static.igem.org/mediawiki/2014/d/d5/Insa_fleche_titre.png" width="20px" />Biofilm production</h1></a><hr/></li>
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          <ul id="contenu3" style="list-style-type: none !important;display:none;">
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              <li><p>
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TEXT
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</p></li>
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    <li><a href="#contenu4" onclick="$('#contenu4').slideToggle('slow')"><h1><img src="https://static.igem.org/mediawiki/2014/d/d5/Insa_fleche_titre.png" width="20px" />Survival after UV and high temperature exposure</h1></a><hr/></li>
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          <ul id="contenu4" style="list-style-type: none !important;display:none;">
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              <li><p>
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<p>To get rid of biosafety issues linked with GMO, we worked on destroying our bacteria after letting them grow in a biofilm. Curli proteins being very resistant to environmental changes, our goal was to obtain a biomaterial made out of modified Curli able to chelate nickel. </p> <br/>
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<p>To find the best  way to degrade bacteria and DNA, the following protocol was used to test the influence of UV light and temperature separately : <br/>
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<ul>
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<li> Wells containing M63 cultures of strain 227 were put under UV light / at 60 or 70°C for different lengths of time. Well contents were then gradually transferred into Eppendorf  and diluted (100, 300, 900 and 2700 times).<br/>
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<li> LB plates (without antibiotic) corresponding to UV/temperature exposure times (+ one plate for control) were then spotted with s227 different concentrations in order to be able to count survival bacteria after incubation at 37°C.<br/>
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<li> Genomic DNA was extracted from s227 concentrated culture. From the solution obtained, Curli promoter(750 bp) was amplified by PCR with Q5 polymerase and designed primers. <br/>
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<li>Epifluorescence observations were made after Back Light coloration with 200µL s227 liquid cultures.</p><br/> <br/>
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</ul>
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<p> <i> UV light influence </i></p><br/>
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<p><img src="https://static.igem.org/mediawiki/2014/3/35/DSCN2175.JPG" alt="control plate" width="200 px"/> <img src="https://static.igem.org/mediawiki/2014/4/4e/DSCN2188.JPG" alt="5min" width="200 px"/> <img src="https://static.igem.org/mediawiki/2014/5/55/DSCN2179.JPG" alt="10min" width="200 px"/> <img src="https://static.igem.org/mediawiki/2014/9/99/DSCN2184.JPG" alt="15 min" width="200 px"/> <img src="https://static.igem.org/mediawiki/2014/5/5f/DSCN2185.JPG" alt="20min" width="200 px"/>  No bacteria grew on LB plate after 15 minutes UV light exposure.<br/>-> <b>Bacterian growth can be stopped this way. </b></p><br/>
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<p> image gel PCR<br/> Bacterian DNA seemed to be degraded after 10 min UV light exposure.<br/>-> <b>In consequence, UV light can be used to destroy DNA.</b> </p><br/>
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<p>3 images 40X Back Light<br/> Still some green-colored bacteria could be seen after 20 min UV exposure. <br/>
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-> <b>UV light isn’t enough to kill bacteria.</b></p><br/><br/>
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<p> <i> Temperature influence </i></p><br/>
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<p> 3 autres images LB plates 60°C <img src="https://static.igem.org/mediawiki/2013/9/97/DSCN2491.JPG" alt="control plate" width="200 px" /><br/> Bacteria grew on LB plates even 45 min after being heated to 60°C. <br/> -> <b>Temperature isn't enough high to kill bacteria.</b></p><br/>
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<p> 4 images LB plates 70°C<br/>No more bacteria on LB plate after 15min at 70°C<br/> -> <b>Bacterian growth can be stopped as well as with UV light.</b></p><br/>
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<p> image gel PCR<br/> No DNA degradation at all.<br/>-> <b>In consequence, temperature doesn't enable to destroy DNA, in contrary to UV light.</b> </p><br/>
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<p>4 images 40X Back Light<br/> No difference of coloration was observed between the control and the samples heated at 70°C : indeed a lot of green-colored bacteria remained after 45 min of heating.<br/> -> <b>Temperature isn’t enough to kill bacteria just like UV light.</b></p><br/>
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<p> To solve this last problem, bacteria were put in contact with ethanol absolute. The Back Light coloration gives the following picture. <br/> image benjamin</p><br/><br/>
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<p>These numerous experiments lead us to developp a protocol in three steps, illustrated by the drawing below :<br/>
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<img src="https://static.igem.org/mediawiki/2013/5/5f/Strat%C3%A9gie_bilan.jpg" alt="schéma bilan" width="600 px" /></p>  
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</p></li>
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    <li><a href="#contenu5" onclick="$('#contenu5').slideToggle('slow')"><h1><img src="https://static.igem.org/mediawiki/2014/d/d5/Insa_fleche_titre.png" width="20px" />Promoter optimization and characterization</h1></a><hr/></li>
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          <ul id="contenu5" style="list-style-type: none !important;display:none;">
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              <li><p>
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<div align="justify">
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<b>Biobrick for promoter characterization</b></br>
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The principal of the construction is shown in the figure below.</br></div>
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<div align="center">
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<img src= "https://static.igem.org/mediawiki/parts/f/fb/2.png"></br></div>
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<div align="justify">
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<p>On a pKK backbone, two essential parts have been assembled: a promoter and a reporter.</br>
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The reporter in this case is always the same: gfp. However, the promoter is different for each construction.</br>
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The first promoter is P70, sequence found in the Resgistry. This construction is called p70::gfp</br>
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The second promoter is PCurli, obtained from a PCR, sequence coding for the inter-genic regulation for curli production. This construction is called pcurli::gfp.</p></br></div>
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                <div class="protocolsButtonCell" onclick="redirectWindow('https://static.igem.org/mediawiki/2014/3/39/CongoRed.pdf')">
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<img src="https://static.igem.org/mediawiki/2014/b/b3/Congored.png" alt="UV" class="pImgOver">
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<img src="https://static.igem.org/mediawiki/2014/a/ad/Fond_picto_protocols.png" alt="Fond" class="pImgUnder">
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</div>
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<div class="protocolsButtonText">
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<span>Congo Red</span>
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Latest revision as of 00:34, 18 October 2014

Curly'on - IGEM 2014 INSA-LYON

ABC Minipreping Fond
ABC Minipreping
Adhesion test Fond
Adhesion test
Crystal violet Fond
Crystal Violet
Confocal analyses Fond
Confocal analyses
Digestion Fond
Digestion
Ligation Fond
Ligation
ICP-MS Fond
Ni Chelation (ICP-MS)
DMG Fond
Ni Chelation (DMG)
Transformation Fond
TSS Transformation
UV Fond
UV & Temperature
UV Fond
Congo Red


Retrieved from "http://2014.igem.org/Team:INSA-Lyon/Protocol"