Team:Bielefeld-CeBiTec/Notebook/Journal/rMFC/Sep

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<h1> September </h1>
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<td width="100px"><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Journal/rMFC/Aug"><img src="https://static.igem.org/mediawiki/2014/b/b9/Bielefeld-CeBiTec_2014-08-14_Arrrow-left.png" width="50px"> </a></td>
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<td style="padding:30px; width:400px"><h1> September </h1></td>
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<td width="100px" align="right"><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Journal/rMFC/Oct"><img src="https://static.igem.org/mediawiki/2014/9/9b/Bielefeld-CeBiTec_2014-08-14_Arrow-right.png" width="50px"> </a>      </td>
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                 <a  style="font-size:24px" href="#"><p style="margin-left:42%">Week 1 &nbsp;&nbsp;&nbsp; 09/01 - 09/07</p></a>
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                 <h6><a  style="font-size:24px" href="#">Week 1 &nbsp;&nbsp;&nbsp; 09/01 - 09/07</a></h6>
             </div>
             </div>
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             <div class="content">
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             <div class="content" style="margin-right:10%; margin-left:10%">
 +
<ul>
 +
<li><b><i>FumA</i></b></li>
 +
<ul>
 +
<li> This week we assembled <i>FumA</i> with different constitutive promotors</li>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>EcoR</i>I</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>Pst</i>I</a> </li>
 +
<ul>
 +
<li>Bands as expected (~2070 bp and ~1800 bp) for pSB1C3_T7_<i>FumA</i></li>
 +
</ul>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a> (Suffix)</li>
 +
<ul>
 +
<li>Backbone (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Spe</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>)</li>
 +
<ul>
 +
<li><a href="http://parts.igem.org/Part:BBa_J23101" target="_blank">BBa_J23101</a></li>
 +
<li><a href="http://parts.igem.org/Part:BBa_J23102" target="_blank">BBa_J23102</a></li>
 +
<li><a href="http://parts.igem.org/Part:BBa_J23104" target="_blank">BBa_J23104</a></li>
 +
</ul>
 +
<li>Insert (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Xba</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>)</li>
 +
<ul>
 +
<li><i>FumA</i></li>
 +
</ul>
 +
</ul>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> of all constructs with electrocompotetent cells</li>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#VF-Primer" target="_blank">VF-Primer</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#VR-Primer" target="_blank">VR-Primer</a>)
 +
            </li>
 +
<ul>
 +
<li>Annealing temperature: 55 °C</li>
 +
<li>Bands as expected (~2100 bp) for all constructs</li>
 +
</ul>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of the positive colonies</li>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>EcoR</i>I</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>Pst</i>I</a> </li>
 +
<ul>
 +
<li>Bands as expected (~2070 bp and ~1800 bp)</li>
 +
</ul>
 +
</ul>
 +
</ul>
 +
</ul>
 +
<br>
 +
<ul>
 +
<li><b><i>ccm</i></b></li>
 +
<ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with <i>ccm</i> and pSB1C3</li>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> of pSB1C3_<i>ccm</i> with electrocompotetent cells</li>
 +
</ul>
 +
 
 +
 
 +
 
 +
 
 +
</ul>
 +
</ul>
 +
<br>
 +
 
 +
 
 +
<ul>
 +
<li><b><i>GSU 3274</i></b></li>
 +
<ul><li> This week we assembled <i>GSU 3274</i> with different promotors</li>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a> (Suffix)</li>
 +
<ul>
 +
<li>Backbone (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Spe</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>)</li>
 +
<ul>
 +
<li>pSB1C3_p<sub>tac</sub></li>
 +
<li>pSB1K3_p<sub>Tet</sub></li>
 +
<li>pSB1A2_T7</i>
 +
</ul>
 +
<li>Insert (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Xba</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>)</li>
 +
<ul>
 +
<li><i>GSU 3274</i></li>
 +
</ul>
 +
</ul>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> of all constructs with electrocompotetent cells</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#VF-Primer" target="_blank">VF-Primer</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#VR-Primer" target="_blank">VR-Primer</a>)
 +
            </li>
 +
<ul>
 +
<li>Annealing temperature: 55°C</li>
 +
<li>Bands as expected (~ bp) for pSB1C3_p<sub>tac</sub> and pSB1A2_T7</li>
 +
</ul>
 +
</ul>
 +
</ul>
 +
</ul>
 +
<br>
 +
<ul>
 +
<li><b>Electrobiochemical reactor system</b></li>
 +
<ul>
 +
<li>This week we arranged the complete <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Results/rMFC/Construction">H-cell reactor </a> for the first time to start first trials. Therefore we prepare the<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Media#H-cell%20buffer" target="_blank"> H-cell buffers</a> and the <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Nafion%C2%AE%20membrane" target="_blank">Nafion® membrane</a> which ensures the division of the two compartments.</li>
 +
</ul>
 +
    <ul><li>Additionally we started to cultivate in the H-cell reactor to evaluate the growth of <i>E. coli</i> under the current intesity which is according to the literature needed to reduce neutral red and bromphenol blue.</li>                 
 +
</ul>
 +
</ul>
 +
<br>
 +
<ul>
 +
<li><b>NAD/NADH Assay</b></li>
 +
<ul>
 +
<li>This week we startet the first test with the <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PromegaGlo%E2%84%A2%20Assay" target="_blank">Promega NAD/NADH-Glo™ Assay</a> to evaluate later cultivations respectively.</li>
 +
                   
 +
</ul>
 +
</ul>
 +
<br>
 +
 
 +
 
 +
 
 +
 
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                 <a  style="font-size:24px" href="#"><p style="margin-left:42%">Week 2 &nbsp;&nbsp;&nbsp; 09/08 - 09/14</p></a>
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                 <h6><a  style="font-size:24px" href="#">Week 2 &nbsp;&nbsp;&nbsp; 09/08 - 09/14</a></h6>
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             </div>
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             <div class="content">
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             <div class="content" style="margin-right:10%; margin-left:10%">
 +
 +
<ul>
 +
<li><b><i>frd (E.coli)</i></b></li>
 +
<ul>
 +
<li>This week we transform pSB1C3_T7_<i>frd</i> with different <i>E.coli</i> strains for the characterization of it respectively</li>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> of pSB1C3_T7_<i>frd</i> with electrocompotetent cells of <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/StrainsAndConstructs" target="_blank">KRX &Delta;dcuB::oprF</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/StrainsAndConstructs" target="_blank">JW4084-1</a> as well as <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/StrainsAndConstructs" target="_blank">JW0506-1</a> from the KEIO collection</li>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#VF-Primer" target="_blank">VF-Primer</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#VR-Primer" target="_blank">VR-Primer</a>)
 +
            </li>
 +
<ul>
 +
<li>Annealing temperature: 55 °C</li>
 +
<li>Bands as expected (~3600 bp)</li>
 +
</ul>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> of all constructs with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>EcoR</i>I</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>Pst</i>I</a> </li>
 +
<ul>
 +
<li>Bands as expected (~3300 bp and ~2070 bp)</li>
 +
</ul>
 +
</ul>
 +
 
 +
<ul>
 +
<li>Expression of <i>frd (E.coli)</i> for SDS-Page
 +
<ul>
 +
<li>Induction was carried out with 0.1 % Rhamnose and 1 mM IPTG</li>
 +
<li>For the release of the periplasmatic protein fraction a <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColdOsmoticShock" target="_blank">cold osmotic shock </a> was performed</li>
 +
                     
 +
</ul>
 +
</ul>
 +
</ul>
 +
<br>
 +
 
 +
 
 +
 
 +
<ul>
 +
<li><b><i>ccm</i></b></li>
 +
<ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pSB1C3_<i>ccm</i></li>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>Not</i>I</a></li>
 +
<ul>
 +
<li>Bands not as expected (~6611 bp)</li>
 +
</ul>
 +
</ul>
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                 <a  style="font-size:24px" href="#"><p style="margin-left:42%">Week 3 &nbsp;&nbsp;&nbsp; 09/15 - 09/21</p></a>
+
                 <h6><a  style="font-size:24px" href="#">Week 3 &nbsp;&nbsp;&nbsp; 09/15 - 09/21</a></h6>
             </div>
             </div>
-
             <div class="content">
+
             <div class="content" style="margin-right:10%; margin-left:10%">
 +
 
 +
<ul>
 +
<li><b><i>ccm</i></b></li>
 +
<ul>
 +
<li> This week we tried to get the <i>ccm</i> into the correct backbone </li>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with <i>ccm</i> and pSB1C3</li>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> with electrocompotetent cells and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaheatshock" target="_blank">Transformation</a> with chemocompetent cells</li>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of <i>ccm</i> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>EcoR</i>I</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>Pst</i>I</a> </li>
 +
<ul>
 +
<li>Bands mainly not as expected (~6281 bp and ~2070 bp ) because additional bands are visible.
 +
<li>Therefore a <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">restriction digestion</a> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>EcoR</i>V</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>Hind</i>III</a> was done</li>
 +
<ul>
 +
<li>Bands not as expected because only two bands were visible</li>
 +
</ul>
 +
</ul>
 +
</ul>
 +
 
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> with new primer(<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#ccm_con1" target="_blank">ccm_con1</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#VR-Primer" target="_blank">VR-Primer</a>)
 +
            </li>
 +
<ul>
 +
<li>Annealing temperature: 55°C</li>
 +
<li>Bands as expected (~880 bp)</li>
 +
</ul>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pSB1C3_<i>ccm</i> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">restriction digestion</a> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>EcoR</i>I</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>Pst</i>I</a> </li>
 +
<ul>
 +
<li>Bands not as expected (~6281 bp and ~2070 bp)</li>
 +
</ul>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> of <i>ccm</i> backbone(<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#pSB1C3_pre_ccm" target="_blank">pSB1C3_pre_ccm</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#pSB1C3_suf_ccm" target="_blank">pSB1C3_suf_ccm</a>)</li>
 +
<ul>
 +
<li>Annealing temperature: 61&deg;C</li>
 +
<li>Bands as expected (~6281 bp)</li>
 +
</ul>
 +
</ul>
 +
</ul>
 +
</ul>
 +
</ul>
 +
<br>
 +
<ul>
 +
<li><b>Electrobiochemical reactor system</b></li>
 +
<ul>
 +
<li> This week we performed several cyclic voltammetric measurements to characterize different electrode materials and mediators. Therefore the system was purged with nitrogen to remove any oxygen.</li>
 +
<li> Measurements were performed under the following conditions and the the following experimental setup</li>
 +
<ul>
 +
<li> Cathode space: <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Media#H-cell%20buffer" target="_blank">cathode buffer</a> or <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Media#M9medium" target="_blank">M9 medium</a></li>
 +
<li> Anode space: <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Media#H-cell%20buffer" target="_blank">anode buffer</a> 
 +
<li> Working electrode: platinic plate or graphite electrode</li>
 +
<li> Counter electrode: platinic wire </li>
 +
<li> Reference electrode: Ag/AgCl- electrode</li>
 +
<li> Scan rate: 10 mV/s</li>
 +
<li> Scan Limit: was varied among the different measurements</li>
 +
<li> Cycle number: 3, 10 or 500</li>
 +
<li> Mediators: neutral red or bromphenol blue</li>
 +
<li> Temperature: 37&deg;C</li>
 +
</ul>
 +
         </div>
         </div>
       </div>
       </div>
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<div class="tab" id="Week4">
             <div class="show">
             <div class="show">
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             </div>
             </div>
             <div class="hide">
             <div class="hide">
-
                 <a  style="font-size:24px" href="#"><p style="margin-left:42%">Week 4 &nbsp;&nbsp;&nbsp; 09/22 - 09/28</p></a>
+
                 <h6><a  style="font-size:24px" href="#">Week 4 &nbsp;&nbsp;&nbsp; 09/22 - 09/28</a></h6>
             </div>
             </div>
-
             <div class="content">
+
             <div class="content" style="margin-right:10%; margin-left:10%">
 +
<ul>
 +
<li><b><i>FumA</i></b></li>
 +
<ul>
 +
<li> This week we removed the the mutation of <i>FumA</i> by PCR </li>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> of <i>FumA</i> using a gradient(<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#FumA_mut_fwd" target="_blank">FumA_mut_fwd, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#FumA_mut_rev" target="_blank">FumA_mut_rev</a>)</li>
 +
<ul>
 +
<li>Annealing temperature: 50&deg;C to 60&deg;C</li>
 +
<li>Bands as expected (~4000 bp)</li>
 +
</ul>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with <i>FumA</i> and pSB1C3 and digestion with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>Dpn</i>I</a> to remove the template
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaheatshock" target="_blank">Transformation</a> with chemocompetent cells</li>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#VF-Primer" target="_blank">VF-Primer</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#VR-Primer" target="_blank">VR-Primer</a>)
 +
            </li>
 +
<ul>
 +
<li>Annealing temperature: 55°C</li>
 +
<li>Bands as expected (~2100 bp)</li>
 +
</ul>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pSB1C3_<i>FumA</i></li>
 +
</ul>
 +
<ul>
 +
<li>
 +
Sequencing of pSB1C3_<i>FumA</i> (Primer: <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#FumA_seq" target="_blank">FumA_seq</a>) confirmed the correct construct</i>
 +
</ul>
 +
</ul>
 +
</ul>
 +
<br>
 +
<ul>
 +
<li><b><i>FumBCD</i></b></li>
 +
<ul>
 +
<li> This week we amplified and transformed <i>FumBCD</i></li>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> of <i>FumBCD</i> of pJet-Vector (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#FumBCD_fwd" target="_blank">FumBCD_fwd</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#FumBCD_rev" target="_blank">FumBCD_rev</a>)</li>
 +
<ul>
 +
<li>Annealing temperature: 55&deg;C</li>
 +
<li>Bands as expected (1579 bp)</li>
 +
</ul>
 +
</ul>
 +
<ul>
 +
<li>PCR products were <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">purified</a> out of the gel</li>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with <i>FumBCD</i> and pSB1C3</li>
 +
</ul>
 +
<ul>
 +
<li> Digestion with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>Dpn</i>I</a> to remove the template</li>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> with electrocompotetent cells and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaheatshock" target="_blank">Transformation</a> with chemocompetent cells</li>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#VF-Primer" target="_blank">VF-Primer</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#VR_primer" target="_blank">VR-Primer</a>)
 +
            </li>
 +
<ul>
 +
<li>Annealing temperature: 55°C</li>
 +
<li>Bands not as expected (~1909 bp)</li>
 +
</ul>
 +
</ul>
         </div>
         </div>
       </div>
       </div>
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-
   <div id="text">
+
   <div id="text" style="text-align:left">
<div class="tab" id="Week5">
<div class="tab" id="Week5">
             <div class="show">
             <div class="show">
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             </div>
             </div>
             <div class="hide">
             <div class="hide">
-
                 <a  style="font-size:24px" href="#"><p style="margin-left:42%">Week 5 09/29 - 10/05</p></a>
+
                 <h6><a  style="font-size:24px" href="#">Week 5 09/29 - 10/05</a></h6>
             </div>
             </div>
-
             <div class="content">
+
             <div class="content" style="margin-right:10%; margin-left:10%">
 +
<ul>
 +
<li><b>Deletion of <i>dcuB</i> and integration of <i>oprF</i> into chromosome</b></li>
 +
<ul>
 +
<li>Controll of the strain <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/StrainsAndConstructs" target="_blank">KRX &Delta;dcuB::oprF</a> with and without <i>frd (E.coli)</i>
 +
</ul>
 +
</ul>
 +
<br>
 +
<ul>
 +
<li><b><i>FumBCD</i></b></li>
 +
<ul>
 +
<li> This week we amplified and transformed <i>FumBCD</i></li>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> of <i>FumBCD</i> of pJet-Vector (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#FumBCD_fwd" target="_blank">FumBCD_fwd</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#FumBCD_rev" target="_blank">FumBCD_rev</a>)</li>
 +
<ul>
 +
<li>Annealing temperature: 55&deg;C</li>
 +
<li>Bands as expected (1579 bp)</li>
 +
</ul>
 +
</ul>
 +
<ul>
 +
<li>PCR products were <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">purified</a> out of the gel</li>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> of <i>FumBCD</i> backbone(<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#pSB1C3_pre_Fum_BCD" target="_blank">pSB1C3_pre_Fum_BCD</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#pSB1C3_suf_Fum_BCD" target="_blank">pSB1C3_suf_Fum_BCD</a>)</li>
 +
<ul>
 +
<li>Annealing temperature: 55&deg;C</li>
 +
<li>Bands (not) as expected (~2070 bp)</li>
 +
</ul>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with <i>FumBCD</i> and pSB1C3</li>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> with electrocompotetent cells and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaheatshock" target="_blank">Transformation</a> with chemocompetent cells</li>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#FumBCD_fwd" target="_blank">FumBCD_fwd</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#FumBCD_rev" target="_blank">FumBCD_rev</a>)
 +
            </li>
 +
<ul>
 +
<li>Annealing temperature: 55°C</li>
 +
<li>Bands as expected (~1579 bp)</li>
 +
</ul>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pSB1C3_<i>FumBCD</i></li>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>EcoR</i>I</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>Pst</i>I</a> </li>
 +
<ul>
 +
<li>Bands not as expected (~1579 bp and ~2070 bp)</li>
 +
</ul>
 +
</ul>
 +
<ul>
 +
<li> Cloning by restriction and ligation </li>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> of <i>FumBCD</i> of pJet-Vector (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#FumBCD_fwd" target="_blank">FumBCD_fwd</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#FumBCD_rev" target="_blank">FumBCD_rev</a>)</li>
 +
<ul>
 +
<li>Annealing temperature: 55&deg;C</li>
 +
<li>Bands as expected (1579 bp)</li>
 +
</ul>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> of <i>FumBCD</i> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>Xba</i>I</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>Spe</i>I</a> </li>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> of the backbone with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>Xba</i>I</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>Spe</i>I</a> </li>
 +
</ul>
 +
<ul>
 +
<li> Ligation of both fragments and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> with electrocompotetent cells as well as <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaheatshock" target="_blank">Transformation</a> with chemocompetent cells</li>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#FumBCD_fwd" target="_blank">FumBCD_fwd</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#FumBCD_rev" target="_blank">FumBCD_rev</a>)
 +
            </li>
 +
<ul>
 +
<li>Annealing temperature: 55°C</li>
 +
<li>Bands as expected (~1579 bp)</li>
 +
</ul>
 +
</ul>
 +
<ul>
 +
<li> Verification of the <i>FumBCD</i> insertion in pSB1C3</li>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#VF-Primer" target="_blank">VF-Primer</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#FumBCD_rev" target="_blank">FumBCD_rev</a>)</li>
 +
<ul>
 +
<li>Annealing temperature: 55&deg;C</li>
 +
<li>Bands as expected (~1694 bp)</li>
 +
</ul>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> of <i>FumBCD</i> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>EcoR</i>I</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>Pst</i>I</a> </li>
 +
<ul>
 +
<li>Bands as expected (~1579 bp)</li>
 +
</ul>
 +
</ul>
 +
</ul>
 +
</ul>
 +
</ul>
 +
<br>
 +
<ul>
 +
<li><b>anaerobic cultivation for characterization of pSB1C3_T7_<i>frd</i></b></li>
 +
<ul>
 +
<li>We tested all combinations of the following media and strains for the characterization of pSB1C3_T7_<i>frd</i></li>
 +
<ul>
 +
<li>Strains</li>
 +
<ul>
 +
<li>KRX wildtype B0015</li>
 +
<li>KRX wildtype with pSB1C3_T7_<i>frd</i></li>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/StrainsAndConstructs" target="_blank">JW0506-1</a>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/StrainsAndConstructs" target="_blank">JW0506-1</a> with pSB1C3_T7_<i>frd</i>
 +
</ul>
 +
</ul>
 +
<ul>
 +
<li>Media</li>
 +
<ul>
 +
<li>M9 with xylose (50 mM)</li>
 +
<li>M9 with xylose (50 mM) and fumarate (50 mM)</li>
 +
<li>M9 with fumarate (50 mM)
 +
</ul>
 +
</ul>
 +
<ul>
 +
<li>Induction was performed by adding 0,1% Rhamnose</li>
 +
<li>To document the growth curve and the consumption of substrate daily samples were taken for OD<sub>600</sub> measurement and HPLC analytics
 +
</ul>
 +
</ul>
 +
</ul>
 +
<br>
 +
<ul>
 +
<li><b>anaerobic cultivation for characterization of the antiporter &delta;dcuB</b></li>
 +
<ul>
 +
<li>We tested all combinations of the following media and strains for the characterization of pSB1C3_T7_<i>frd</i></li>
 +
<ul>
 +
<li>Strains</li>
 +
<ul>
 +
<li>KRX wildtype B0015</li>
 +
<li>KRX wildtype with pSB1C3_T7_<i>frd</i>
 +
<li>KRX &Delta;dcuB::oprF</li>
 +
<li>KRX &Delta;dcuB::oprF with pSB1C3_T7_<i>frd</i></li>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/StrainsAndConstructs" target="_blank">JW4084-1</a>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/StrainsAndConstructs" target="_blank">JW4084-1</a> with pSB1C3_T7_<i>frd</i></li>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/StrainsAndConstructs" target="_blank">JW0506-1</a>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/StrainsAndConstructs" target="_blank">JW0506-1</a> with pSB1C3_T7_<i>frd</i></li>
 +
 
 +
</ul>
 +
</ul>
 +
<ul>
 +
<li>Media</li>
 +
<ul>
 +
<li>M9 with xylose (50 mM)</li>
 +
<li>M9 with xylose (50 mM) and fumarate (50 mM)</li>
 +
<li>M9 with fumarate (50 mM)
 +
</ul>
 +
</ul>
 +
<ul>
 +
<li>Induction was performed by adding 0,1% Rhamnose</li>
 +
<li>To document the growth curve and the consumption of substrate daily samples were taken for OD<sub>600</sub> measurement and HPLC analytics
 +
 
         </div>
         </div>
       </div>
       </div>

Latest revision as of 19:46, 15 October 2014


September

Week 1     09/01 - 09/07
Week 1     09/01 - 09/07


  • GSU 3274
    • This week we assembled GSU 3274 with different promotors

  • Electrobiochemical reactor system
    • Additionally we started to cultivate in the H-cell reactor to evaluate the growth of E. coli under the current intesity which is according to the literature needed to reduce neutral red and bromphenol blue.


Week 2     09/08 - 09/14
Week 2     09/08 - 09/14
Week 3     09/15 - 09/21
Week 3     09/15 - 09/21

  • Electrobiochemical reactor system
    • This week we performed several cyclic voltammetric measurements to characterize different electrode materials and mediators. Therefore the system was purged with nitrogen to remove any oxygen.
    • Measurements were performed under the following conditions and the the following experimental setup
      • Cathode space: cathode buffer or M9 medium
      • Anode space: anode buffer
      • Working electrode: platinic plate or graphite electrode
      • Counter electrode: platinic wire
      • Reference electrode: Ag/AgCl- electrode
      • Scan rate: 10 mV/s
      • Scan Limit: was varied among the different measurements
      • Cycle number: 3, 10 or 500
      • Mediators: neutral red or bromphenol blue
      • Temperature: 37°C
Week 4     09/22 - 09/28
Week 4     09/22 - 09/28

Week 5     09/29 - 10/05
Week 5 09/29 - 10/05
  • Deletion of dcuB and integration of oprF into chromosome

  • FumBCD

    • anaerobic cultivation for characterization of pSB1C3_T7_frd
      • We tested all combinations of the following media and strains for the characterization of pSB1C3_T7_frd
        • Strains
          • KRX wildtype B0015
          • KRX wildtype with pSB1C3_T7_frd
          • JW0506-1
          • JW0506-1 with pSB1C3_T7_frd
        • Media
          • M9 with xylose (50 mM)
          • M9 with xylose (50 mM) and fumarate (50 mM)
          • M9 with fumarate (50 mM)
        • Induction was performed by adding 0,1% Rhamnose
        • To document the growth curve and the consumption of substrate daily samples were taken for OD600 measurement and HPLC analytics

    • anaerobic cultivation for characterization of the antiporter δdcuB
      • We tested all combinations of the following media and strains for the characterization of pSB1C3_T7_frd
        • Strains
          • KRX wildtype B0015
          • KRX wildtype with pSB1C3_T7_frd
          • KRX ΔdcuB::oprF
          • KRX ΔdcuB::oprF with pSB1C3_T7_frd
          • JW4084-1
          • JW4084-1 with pSB1C3_T7_frd
          • JW0506-1
          • JW0506-1 with pSB1C3_T7_frd
        • Media
          • M9 with xylose (50 mM)
          • M9 with xylose (50 mM) and fumarate (50 mM)
          • M9 with fumarate (50 mM)
        • Induction was performed by adding 0,1% Rhamnose
        • To document the growth curve and the consumption of substrate daily samples were taken for OD600 measurement and HPLC analytics

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