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

From 2014.igem.org

(Difference between revisions)
 
(35 intermediate revisions not shown)
Line 50: Line 50:
             <div class="content" style="margin-right:10%; margin-left:10%">
             <div class="content" style="margin-right:10%; margin-left:10%">
<ul>
<ul>
-
<li><b><i>pR6K</i></b></li>
+
<li><b>Deletion of <i>dcuB</i> and integration of <i>oprF</i> into chromosome</b></li>
 +
<ul><li>This week we amplified and purified the <i>pR6K</i>-cassette again in order to connect it to <i>oprF</i> 
 +
<ul>
 +
<li><i>pR6K</i></li>
<ul>
<ul>
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> of <i>pR6K</i>-cassette</li>
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> of <i>pR6K</i>-cassette</li>
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of <i>pR6K</i>-cassette and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">Purification</a> out of the gel</li> </li>
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of <i>pR6K</i>-cassette and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">Purification</a> out of the gel</li> </li>
</ul>
</ul>
-
<li><b>Connection of the <i>pR6K</i>-cassette and <i>oprF</i></b></li>
+
<div class="element" style="height:350px; width:120px; text-align:center">
 +
                      <a href="https://static.igem.org/mediawiki/2014/3/33/Bielefeld_CeBiTec_2014-09-24_csoS1-4_cPCR_08_22.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/3/37/Bielefeld-CeBiTec_2014-10-14_PR6K_PCR_08_19.png" height="230px"></a><br><font size="1">Agarose gel from colony PCR. As a Ladder we used <a href="http://www.thermoscientificbio.com/nucleic-acid-electrophoresis/generuler-1-kb-dna-ladder-ready-to-use-250-to-10000-bp">GeneRuler™ 1 kb DNA Ladder from Thermo Scientific</a>. </font>
 +
                    </div>
 +
 
 +
</ul>
 +
<br>
 +
 
 +
<li>Connection of the <i>pR6K</i>-cassette and <i>oprF</i></li>
<ul>
<ul>
Line 64: Line 74:
<li>PCR product was <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">purified</a> out of the gel</li>
<li>PCR product was <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">purified</a> out of the gel</li>
</ul>
</ul>
-
             
 
-
</ul>
 
-
</ul>
 
<ul>
<ul>
 +
  <div class="element" style="height:350px; width:120px; text-align:center">
 +
                      <a href="https://static.igem.org/mediawiki/2014/3/33/Bielefeld_CeBiTec_2014-09-24_csoS1-4_cPCR_08_22.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/b/b3/Bielefeld-CeBiTec_2014-10-14_Knockout_deletion-cassette_PCR_08_09.png" height="230px"></a><br><font size="1">Agarose gel from colony PCR. As a Ladder we used <a href="http://www.thermoscientificbio.com/nucleic-acid-electrophoresis/generuler-1-kb-dna-ladder-ready-to-use-250-to-10000-bp">GeneRuler™ 1 kb DNA Ladder from Thermo Scientific</a>. </font>
 +
                    </div>
 +
           
 +
 +
</ul>
 +
</ul>
 +
</ul>
 +
</ul>
 +
 +
<br>
 +
<ul>
 +
<li><b><i>Fum A</i></b></li>
<li><b><i>Fum A</i></b></li>
<ul>
<ul>
-
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of <i>Fum A</i> </li>
+
<li>This week we assembled <i>FumA</i> it with the T7-Promotor as well as with several Anderson-Promotor using Biobrick-Assembly</li>
-
</ul>
+
<ul>
<ul>
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a> (Suffix)</li>
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a> (Suffix)</li>
<ul>
<ul>
-
<li>Backbone (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" 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>
+
<li>Backbone (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" 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>
<ul>
<li>pSB1A2_T7</li>
<li>pSB1A2_T7</li>
Line 86: Line 105:
</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#RestrictionEnzymes" 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#RestrictionEnzymes" 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>Fum A</i></li>
 +
</ul>
 +
</ul>
 +
</ul>
 +
</ul>
 +
</ul>
 +
<br>
 +
<ul>
 +
<li><b>Cultivation of <i>E.coli</i> with neutral red and bromphenol blue</b></li>
 +
<ul><li>This week we tested if neutral red and bromphenol blue have an influence on the growth of <i>E. coli</i>. Therefore we cultivated <i>E.coli</i> wildtype in <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Media#M9medium" target="_blank">M9 medium</a> with glucose supplemented with 100 µM of neutral red or bromphenol blue. Samples were taken for OD<sub>600</sub> measurement to document the growth curve.</li>
 +
</ul>
 +
</ul>
         </div>
         </div>
       </div>
       </div>
Line 104: Line 144:
             </div>
             </div>
             <div class="content" style="margin-right:10%; margin-left:10%">
             <div class="content" style="margin-right:10%; margin-left:10%">
-
        </div>
+
<ul>
 +
<li><b>Deletion of <i>dcuB</i> and integration of <i>oprF</i> into chromosome</b></li>
 +
<ul>
 +
<li>This week we checked our first colonies to verify the deletion of <i>dcuB</i></li>
 +
<ul>
 +
<li><i>pR6K</i></li>
 +
<ul>
 +
<li> The <i>pR6K</i>-cassette for the <i>dcuB</i> deletion was <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">purified</a> out of the gel</li>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> of <i>pR6K</i>-cassette for the deletion of <i>dcuB</i> with electrocompotetent cells</li>
 +
</ul>
 +
</ul>
 +
</ul>
 +
<ul>
 +
<li>pRedET</i>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> of RedET plasmid with electrocompotetent cells</li>
 +
</ul>
 +
</ul>
 +
<ul>
 +
<li><i>dcuB</i></li>
 +
 
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> to verify the deletion of <i>dcuB</i> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#dcuB_del_kon1" target="_blank">dcuB_del_kon1</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#dcuB_del_kon2" target="_blank">dcuB_del_kon2</a>)
 +
            </li>
 +
<ul>
 +
<li>Annealing temperature: 55 °C</li>
 +
<li>Bands not as expected (~2390 bp)</li>
 +
</ul>
 +
</ul>
 +
</ul>
 +
</ul>
 +
</ul>
 +
<br>
 +
<ul>
 +
<li><b><i>Fum A</i></b></li>
 +
<ul>
 +
<li> This week we assembled <i>FumA</i> with different promotor</li>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> of
 +
<ul>
 +
<li><a href="http://parts.igem.org/Part:BBa_J23101" target="_blank">BBa_J23101</a>_<i>FumA</i></li>
 +
<li><a href="http://parts.igem.org/Part:BBa_J23102" target="_blank">BBa_J23102</a>_ <i>Fum A </i></li>
 +
<li><a href="http://parts.igem.org/Part:BBa_J23104" target="_blank">BBa_J23104</a>_<i>FumA</i></li>
 +
<li>pSB1A2_T7 with electrocompotetent cells</li>
 +
</ul>
 +
</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>Band as expected (~2200 bp)</li>
 +
</ul>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of <a href="http://parts.igem.org/Part:BBa_J23102" target="_blank">BBa_J23102</a>_ <i>Fum A </i> </li>
 +
</ul>
 +
<ul>
 +
<li>Sequencing of
 +
<ul>
 +
<li><a href="http://parts.igem.org/Part:BBa_J23101" target="_blank">BBa_J23101</a>_<i>FumA</i></li>
 +
<li><a href="http://parts.igem.org/Part:BBa_J23102" target="_blank">BBa_J23102</a>_<i>FumA</i></li>
 +
<li><a href="http://parts.igem.org/Part:BBa_J23104" target="_blank">BBa_J23104</a>_<i>FumA</i></li>
 +
<li>pSB1A2_T7_<i>FumA</i> showed different mutations
 +
</ul>
 +
</ul>
 +
</ul>
 +
<br>
 +
<ul>
 +
<li><b><i>Fum BCD</i></b></li>
 +
<ul>
 +
<li>This week wie amplified <i>FumBCD</i> and its backbone </li>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> of <i>Fum BCD</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_pre_Fum_BCD</a>)</li>
 +
<ul>
 +
<li>Annealing temperature: 55 &deg;C</li>
 +
<li>Bands as expected (2070 bp)</li>
 +
</ul>
 +
</ul>
 +
<ul>
 +
<li> The <i>Fum BCD</i> backbone was <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>Fum BCD</i> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Fum_BCD_fwd" target="_blank">Fum_BCD_fwd </a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Fum_BCD_rev " target="_blank">Fum_BCD_rev</a>)</li>
 +
<ul>
 +
<li>Annealing temperature: 55 &deg;C</li>
 +
<li>Bands not as expected (1579 bp)</li>
 +
</ul>
 +
</ul>
 +
</ul>
 +
</ul>
 +
<br>
 +
<ul>
 +
<li><b><i>ccm</i></b></li>
 +
<ul>
 +
<li>This week we amplified the <i>ccm</i>-cassette and its backbone</li>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> of the ccm-backbone (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#pSB1C3_suf_ccm" target="_blank">pSB1C3_suf_ccm</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#pSB1C3_pre_ccm" target="_blank">pSB1C3_pre_ccm</a>)</li>
 +
<ul>
 +
<li>Annealing temperature: 65 &deg;C</li>
 +
<li>Bands as expected (~2070 bp) but slightly visible because of inappropriate annealing temperature</li>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> was repeated with an annealing temperature of 55 &deg;C </li>
 +
<li>Both 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>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> of <i>ccm</i>-cassette using a gradient (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#ccm_fwd" target="_blank">ccm_fwd</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#ccm_rev" target="_blank">ccm_rev</a>)</li>
 +
<ul>
 +
<li>Annealing temperature: 61 &deg;C appeared as the optimum</li>
 +
<li>Bands as expected (~6281 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>-cassette (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#ccm_fwd" target="_blank">ccm_fwd</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#ccm_rev" target="_blank">ccm_rev</a>)</li>
 +
<ul>
 +
<li>Annealing temperature: 61 &deg;C</li>
 +
<li>Bands as expected (~6281 bp)</li>
 +
<li>PCR product was <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">purified</a></li>
 +
 
 +
</ul>
 +
</ul>
 +
 
 +
<div class="element" style="height:350px; width:120px; text-align:center">
 +
                      <a href="https://static.igem.org/mediawiki/2014/3/33/Bielefeld_CeBiTec_2014-09-24_csoS1-4_cPCR_08_22.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/f/f8/Bielefeld-CeBiTec_2014-10-14_Ccm_cassette_PCR_08_16.png" height="230px"></a><br><font size="1">Agarose gel from colony PCR. As a Ladder we used <a href="http://www.thermoscientificbio.com/nucleic-acid-electrophoresis/generuler-1-kb-dna-ladder-ready-to-use-250-to-10000-bp">GeneRuler™ 1 kb DNA Ladder from Thermo Scientific</a>. </font>
 +
                    </div>
 +
</ul>
 +
</ul>
 +
<br>
 +
<ul>
 +
<li><b><i>GSU 3274</i></b></li>
 +
<ul>
 +
<li> This week we amplified the <i>GSU 3274</i> cytochrome and its backbone</li>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification </a> of <i>GSU 3274</i>-backbone (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#pSB1C3_pre_pccH" target="_blank">pSB1C3_pre_pccH</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#pSB1C3_suf_pccH" target="_blank">pSB1C3_suf_pccH</a>)</li>
 +
<ul>
 +
<li>Annealing temperature: 65 &deg;C</li>
 +
<li>Bands not as expected (~2070 bp) because of inappropriate annealing temperature</li>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> was repeated with an annealing temperature of 55 &deg;C </li>
 +
<ul>
 +
<li>Bands as expected (~2070 bp)</li>
 +
</ul>
 +
</ul>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> of <i>GSU 3274</i> using a gradient (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#pccH_fwd" target="_blank">pccH_fwd</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#pccH_rev" target="_blank">pccH_rev</a>)</li>
 +
<ul>
 +
<li>Annealing temperature: 55 &deg;C appeared as the optimum</li>
 +
<li>Bands as expected (~453 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>GSU 3274 </i> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#pccH_fwd" target="_blank">pccH_fwd</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#pccH_rev" target="_blank">pccH_rev</a>)</li>
 +
<ul>
 +
<li>Annealing temperature: 55 &deg;C</li>
 +
<li>Bands as expected (~453 bp)</li>
 +
<li> genomic DNA of <i>G. sulfurreducens</i> as well as the PCR product of the gradient-PCR was used as template</li>
 +
<li>PCR product was <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">purified</a></li>
 +
 
 +
</ul>
 +
</ul>
 +
</ul>
 +
<div class="element" style="height:350px; width:120px; text-align:center">
 +
                      <a href="https://static.igem.org/mediawiki/2014/3/33/Bielefeld_CeBiTec_2014-09-24_csoS1-4_cPCR_08_22.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/3/3d/Bielefeld-CeBiTec_2014-10-14_PccH_PCR_08_17.png" height="230px"></a><br><font size="1">Agarose gel from colony PCR. As a Ladder we used <a href="http://www.thermoscientificbio.com/nucleic-acid-electrophoresis/generuler-1-kb-dna-ladder-ready-to-use-250-to-10000-bp">GeneRuler™ 1 kb DNA Ladder from Thermo Scientific</a>. </font>
 +
                    </div>
 +
 
 +
        </div>
       </div>
       </div>
     </div>
     </div>
Line 120: Line 326:
             </div>
             </div>
             <div class="content" style="margin-right:10%; margin-left:10%">
             <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> This week we contninued to test colonies for the verification of the deletion if <i>dcuB</i>
 +
<ul>
 +
<li>Connection of <i>pR6K</i>-cassette and <i>oprF</i>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> to connect the <i>pR6K</i>-cassette and <i>oprF</i>
 +
      <ul>
 +
<li>Bands as expected (~3300 bp)</li>
 +
</ul>
 +
<li>PCR product was <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#ColonyPCR" target="_blank">Colony PCR</a> to verify the deletion of <i>dcuB</i> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#dcuB_del_kon1" target="_blank">dcuB_del_kon1</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#dcuB_del_kon2" target="_blank">dcuB_del_kon2</a>)
 +
            </li>
 +
<ul>
 +
<li>Annealing temperature: 55 °C</li>
 +
<li>Bands not as expected (~4046 bp)</li>
 +
</ul>
 +
</ul>
 +
</ul>
 +
</ul>
 +
</ul>
 +
<br>
 +
<ul>
 +
<li><b><i>Fum A</i></b></li>
 +
<ul><li> This week wie tested some colonies for <i>FumA</i></li>
 +
<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 (1843 bp)</li>
 +
</ul>
 +
</ul>
 +
</ul>
 +
</ul>
 +
<br>
 +
<ul>
 +
<li><b><i>frd (E. coli)</i></b></li>
 +
<ul>
 +
<li> This week we amplified and transformed <i>frd (E. coli)</i></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#T7_frd_Ec_fwd" target="_blank">T7_frd_Ec_fwd</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#T7_frd_Ec_rev" target="_blank">T7_frd_Ec_rev</a>)</li>
 +
<ul>
 +
<li>Annealing temperature: 55 &deg;C</li>
 +
<li>Bands as expected (~3300 bp)</li>
 +
<li>Additionally there were undefined bands at ~1200 bp </li>
 +
<li> illiegal restriction sites were not removed yet </li>
 +
</ul>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with <i>frd (E. coli)</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 of pSB1C3_<i>frd</i></li>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> of pSB1C3_<i>frd</i> (<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 (~3600 bp)</li>
 +
</ul>
 +
</ul>
 +
</ul>
 +
</ul>
 +
<br>
 +
<ul>
 +
<li><b><i>ccm</i></b></li>
 +
<ul>
 +
<li> This week we transformed the <i>ccm</i>-cassette and tested colonies for it</li>
 +
<ul>
 +
<li>DpnI-digest of <i>ccm</i>-backbone</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> with electrocompotetent cells of pSB1C3_<i>ccm</i></li>
 +
</ul>
 +
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> of pSB1C3_<i>ccm</i> (<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 (~6611 bp) probably because it was not completely amplified because of its length</li>
 +
<li>To check whether one of the colonies is positiv a <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">restriction digestion</a> was done</li>
 +
</ul>
 +
</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 (~2070 bp and 6281 bp)</li>
 +
</ul>
 +
</ul>
 +
</ul>
 +
<br>
 +
<ul>
 +
<li><b><i>GSU 3274</i></b></li>
 +
<ul>
 +
<li>This week we transformed the <i>GSU 3274</i> and tested colonies for it</li>
 +
<ul>
 +
<li>DpnI-digest of <i>GSU 3274</i>-backbone</li>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with <i>GSU 3274</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 of pSB1C3_<i>GSU 3274</i></li>
 +
</ul>
 +
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> of pSB1C3_<i>GSU 3274</i> (<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 (~783 bp)</li>
 +
</ul>
 +
</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 (~2070 bp and 453 bp)</li>
 +
</ul>
 +
</ul>
 +
</ul>
 +
 +
 +
 +
         </div>
         </div>
       </div>
       </div>
Line 136: Line 475:
             </div>
             </div>
             <div class="content" style="margin-right:10%; margin-left:10%">
             <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>This week we tested the next colonies for the deletion of <i>dcuB</i> and performed the NPN-assay to verify the integration of <i>oprF</i> into chromosome
 +
<ul>
 +
<li><i>oprF</i></li>
 +
<ul>
 +
<li>NPN-uptake assay for porine verification</li>
 +
</ul>
 +
</ul>
 +
<ul>
 +
<li><i>dcuB</i></li>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> to verify the deletion of dcuB (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#dcuB_del_kon1" target="_blank">dcuB_del_kon1</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#dcuB_del_kon2" target="_blank">dcuB_del_kon2</a>)
 +
            </li>
 +
<ul>
 +
<li>Annealing temperature: 55°C</li>
 +
<li>Bands as expected (~4046 bp)</li>
 +
<li> another <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> to verify the positive colony
 +
</ul>
 +
</ul>
 +
</ul>
 +
</ul>
 +
</ul>
 +
<br>
 +
<ul>
 +
<li><b><i>FumA</i></b></li>
 +
<ul>
 +
<li>This week we assembled <i>FumA</i> with different promotor</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><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>
 +
<li>pSB1A2_T7
 +
</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 contrsucts with electrocompotetent cells</li>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> with all contructs (<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 <a href="http://parts.igem.org/Part:BBa_J23101" target="_blank"> BBa_J23101</a>_<i>FumA</i> and pSB1A2_T7_<i>FumA</i>
 +
</ul>
 +
</ul>
 +
</ul>
 +
</ul>
 +
<br>
 +
<ul>
 +
<li><b><i>frd (E. coli)</i></b></li>
 +
<ul>
 +
<li> This week we transformed <i>frd</i> and checked several colonies for it
 +
<ul>
 +
<li> DpnI digest of Gibson-Assembly of <i>frd (E. coli)</i> to remove the template</li>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> of Gibson-Assembly with electrocompotetent cells</li>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR </a> of pSB1C3_<i>frd</i> (<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) but there were several other bands too </li>
 +
<li>Therefore another <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> with electrocompotetent cells was done</li>
 +
</ul>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR </a> of pSB1C3_<i>frd</i> (<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 (~ 3600 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>frd</i> backbone (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#pSB1C3_frd_pre" target="_blank">pSB1C3_frd_pre</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#pSB1C3_frd_suf" target="_blank">pSB1C3_frd_suf</a>)</li>
 +
<ul>
 +
<li>Annealing temperature: 55 &deg;C</li>
 +
<li>Bands as expected (~ 2070 bp)</li>
 +
</ul>
 +
</ul>
 +
<ul>
 +
<li><i>frd</i> backbone was <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>frd</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>frd</i> with chemocompetent cells</li>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> of pSB1C3_<i>frd</i> (<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> 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 3300 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>frd</i></li>
 +
</ul>
 +
<ul>
 +
<li>sequencing of pSB1C3_<i>frd</i> confirmed the correct construct</li>
 +
</ul>
 +
<ul>
 +
<ul>
 +
<li>next the illegal restriction sites had to be removed</li>
 +
</ul>
 +
</ul>
 +
</ul>
 +
</ul>
 +
<br>
 +
<ul><li><b><i>ccm</i></b></li>
 +
<ul>
 +
<li> This week we performed Eckhardt gels as a new method besides <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> to identify positiv colonies
 +
<ul>
 +
<li>Eckhardt gel of <i>ccm</i> was performed two times</li>
 +
<ul>
 +
<li> Bands not impeccable to identify (~6230 bp) and therefore a <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">restriction digestion</a> was done</li>
 +
<ul><li>Bands not as expected (~2070 bp and ~6239 bp) </li>
 +
</ul>
 +
</ul>
 +
</ul>
 +
</ul>
 +
</ul>
 +
<br>
 +
<ul><li><b><i>GSU 3274</i></b></li>
 +
<ul>
 +
<li> This week we tested colonies for <i>GSU 3274</i>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> of <i>GSU 3274</i> (<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 (~783 bp)</li>
 +
</ul>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of <i>GSU 3274</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>Pst</i>I</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>EcoR</i>I</a> </li>
 +
<ul>
 +
<li>Bands as expected (~20170 bp and 453 bp)</li>
 +
</ul>
 +
</ul>
 +
 +
 +
 +
 +
         </div>
         </div>
       </div>
       </div>

Latest revision as of 19:52, 16 October 2014


August

Week 1     08/04 - 08/10
Week 1     08/04 - 08/10


  • Cultivation of E.coli with neutral red and bromphenol blue
    • This week we tested if neutral red and bromphenol blue have an influence on the growth of E. coli. Therefore we cultivated E.coli wildtype in M9 medium with glucose supplemented with 100 µM of neutral red or bromphenol blue. Samples were taken for OD600 measurement to document the growth curve.
Week 2     08/11 - 08/17
Week 2     08/11 - 08/17
  • Deletion of dcuB and integration of oprF into chromosome
    • This week we checked our first colonies to verify the deletion of dcuB
      • pR6K
        • The pR6K-cassette for the dcuB deletion was purified out of the gel
          • Transformation of pR6K-cassette for the deletion of dcuB with electrocompotetent cells
      • pRedET

Week 3     08/18 - 08/24
Week 3     08/18 - 08/24
  • Deletion of dcuB and integration of oprF into chromosome
    • This week we contninued to test colonies for the verification of the deletion if dcuB

  • Fum A


Week 4     08/25 - 08/31
Week 4     08/25 - 08/31
  • Deletion of dcuB and integration of oprF into chromosome
    • This week we tested the next colonies for the deletion of dcuB and performed the NPN-assay to verify the integration of oprF into chromosome
      • oprF
        • NPN-uptake assay for porine verification



  • ccm
    • This week we performed Eckhardt gels as a new method besides Colony PCR to identify positiv colonies
      • Eckhardt gel of ccm was performed two times
        • Bands not impeccable to identify (~6230 bp) and therefore a restriction digestion was done
          • Bands not as expected (~2070 bp and ~6239 bp)

Retrieved from "http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Journal/rMFC/Aug"