Difference between revisions of "Team:Dundee/labjournal manu"

 
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         <p class="journal-content">This week was all about getting started with the newly arrived biobrick <a href="http://parts.igem.org/Part:BBa_K1058008">BBa_K1058008.</a></p>
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         <p class="journal-content">This week was all about getting started with the newly arrived biobrick <a href="http://parts.igem.org/Part:BBa_K1058008" target="_blank">BBa_K1058008</a></p>
 
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             <span class="box-title">Day 1 - 22/06: Plated <a href="http://parts.igem.org/Part:BBa_K1058008" target="_blank">BBa_K1058008</a> from agar stab.</span> <!--Title of collapsible box-->
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             <span class="box-title">22/06: Plated <a href="http://parts.igem.org/Part:BBa_K1058008" target="_blank">BBa_K1058008</a> from agar stab.</span> <!--Title of collapsible box-->
 
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             <span class="box-title">Day 2 - 23/06: Purified <a href="http://parts.igem.org/Part:BBa_K1058008" target="_blank">BBa_K1058008</a> from liquid culture.</span> <!--Title of collapsible box-->
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             <span class="box-title">23/06: Purified <a href="http://parts.igem.org/Part:BBa_K1058008" target="_blank">BBa_K1058008</a> from liquid culture.</span> <!--Title of collapsible box-->
 
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                   <p><b>Results Sequencing: </b><a data-toggle="modal" data-target="#BBa_K1058008-figure1-modal" class="journal-protocol"> Figure 1 </a></p>
 
                   <p><b>Results Sequencing: </b><a data-toggle="modal" data-target="#BBa_K1058008-figure1-modal" class="journal-protocol"> Figure 1 </a></p>
  
                   <p><b>Next Steps:</b> PCR of promoter region (<i>pChr</i>) and repressor region (<i>ChrB</i>) of <a href="http://parts.igem.org/Part:BBa_K1058008">BBa_K1058008</a> for storage in individual biobricks.</p>
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                   <p><b>Next Steps:</b> PCR of promoter region (P<i>chr</i>) and repressor region (<i>chrB</i>) of <a href="http://parts.igem.org/Part:BBa_K1058008" target="_blank">BBa_K1058008</a> for storage in individual biobricks.</p>
 
                  
 
                  
  
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         <div class="labtitle">Week Beginning 29/06</div>
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         <div class="labtitle">Week Beginning 29/06/2015</div>
 
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         <p class="journal-content"> During this week, <a href="http://parts.igem.org/Part:BBa_K1058008" target="_blank">BBa_K1058008</a> was disassembled into its constituent parts, <i>pChr</i>, and <i>ChrB</i>.</p>
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         <p class="journal-content"> During this week, <a href="http://parts.igem.org/Part:BBa_K1058008" target="_blank">BBa_K1058008</a> was disassembled into its constituent parts, P<i>chr</i>, and <i>chrB</i>.</p>
 
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             <span class="box-title">Day 1 - 29/06: PCR-amplification of <i>pChr</i> and <i>ChrB</i> from <a href="http://parts.igem.org/Part:BBa_K1058008" target="_blank">BBa_K1058008</a>.</span> <!--Title of collapsible box-->
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             <span class="box-title">29/06: PCR-amplification of <i>pChr</i> and <i>ChrB</i> from <a href="http://parts.igem.org/Part:BBa_K1058008" target="_blank">BBa_K1058008</a>.</span> <!--Title of collapsible box-->
 
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                   <p><b>Next Steps:</b> Repeat of PCR of <i>ChrB</i>. Purification of the Plasmid, containing <a href="http://parts.igem.org/Part:BBa_K1058008">BBa_K1058008</a> and confirmation of size and sequence.</p>
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                   <p><b>Next Steps:</b> Repeat of PCR of <i>ChrB</i>. Purification of the Plasmid, containing <a href="http://parts.igem.org/Part:BBa_K1058008" target="_blank">BBa_K1058008</a> and confirmation of size and sequence.</p>
 
                    
 
                    
 
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         <div class="labtitle">Week Beginning 06/07</div>
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         <div class="labtitle">Week Beginning 06/07/2015</div>
 
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             <span class="box-title">Day 1 - 06/07: Repeated amplification of <i>ChrB</i>. Ligation and transformation of <i>pChr</i> and <i>ChrB</i>.</span> <!--Title of collapsible box-->
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             <span class="box-title">06/07: Repeated amplification of <i>ChrB</i>. Ligation and transformation of <i>pChr</i> and <i>ChrB</i>.</span> <!--Title of collapsible box-->
 
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                   <p><b>Aim of experiment:</b> To repeat PCR of ChrB for subsequent ligation into pSB1C3. Ligation of <i>pChr</i> and <i>ChrB</i> into pSB1C3. Transformation of each into a non-pathogenic lab strain of <i>Escherichia coli</i>.
 
                   <p><b>Aim of experiment:</b> To repeat PCR of ChrB for subsequent ligation into pSB1C3. Ligation of <i>pChr</i> and <i>ChrB</i> into pSB1C3. Transformation of each into a non-pathogenic lab strain of <i>Escherichia coli</i>.
                   Overnight culture of <i>E. coli DH5-alpha</i>, containing GFPmut2, as a fluorescent reporter for our chromate sensing system</p>
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                   Overnight culture of <i>E. coli</i> DH5-alpha, containing GFPmut2, as a fluorescent reporter for our chromate sensing system</p>
  
 
                   <p><b>Protocols Used:</b>
 
                   <p><b>Protocols Used:</b>
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                   <a data-toggle="modal" data-target="#dundee15-chr-igem023-modal" class="journal-protocol"> Figure 3 </a></p>  
 
                   <a data-toggle="modal" data-target="#dundee15-chr-igem023-modal" class="journal-protocol"> Figure 3 </a></p>  
 
                   <p>2 bands were extracted from the gel for further characterisation, the strongest band, hence called <i>ChrBs</i>, and the band directly above, hence called <i>ChrBw</i>. <i>pChr</i>, <i>ChrBs</i>, and <i>ChrBw</i> were ligated into pSB1C3.
 
                   <p>2 bands were extracted from the gel for further characterisation, the strongest band, hence called <i>ChrBs</i>, and the band directly above, hence called <i>ChrBw</i>. <i>pChr</i>, <i>ChrBs</i>, and <i>ChrBw</i> were ligated into pSB1C3.
                   pSB1C3-pChr, pSB1C3-ChrBs, and pSB1C3-ChrBw were transformed into <i>E. coli MC1061.</i></p>
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                   pSB1C3-pChr, pSB1C3-ChrBs, and pSB1C3-ChrBw were transformed into <i>E. coli</i> MC1061.</p>
  
  
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             <span class="box-title">Day 2 - 07/07: Purification of <i>GFP</i> and overnight cultures of pSB1C3-pChr, pSB1C3-ChrBs, and pSB1C3-ChrBw</span> <!--Title of collapsible box-->
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             <span class="box-title">07/07: Purification of <i>gfp</i> and overnight cultures of pSB1C3-P<i>chr</i>, pSB1C3-<i>chrB</i> (s), and pSB1C3-<i>chrB</i> (w)</span> <!--Title of collapsible box-->
 
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                   <p><b>Aim of experiment:</b> Purification of GFPmut2 from overnight cultures. Overnight cultures of recombinant <i>E. coli MC1061</i>, containing pSB1C3-pChr, pSB1C3-ChrBs, and pSB1C3-ChrBw respectively for further size and sequence confirmation. Overnight culture of pUniprom for subsequent ligation of ChrB</p>
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                   <p><b>Aim of experiment:</b> Purification of GFPmut2 from overnight cultures. Overnight cultures of recombinant <i>E. coli</i> MC1061, containing pSB1C3-P<i>chr</i>, pSB1C3-<i>chrB</i> (s), and pSB1C3-<i>chrB</i> (w) respectively for further size and sequence confirmation. Overnight culture of pUniprom for subsequent ligation of <i>chrB</i></p>
  
 
                   <p><b>Protocols Used:</b>
 
                   <p><b>Protocols Used:</b>
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                   <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15-chr-igem024-modal" class="journal-protocol"> Figure 5 </a> </p>
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                   <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15-chr-igem024-modal" class="journal-protocol"> Figure 4 </a> </p>
  
  
                   <p><b>Next Steps:</b> Repeat of amplification of <i>ChrB</i>. Sequence and size confirmation of pSB1C3-pChr, pSB1C3-ChrBs, and pSB1C3-ChrBw.</p>
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                   <p><b>Next Steps:</b> Repeat of amplification of <i>ChrB</i>. Sequence and size confirmation of pSB1C3-P<i>chr</i>, pSB1C3-<i>chrB</i> (s), and pSB1C3-<i>chrB</i> (w).</p>
 
                    
 
                    
 
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             <span class="box-title">Day 3 - 08/07: Purification of plasmids pSB1C3-pChr, pSB1C3-ChrBs, and pSB1C3-ChrBw. Size and sequence confirmation.</span> <!--Title of collapsible box-->
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             <span class="box-title">08/07: Purification of plasmids pSB1C3-P<i>chr</i>, pSB1C3-<i>chrB</i> (s), and pSB1C3-<i>chrB</i> (w). Size and sequence confirmation.</span> <!--Title of collapsible box-->
 
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                     <a data-toggle="modal" data-target="#restriction-digest-modal" class="journal-protocol">Restriction digest, </a>
 
                     <a data-toggle="modal" data-target="#restriction-digest-modal" class="journal-protocol">Restriction digest, </a>
 
                     <a data-toggle="modal" data-target="#sequencing-modal" class="journal-protocol">Sequencing</a>
 
                     <a data-toggle="modal" data-target="#sequencing-modal" class="journal-protocol">Sequencing</a>
                     <p><a data-toggle="modal" data-target="#restriction-digest-modal" class="journal-protocol"> Restriction digest</a> of gel purified PCR-products of pChr, ChrB, and GFPmut2, using EcoRI/SpeI, BamHI/HindIII, and XbaI/PstI respecitvely.</p>
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                     <p><a data-toggle="modal" data-target="#restriction-digest-modal" class="journal-protocol"> Restriction digest</a> of gel purified PCR-products of P<i>chr</i>, <i>chrB</i>, and <i>gfp</i>, using EcoRI/SpeI, BamHI/HindIII, and XbaI/PstI respecitvely.</p>
 
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                   <p><b>Results:</b><p><a data-toggle="modal" data-target="#dundee15-chr-igem026-modal" class="journal-protocol"> Figure 6 </a> - Size confirmation of <i>pChr</i> and <i>ChrB</i>.</p>
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                   <p><b>Results:</b><p><a data-toggle="modal" data-target="#dundee15-chr-igem026-modal" class="journal-protocol"> Figure 5 </a> - Size confirmation of <i>pChr</i> and <i>ChrB</i>.</p>
                   <p><a data-toggle="modal" data-target="#dundee15-chr-pChr1-modal" class="journal-protocol"> Figure 7 </a> - Sequence confirmation of <i>pChr</i></p>
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                   <p><a data-toggle="modal" data-target="#dundee15-chr-pChr1-modal" class="journal-protocol"> Figure 6 </a> - Sequence confirmation of P<i>chr</i></p>
                   <p><a data-toggle="modal" data-target="#dundee15-chr-ChrBw4-modal" class="journal-protocol"> Figure 8 </a> - Sequence confirmation of <i>ChrB</i>.</p>
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                   <p><a data-toggle="modal" data-target="#dundee15-chr-ChrBw4-modal" class="journal-protocol"> Figure 7 </a> - Sequence confirmation of <i>chrB</i>.</p>
 
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                   <p><b>Next Steps:</b> Purification and digest of pUniprom for insertion of <i>ChrB</i>.</p>
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                   <p><b>Next Steps:</b> Purification and digest of pUniprom for insertion of <i>chrB</i>.</p>
 
                    
 
                    
 
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             <span class="box-title">Day 4 - 09/07: Digested and cleaned up pUniprom for insertion of <i>ChrB</i>. Amplification of optimised <i>ChrB</i> with primers specific for the insertion into pUniprom.</span> <!--Title of collapsible box-->
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             <span class="box-title">09/07: Digested and cleaned up pUniprom for insertion of <i>chrB</i>. Amplification of optimised <i>chrB</i> with primers specific for the insertion into pUniprom.</span> <!--Title of collapsible box-->
 
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                   <p><b>Aim of experiment:</b> To prepare pUniprom for insertion of <i>ChrB</i> by digesting it with BamHI and HindIII. To amplify <i>ChrB</i> version with first 15 codons optimised for insertion into pUniprom.</p>
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                   <p><b>Aim of experiment:</b> To prepare pUniprom for insertion of <i>chrB</i> by digesting it with BamHI and HindIII. To amplify <i>chrB</i> version with first 15 codons optimised for insertion into pUniprom.</p>
  
 
                   <p><b>Protocols Used:</b>
 
                   <p><b>Protocols Used:</b>
                     <p><a data-toggle="modal" data-target="#restriction-digest-modal" class="journal-protocol">Restriction digest</a> and
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                     <p><a data-toggle="modal" data-target="#restriction-digest-modal" class="journal-protocol"> Restriction digest</a> and
 
                     <a data-toggle="modal" data-target="#gelextraction-modal" class="journal-protocol">Gel extraction</a> of pUniprom</p>
 
                     <a data-toggle="modal" data-target="#gelextraction-modal" class="journal-protocol">Gel extraction</a> of pUniprom</p>
 
                     <p><a data-toggle="modal" data-target="#PCR-modal" class="journal-protocol">PCR</a> and
 
                     <p><a data-toggle="modal" data-target="#PCR-modal" class="journal-protocol">PCR</a> and
                     <a data-toggle="modal" data-target="#gelextraction-modal" class="journal-protocol"> Gel extraction </a> of optimised <i>ChrB</i></p>
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                     <a data-toggle="modal" data-target="#gelextraction-modal" class="journal-protocol"> Gel extraction </a> of <i>chrB</i> (opt)</p>
 
                   </p>
 
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                   <p><b>Results:</b><p><a data-toggle="modal" data-target="#dundee15-chr-igem034-modal" class="journal-protocol"> Figure 9 </a> Gel with pUniprom</p>
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                   <p><b>Results:</b><p><a data-toggle="modal" data-target="#dundee15-chr-igem034-modal" class="journal-protocol"> Figure 8 </a> Gel with pUniprom</p>
                   <p><a data-toggle="modal" data-target="#dundee15-chr-igem032-modal" class="journal-protocol"> Figure 10 </a>Gel with ChrB</p></p>
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                   <p><a data-toggle="modal" data-target="#dundee15-chr-igem032-modal" class="journal-protocol"> Figure 9 </a>Gel with <i>chrB</i></p></p>
  
  
                   <p><b>Next Steps:</b> Ligation of <i>ChrB</i> and codon optimised <i>ChrB</i> into pUniprom.</p>
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                   <p><b>Next Steps:</b> Ligation of <i>chrB</i> and <i>chrB</i> (opt) into pUniprom.</p>
 
                    
 
                    
 
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             <span class="box-title">Day 5 - 10/07: Ligations of <i>ChrB</i> and optimised <i>ChrB</i> into pUniprom, and of <i>pChr</i> and <i>GFPmut2</i> into pSB1C3.</span> <!--Title of collapsible box-->
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             <span class="box-title">10/07: Ligations of <i>chrB</i> and <i>chrB</i> (opt) into pUniprom, and of P<i>chr</i> and <i>gfp</i> into pSB1C3.</span> <!--Title of collapsible box-->
 
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                   <p><b>Aim of experiment:</b> Ligation of <i>ChrB</i> and optimised <i>ChrB</i> into pUniprom via BamHI and HindIII restriction sites. Ligation of <i>pChr</i> and <i>GFPmut2</i> to each other via SpeI/XbaI-link and into pSB1C3 via EcoRI and PstI restriction sites.</p>
+
                   <p><b>Aim of experiment:</b> Ligation of <i>chrB</i> and optimised <i>chrB</i> into pUniprom via BamHI and HindIII restriction sites. Ligation of P<i>chr</i> and <i>gfp</i> to each other via SpeI/XbaI-link and into pSB1C3 via EcoRI and PstI restriction sites.</p>
  
 
                   <p><b>Protocols Used:</b>
 
                   <p><b>Protocols Used:</b>
                     <a data-toggle="modal" data-target="#ligation-modal" class="journal-protocol">Ligations</a>
+
                     <a data-toggle="modal" data-target="#ligation-modal" class="journal-protocol"> Ligations</a>
 
                   </p>
 
                   </p>
  
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         <div class="labtitle">Week Beginning 13/07</div>
+
         <div class="labtitle">Week Beginning 13/07/2015</div>
 
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             <span class="box-title">Day 1 - 13/07: Transformation of pUniprom-ChrB, pUniprom-ChrBopt, and pSB1C3-pChr-GFP.</span>  
+
             <span class="box-title">13/07: Transformation of pUniprom-<i>chrB</i>, pUniprom-<i>chrB</i> (opt), and pSB1C3-P<i>chr-gfp</i>.</span>  
 
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                   <p><b>Aim of experiment:</b> Transformation of pUniprom-ChrB, pUniprom-ChrBopt, and pSB1C3-pChr-GFP into <i>E. coli JM110</i> chassis. Setting up a backup restriction digest of pSB1C3 with EcoRI and SpeI in case the double ligation pSB1C3-pChr-GFP is unsuccessful.</p>
+
                   <p><b>Aim of experiment:</b> Transformation of pUniprom-<i>chrB</i>, pUniprom-<i>chrB</i> (opt), and pSB1C3-P<i>chr-gfp</i> into <i>E. coli</i> JM110 chassis. Setting up a backup restriction digest of pSB1C3 with EcoRI and SpeI in case the double ligation pSB1C3-P<i>chr-gfp</i> is unsuccessful.</p>
  
 
                   <p><b>Protocols Used:</b>
 
                   <p><b>Protocols Used:</b>
                     <a data-toggle="modal" data-target="#transformation-modal" class="journal-protocol">Transformations, </a>
+
                     <a data-toggle="modal" data-target="#transformation-modal" class="journal-protocol"> Transformations, </a>
 
                     <a data-toggle="modal" data-target="#restriction-digest-modal" class="journal-protocol">Restriction digest</a>
 
                     <a data-toggle="modal" data-target="#restriction-digest-modal" class="journal-protocol">Restriction digest</a>
 
                   </p>
 
                   </p>
  
                   <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15-chr-first-transformation-modal" class="journal-protocol"> Figure 11 </a></p>
+
                   <p><b>Results:</b>
 +
                    <p><a data-toggle="modal" data-target="#dundee15-chr-first-transformation-modal" class="journal-protocol"> Figure 10 </a>Plates after transformation</p>
 +
                    <p><a data-toggle="modal" data-target="#dundee15-chr-igem035-modal" class="journal-protocol"> Figure 11 </a> Gel after digest of pSB1C3</p>
 +
                  </p>
  
 
                   <p><b>Next Steps:</b> Purification of recombinant vectors and confirmation of size and sequence. Store digested pSB1C3 at -20<sup>o</sup>C.</p>
 
                   <p><b>Next Steps:</b> Purification of recombinant vectors and confirmation of size and sequence. Store digested pSB1C3 at -20<sup>o</sup>C.</p>
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             <span class="box-title">Day 2 - 14/07: Set up cultures for subsequent purification of recombinant vectors for size and sequence confirmation.</span>
+
             <span class="box-title">14/07: Set up cultures for subsequent purification of recombinant vectors for size and sequence confirmation.</span>
 
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                   <p><b>Aim of experiment:</b> Purification of recombinant vectors, pUniprom-ChrB, pUniprom-ChrBopt, and pSB1C3-pChr-GFP, for size and sequence confirmation. Patch plating of the same colonies for storage and retrieval once size and sequence have been confirmed.</p>
+
                   <p><b>Aim of experiment:</b> Purification of recombinant vectors, pUniprom-<i>chrB</i>, pUniprom-<i>chrB</i> (opt), and pSB1C3-P<i>chr-gfp</i>, for size and sequence confirmation. Patch plating of the same colonies for storage and retrieval once size and sequence have been confirmed.</p>
  
 
                   <p><b>Protocols Used:</b>
 
                   <p><b>Protocols Used:</b>
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             <span class="box-title">Day 3 - 15/07: Purified pUniprom-ChrB, pUniprom-ChrBopt, and pSB1C3-pChr-GFP from overnight cultures</span> <!--Title of collapsible box-->
+
             <span class="box-title">15/07: Purified pUniprom-<i>chrB</i>, pUniprom-<i>chrB</i> (opt), and pSB1C3-P<i>chr-gfp</i> from overnight cultures</span> <!--Title of collapsible box-->
 
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                   <p><b>Aim of experiment:</b> Purification of pUniprom-ChrB, pUniprom-ChrBopt, and pSB1C3-pChr-GFP from overnight for confirmation of size and sequence. Restriction digest of pUnipromChrB and optimised pUniprom-ChrB optimised with BamHI and HindIII and of pSB1C3-pChr-GFP with EcoRI/PstI for size confirmation. </p>
+
                   <p><b>Aim of experiment:</b> Purification of pUniprom-<i>chrB</i>, pUniprom-<i>chrB</i> (opt), and pSB1C3-P<i>chr-gfp</i> from overnight for confirmation of size and sequence. Restriction digest of pUniprom-<i>chrB</i>, pUniprom-<i>chrB</i> (opt) with BamHI and HindIII and of pSB1C3-P<i>chr-gfp</i> with EcoRI/PstI for size confirmation. </p>
  
 
                   <p><b>Protocols Used:</b>
 
                   <p><b>Protocols Used:</b>
                     <a data-toggle="modal" data-target="#miniprep-modal" class="journal-protocol">Miniprep, </a>
+
                     <a data-toggle="modal" data-target="#miniprep-modal" class="journal-protocol"> Miniprep, </a>
 
                     <a data-toggle="modal" data-target="#restriction-digest-modal" class="journal-protocol">Restriction digest, </a>
 
                     <a data-toggle="modal" data-target="#restriction-digest-modal" class="journal-protocol">Restriction digest, </a>
 
                     <a data-toggle="modal" data-target="#colony-modal" class="journal-protocol">Colony PCR, </a>
 
                     <a data-toggle="modal" data-target="#colony-modal" class="journal-protocol">Colony PCR, </a>
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                   <p><b>Results:</b>
 
                   <p><b>Results:</b>
                     <p><a data-toggle="modal" data-target="#dundee15-chr-igem036-modal" class="journal-protocol"> Figure 12 </a> Size confirmation of <i>ChrB</i> and <i>ChrBopt</i>.</p>
+
                     <p><a data-toggle="modal" data-target="#dundee15-chr-igem036-modal" class="journal-protocol"> Figure 12 </a> Size confirmation of <i>chrB</i> and <i>chrB</i> (opt).</p>
                     <a data-toggle="modal" data-target="#dundee15-chr-igem037-modal" class="journal-protocol"> Figure 13 </a> Gel after colony PCR. Colonies 2, 4, 9, and 12 were selected for sequencing.</p>
+
                     <a data-toggle="modal" data-target="#dundee15-chr-igem037-modal" class="journal-protocol"> Figure 13 </a> Gel after colony PCR of <i>chrB</i> (opt).</p>
                     </p><a data-toggle="modal" data-target="#dundee15_chr_pSB1C3-pChr-GFP_reverse" class="journal-protocol"> Figure 14 </a> Sequencing result of pSB1C3-pChr-GFPmut2_1. The Result for pSB1C3-pChr-GFPmut2_2 was not as expected, albeit in the correct orientation.</p>
+
                     </p><a data-toggle="modal" data-target="#dundee15_chr_pSB1C3-pChr-GFP_reverse" class="journal-protocol"> Figure 14 </a> Sequencing result of pSB1C3-P<i>chr-gfp</i> (colony 1).</p>
 
                   </p>
 
                   </p>
 
                    
 
                    
                   <p><b>Next Steps:</b> Confirmation of sequence of <i>ChrB-opt.</i></p>
+
                   <p><b>Next Steps:</b> Confirmation of sequence of <i>chrB</i> (opt)</p>
  
 
                    
 
                    
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             <span class="box-title">Day 4 - 16/07: Set up overnight cultures of colonies chosen after colony-PCR for subsequent purification and sequence confirmation.</span> <!--Title of collapsible box-->
+
             <span class="box-title">16/07: Set up overnight cultures of <i>chrB</i> (opt) - colonies chosen after colony-PCR for subsequent purification and sequence confirmation.</span> <!--Title of collapsible box-->
 
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                   <p><b>Aim of experiment:</b> Set up overnight cultures of <i>ChrBopt</i> colonies 2, 4, 9, and 12 for sequencing.</p>
+
                   <p><b>Aim of experiment:</b> Set up overnight cultures of <i>chrB</i> (opt) colonies 2, 4, 9, and 12 for sequencing.</p>
  
 
                   <p><b>Protocols Used:</b>
 
                   <p><b>Protocols Used:</b>
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             <span class="box-title">Day 5 - 17/07: Purified and sequenced chosen <i>ChrBopt</i>-colonies.</span> <!--Title of collapsible box-->
+
             <span class="box-title">17/07: Purified and sequenced chosen <i>chrB</i> (opt) - colonies.</span> <!--Title of collapsible box-->
 
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                   </p>
 
                   </p>
  
                   <p><b>Results:</b> <i>ChrBopt</i> showed HindIII restriction sites and was hence cleaved into partial inserts.</p>
+
                   <p><b>Results:</b> <i>chrB</i> (opt) showed HindIII restriction sites and was hence cleaved into partial inserts.</p>
  
                   <p><b>Next Steps:</b> Use alternative cloning protocol for <i>ChrB</i> and <i>ChrBopt</i>.</p>
+
                   <p><b>Next Steps:</b> Use alternative cloning protocol for <i>chrB</i> and <i>chrB</i> (opt).</p>
  
 
                    
 
                    
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         <div class="labtitle">Week Beginning 20/07</div>
+
         <div class="labtitle">Week Beginning 20/07/2015</div>
 
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         <p class="journal-content">During this week an alternative cloning strategy for <i>ChrB</i> and <i>ChrBopt</i> was employed. Furthermore, the pSB1C3-pChr-GFP construct was cloned in a stepwise fashion.</p>
+
         <p class="journal-content">During this week an alternative cloning strategy for <i>chrB</i> and <i>chrB</i> (opt) was employed. Furthermore, the pSB1C3-P<i>chr-gfp</i> - construct was cloned in a stepwise fashion.</p>
 
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             <span class="box-title">Day 1 - 20/07: Went back to the original plate of pSB1C3-pChr-GFP for visualising colonies that do or do not express GFP. </span>
+
             <span class="box-title">20/07: Went back to the original plate of pSB1C3-P<i>chr-gfp</i> for visualising colonies that do or do not express GFP. </span>
 
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                   <p><b>Aim of experiment:</b>To compare colonies on the patch plate of pSB1C3-pChr-GFP by differentially visualising colonies that do or do not express GFP under UV light. The background of this experiment was that if there is one colony that has both inserts in the correct orientation with respect to each other, then there is perhaps one that also has them with the correct orientation with respect to the plasmid. Colonies identified as expressing GFP were patch plated and grown in liquid cultures over night in order to confirm the sequence of the insert.</p>
+
                   <p><b>Aim of experiment:</b> To compare colonies on the patch plate of pSB1C3-P<i>chr-gfp</i> by differentially visualising colonies that do or do not express GFP under UV light. The background of this experiment was that if there is one colony that has both inserts in the correct orientation with respect to each other, then there is perhaps one that also has them with the correct orientation with respect to the plasmid. Colonies identified as expressing GFP were patch plated and grown in liquid cultures over night in order to confirm the sequence of the insert.</p>
  
 
                   <p><b>Protocols Used:</b>
 
                   <p><b>Protocols Used:</b>
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                   <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15_chr_pChrGFPplates_revisited" class="journal-protocol"> Figure 15</a> pSB1C3-pChr-GFP in <i>E. coli JM110</i>.</p>
+
                   <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15_chr_pChrGFPplates_revisited" class="journal-protocol"> Figure 15</a> pSB1C3-P<i>chr-gfp</i> in <i>E. coli</i> JM110.</p>
  
 
                   <p><b>Next Steps:</b> Sequence confirmation of newly selected colonies.</p>
 
                   <p><b>Next Steps:</b> Sequence confirmation of newly selected colonies.</p>
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             <span class="box-title">Day 2 - 21/07: Purified plasmids from new colonies and confirmed sequence. Restriction digest of pUniprom with BamHI and PstI.</span> <!--Title of collapsible box-->
+
             <span class="box-title">21/07: Purified plasmids from new colonies and confirmed sequence. Restriction digest of pUniprom with BamHI and PstI.</span> <!--Title of collapsible box-->
 
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                   <p><b>Aim of experiment:</b>Purification of newly selected colonies, 1, 5, 6, and 7, containing pSB1C3-pChr-GFP for subsequent sequence confirmation. Restriction digest of pUniprom with BamHI and PstI to employ an alternative cloning strategy of <i>ChrB</i> and <i>ChrBopt</i>.</p>
+
                   <p><b>Aim of experiment:</b> Purification of newly selected pSB1C3-P<i>chr-gfp</i> - colonies 1, 5, 6, and 7, for subsequent sequence confirmation. Restriction digest of pUniprom with BamHI and PstI to employ an alternative cloning strategy of <i>chrB</i> and <i>chrB</i> (opt).</p>
  
 
                   <p><b>Protocols Used:</b>
 
                   <p><b>Protocols Used:</b>
 
                     <p><a data-toggle="modal" data-target="#miniprep-modal" class="journal-protocol"> Miniprep </a> and
 
                     <p><a data-toggle="modal" data-target="#miniprep-modal" class="journal-protocol"> Miniprep </a> and
                     <a data-toggle="modal" data-target="#sequencing-modal" class="journal-protocol">Sequencing </a> of pChrGFP insert.</p>
+
                     <a data-toggle="modal" data-target="#sequencing-modal" class="journal-protocol">Sequencing </a> of P<i>chr-gfp</i> - insert.</p>
 
                     <p><a data-toggle="modal" data-target="#restriction-digest-modal" class="journal-protocol">Restriction digest</a> of pUniprom.
 
                     <p><a data-toggle="modal" data-target="#restriction-digest-modal" class="journal-protocol">Restriction digest</a> of pUniprom.
 
                   </p>
 
                   </p>
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                   <p><a data-toggle="modal" data-target="#dundee15_chr_igem039" class="journal-protocol"> Figure 17 </a> Gel after restriction digest of pUniprom.</p>
 
                   <p><a data-toggle="modal" data-target="#dundee15_chr_igem039" class="journal-protocol"> Figure 17 </a> Gel after restriction digest of pUniprom.</p>
  
                   <p><b>Next Steps:</b>Stepwise ligation of <i>pChr</i> and <i>GFP</i> into pSB1C3 in order to clone the inserts in the correct orientation with respect to the plasmid. Insertion of <i>ChrB</i> and <i>ChrBopt</i> into pUniprom via BamHI and PstI links.</p>                   
+
                   <p><b>Next steps:</b> Stepwise ligation of P<i>chr</i> and <i>gfp</i> into pSB1C3 in order to clone the inserts in the correct orientation with respect to the plasmid. Insertion of <i>chrB</i> and <i>chrB</i> (opt) into pUniprom via BamHI and PstI links.</p>                   
 
                 </div>
 
                 </div>
 
           </div>
 
           </div>
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             <span class="box-title">Day 3 - 22/07: Ligated <i>pChr</i> into pSB1C3 via EcoRI and SpeI sites and cloned the recombinant plasmid into <i>E. coli JM110</i>.</span>
+
             <span class="box-title">22/07: Ligated P<i>chr</i> into pSB1C3 via EcoRI and SpeI sites and cloned the recombinant plasmid into <i>E. coli</i> JM110.</span>
 
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                   <p><b>Aim of experiment:</b> Ligation of <i>pChr</i> into pSB1C3 via EcoRI SpeI link. This is the first step of a 2-step cloning strategy with the aim of cloning <i>pChr</i> and <i>GFP</i> into pSB1C3 in the correct orientation with respect to the plasmid.</p>
+
                   <p><b>Aim of experiment:</b> Ligation of P<i>chr</i> into pSB1C3 via EcoRI and SpeI sites. This is the first step of a 2-step cloning strategy with the aim of cloning P<i>chr</i> and <i>gfp</i> into pSB1C3 in the correct orientation with respect to the plasmid.</p>
  
 
                   <p><b>Protocols Used:</b>
 
                   <p><b>Protocols Used:</b>
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                   </p>
 
                   </p>
  
                   <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15_chr_150723_pSB1C3-pChr" class="journal-protocol"> Figure 18 </a> Plates after transformation of pChr into pSB1C3.</p>
+
                   <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15_chr_150723_pSB1C3-pChr" class="journal-protocol"> Figure 18 </a> Plates after transformation of P<i>chr</i> into pSB1C3.</p>
  
                   <p><b>Next Steps:</b> Sequence confirmation of pChr in pSB1C3.</p>
+
                   <p><b>Next Steps:</b> Sequence confirmation of P<i>chr</i> in pSB1C3.</p>
 
                
 
                
 
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                 </div>
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             <span class="box-title">Day 4 - 23/07: Set up overnight cultures of pSB1C3-pChr colonies.</span>
+
             <span class="box-title">23/07: Set up overnight cultures of pSB1C3-P<i>chr</i> colonies.</span>
 
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                   <p><b>Aim of experiment:</b> Set up overnight cultures of pSB1C3-pChr colonies to prepare pSB1C3-pChr for sequence confirmation</p>
+
                   <p><b>Aim of experiment:</b> Set up overnight cultures of pSB1C3-P<i>chr</i> colonies to prepare pSB1C3-P<i>chr</i> for sequence confirmation</p>
  
 
                   <p><b>Protocols Used:</b>
 
                   <p><b>Protocols Used:</b>
                     <a data-toggle="modal" data-target="#overnightculture-modal" class="journal-protocol">Overnight culture, </a>
+
                     <a data-toggle="modal" data-target="#overnightculture-modal" class="journal-protocol"> Overnight culture, </a>
 
                     <a data-toggle="modal" data-target="#plating-modal" class="journal-protocol">Patch-plating</a>
 
                     <a data-toggle="modal" data-target="#plating-modal" class="journal-protocol">Patch-plating</a>
 
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                   </p>
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             <span class="box-title">Day 5 - 24/07: Purified pSB1C3-pChr for sequence confirmation.</span> <!--Title of collapsible box-->
+
             <span class="box-title">24/07: Purified pSB1C3-P<i>chr</i> for sequence confirmation.</span> <!--Title of collapsible box-->
 
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                 <div id="collapsiblew5-5" class="collapse box-content">
  
  
                   <p><b>Aim of experiment:</b> Purification of pSB1C3-pChr for sequence confirmation before ligating <i>GFP</i> into it.</p>
+
                   <p><b>Aim of experiment:</b> Purification of pSB1C3-P<i>chr</i> for sequence confirmation before ligating <i>gfp</i> into it.</p>
  
 
                   <p><b>Protocols Used:</b>
 
                   <p><b>Protocols Used:</b>
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                   </p>
 
                   </p>
  
                   <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15_chr_150727_pSB1C3-pChr_alignment" class="journal-protocol"> Figure 19 </a> Alignment of sequenced pSB1C3-pChr vectors with pChr sequence.</p>
+
                   <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15_chr_150727_pSB1C3-pChr_alignment" class="journal-protocol"> Figure 19 </a> Alignment of sequenced pSB1C3-P<i>chr</i> vectors with P<i>chr</i> sequence.</p>
  
                   <p><b>Next Steps:</b> Digest of pSB1C3-pChr with SpeI/PstI and ligation of GFPmut2.</p>
+
                   <p><b>Next Steps:</b> Digest of pSB1C3-P<i>chr</i> with SpeI/PstI and ligation of P<i>gfp</i></p>
 
                    
 
                    
  
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             <span class="box-title">Day 6 - 25/07: PCR-amplified <i>ChrB</i> and <i>ChrBopt</i>.</span> <!--Title of collapsible box-->
+
             <span class="box-title">25/07: PCR-amplified <i>chrB</i> and <i>chrB</i> (opt).</span> <!--Title of collapsible box-->
 
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                   <p><b>Aim of experiment:</b> Amplification of <i>ChrB</i> and <i>ChrBopt</i> for insertion into pUniprom. Restriction digest of the PCR product with BamHI and PstI for producing compatible sticky ends</p>
+
                   <p><b>Aim of experiment:</b> Amplification of <i>chrB</i> and <i>chrB</i> (opt) for insertion into pUniprom. Restriction digest of the PCR product with BamHI and PstI for producing compatible sticky ends</p>
  
 
                   <p><b>Protocols Used:</b>
 
                   <p><b>Protocols Used:</b>
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                   <p><b>Results:</b> N/A</p>
 
                   <p><b>Results:</b> N/A</p>
  
                   <p><b>Next Steps:</b> Ligation and Transformation of <i>ChrB</i> and <i>ChrBopt</i>.</p>
+
                   <p><b>Next Steps:</b> Ligation and Transformation of <i>chrB</i> and <i>chrB</i> (opt).</p>
  
 
                    
 
                    
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             <span class="box-title">Day 7 - 26/07: Ligated <i>ChrB</i> and <i>ChrBopt</i> into pUniprom.</span> <!--Title of collapsible box-->
+
             <span class="box-title">26/07: Ligated <i>chrB</i> and <i>chrB</i> (opt) into pUniprom.</span> <!--Title of collapsible box-->
 
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                   <p><b>Aim of experiment:</b> Insertion of ChrB and optimised ChrB into pUniprom vector.</p>
+
                   <p><b>Aim of experiment:</b> Insertion of <i>chrB</i> and <i>chrB</i> (opt) into pUniprom.</p>
  
 
                   <p><b>Protocols Used:</b>
 
                   <p><b>Protocols Used:</b>
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         <div class="labtitle">Week Beginning 27/07</div>
+
         <div class="labtitle">Week Beginning 27/07/2015</div>
 
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             <span class="box-title">Day 1 - 27/07: Transformed <i>ChrB</i> and <i>ChrBopt</i> into <i>E. coli JM110</i></span>
+
             <span class="box-title">27/07: Transformed <i>chrB</i> and <i>chrB</i> (opt) into <i>E. coli</i> JM110</span>
 
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                   <p><b>Aim of experiment:</b>Transformation of the repressors, <i>ChrB</i> and <i>ChrBopt</i>, into <i>JM110</i></p>
+
                   <p><b>Aim of experiment:</b> Transformation of the repressors, <i>chrB</i> and <i>chrB</i> (opt), into JM110</p>
  
 
                   <p><b>Protocols Used:</b>
 
                   <p><b>Protocols Used:</b>
                     <a data-toggle="modal" data-target="#transformation-modal" class="journal-protocol">Transformation</a>  
+
                     <a data-toggle="modal" data-target="#transformation-modal" class="journal-protocol"> Transformation</a>  
 
                   </p>
 
                   </p>
  
 
                   <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15_chr_150728_pUniprom-ChrB" class="journal-protocol"> Figure 20</a> Plates after transformation.</p>
 
                   <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15_chr_150728_pUniprom-ChrB" class="journal-protocol"> Figure 20</a> Plates after transformation.</p>
  
                   <p><b>Next Steps:</b> Purification of pUniprom-ChrB and pUniprom-ChrBopt for sequence confirmation.</p>
+
                   <p><b>Next Steps:</b> Purification of pUniprom-<i>chrB</i> and pUniprom-<i>chrB</i> (opt) for sequence confirmation.</p>
  
 
                    
 
                    
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             <span class="box-title">Day 2 - 28/07: Digested pSB1C3-pChr for insertion of <i>GFP</i></span>
+
             <span class="box-title">28/07: Digested pSB1C3-P<i>chr</i> for insertion of <i>gfp</i></span>
 
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                   <p><b>Aim of experiment:</b> Digesting sequence confirmed pSB1C3-pChr with SpeI and PstI for subsequent ligation with <i>GFP</i>. Overnight cultures of <i>ChrB</i> and <i>ChrBopt</i> for subsequent plasmid purification, size confirmation, and sequence confirmation.</p>
+
                   <p><b>Aim of experiment:</b> Digesting sequence confirmed pSB1C3-P<i>chr</i> with SpeI and PstI for subsequent ligation with <i>gfp</i>. Overnight cultures of <i>chrB</i> and <i>chrB</i> (opt) for subsequent plasmid purification, size confirmation, and sequence confirmation.</p>
  
 
                   <p><b>Protocols Used:</b>
 
                   <p><b>Protocols Used:</b>
                     <p><a data-toggle="modal" data-target="#restriction-digest-modal" class="journal-protocol">Restriction digest, </a>
+
                     <p><a data-toggle="modal" data-target="#restriction-digest-modal" class="journal-protocol"> Restriction digest, </a>
                     <a data-toggle="modal" data-target="#ligation-modal" class="journal-protocol">Ligation</a> of <i>GFP</i> into pSB1C3-pChr.</p>
+
                     <a data-toggle="modal" data-target="#ligation-modal" class="journal-protocol">Ligation</a> of <i>gfp</i> into pSB1C3-P<i>chr</i>.</p>
                     <p><a data-toggle="modal" data-target="#overnightculture-modal" class="journal-protocol">Overnight culture</a> of of <i>ChrB</i> and <i>ChrBopt</i>.</p>
+
                     <p><a data-toggle="modal" data-target="#overnightculture-modal" class="journal-protocol">Overnight culture</a> of of <i>chrB</i> and <i>chrB</i> (opt).</p>
 
                   </p>
 
                   </p>
 
                  <p><b>Next Steps:</b> Transformation of recombinant vector for confirmation of size and sequence.</p>
 
  
 
                   <p><b>Results:</b> N/A</p>
 
                   <p><b>Results:</b> N/A</p>
  
                   <p><b>Next Steps:</b> Transformation of pSB1C3-pChr-GFP for size and sequence confirmation. Purification of <i>ChrB</i> and <i>ChrBopt</i> for sequence confirmation.</p>
+
                   <p><b>Next Steps:</b> Transformation of pSB1C3-P<i>chr-gfp</i> for size and sequence confirmation. Purification of <i>chrB</i> and <i>chrB</i> (opt) for sequence confirmation.</p>
  
 
                    
 
                    
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             <span class="box-title">Day 3 - 29/07: Purified <i>ChrB</i> and <i>ChrBopt</i> for size and sequence confirmation.</span>
+
             <span class="box-title">29/07: Purified <i>chrB</i> and <i>chrB</i> (opt) for size and sequence confirmation.</span>
 
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                   <p><b>Aim of experiment:</b> Purification of <i>ChrB</i> and <i>ChrBopt</i> for confirmation of size and sequence.</p>
+
                   <p><b>Aim of experiment:</b> Purification of <i>chrB</i> and <i>chrB</i> (opt) for confirmation of size and sequence.</p>
  
 
                   <p><b>Protocols Used:</b>
 
                   <p><b>Protocols Used:</b>
                     <a data-toggle="modal" data-target="#miniprep-modal" class="journal-protocol">Plasmid purification, </a>
+
                     <a data-toggle="modal" data-target="#miniprep-modal" class="journal-protocol"> Plasmid purification, </a>
 
                     <a data-toggle="modal" data-target="#restriction-digest-modal" class="journal-protocol">Restriction digest, </a>
 
                     <a data-toggle="modal" data-target="#restriction-digest-modal" class="journal-protocol">Restriction digest, </a>
 
                     <a data-toggle="modal" data-target="#sequencing-modal" class="journal-protocol">Sequencing</a>
 
                     <a data-toggle="modal" data-target="#sequencing-modal" class="journal-protocol">Sequencing</a>
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                   </p>
 
                   </p>
  
                   <p><b>Next Steps:</b> Confirmation of expression of <i>ChrB</i> and <i>ChrBopt</i> by western blotting.</p>
+
                   <p><b>Next Steps:</b> Confirmation of expression of <i>chrB</i> and <i>chrB</i> (opt) by western blotting.</p>
  
 
                    
 
                    
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             <span class="box-title">Day 4 - 30/07: Repeated transformation of pSB1C3-pChr-GFP into <i>E.coli JM110</i></span>
+
             <span class="box-title">30/07: Repeated transformation of pSB1C3-P<i>chr-gfp</i> into <i>E.coli JM110</i></span>
 
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                   <p><b>Aim of experiment:</b> Digest of pSB1C3-pChr with SpeI and PstI for insertion of GFP. Repeat of unsuccessful transformation from 28/07.</p>
+
                   <p><b>Aim of experiment:</b> Digest of pSB1C3-P<i>chr</i> with SpeI and PstI for insertion of GFP. Repeat of unsuccessful transformation from 28/07.</p>
  
 
                   <p><b>Protocols Used: </b>
 
                   <p><b>Protocols Used: </b>
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                   <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15_chr_150731_pChrGFP-ligations" class="journal-protocol"> Figure 23 </a>Plates after transformation</p>
 
                   <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15_chr_150731_pChrGFP-ligations" class="journal-protocol"> Figure 23 </a>Plates after transformation</p>
  
                   <p><b>Next Steps:</b> Overnight cultures for purification of the pSB1C3-pChr-GFP for confirmation of insert size and sequence.</p>
+
                   <p><b>Next Steps:</b> Overnight cultures for purification of the pSB1C3-P<i>chr-gfp</i> for confirmation of insert size and sequence.</p>
  
 
                    
 
                    
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             <span class="box-title">Day 5 - 31/07: Set up overnight cultures for confirmation of pSB1C3-pChr-GFP.</span> <!--Title of collapsible box-->
+
             <span class="box-title">31/07: Set up overnight cultures for confirmation of pSB1C3-P<i>chr-gfp</i>.</span> <!--Title of collapsible box-->
 
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                   <p><b>Aim of experiment:</b> Overnight cultures for subsequent plasmid purification of pSB1C3-pChr-GFP.</p>
+
                   <p><b>Aim of experiment:</b> Overnight cultures for subsequent plasmid purification of pSB1C3-P<i>chr-gfp</i>.</p>
  
 
                   <p><b>Protocols Used:</b>
 
                   <p><b>Protocols Used:</b>
                     <a data-toggle="modal" data-target="#overnightculture-modal" class="journal-protocol">Overnight culture</a>
+
                     <a data-toggle="modal" data-target="#overnightculture-modal" class="journal-protocol"> Overnight culture</a>
 
                   </p>
 
                   </p>
  
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         <div class="box">
             <span class="box-title">Day 6 - 01/08: Purified pSB1C3-pChr-GFP for size confirmation and sequencing.</span>
+
             <span class="box-title">01/08: Purified pSB1C3-P<i>chr-gfp</i> for size confirmation and sequencing.</span>
 
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                 <div id="collapsiblew6-6" class="collapse box-content">
  
  
                   <p><b>Aim of experiment:</b> Purification of pSB1C3-pChr-GFP for confirmation of insert size and sequence.</p>
+
                   <p><b>Aim of experiment:</b> Purification of pSB1C3-P<i>chr-gfp</i> for confirmation of insert size and sequence.</p>
  
 
                   <p><b>Protocols Used:</b>
 
                   <p><b>Protocols Used:</b>
                     <a data-toggle="modal" data-target="#miniprep-modal" class="journal-protocol">Plasmid purification, </a>
+
                     <a data-toggle="modal" data-target="#miniprep-modal" class="journal-protocol"> Plasmid purification, </a>
 
                     <a data-toggle="modal" data-target="#restriction-digest-modal" class="journal-protocol">Restriction digest, </a>
 
                     <a data-toggle="modal" data-target="#restriction-digest-modal" class="journal-protocol">Restriction digest, </a>
 
                     <a data-toggle="modal" data-target="#sequencing-modal" class="journal-protocol">Sequencing</a>
 
                     <a data-toggle="modal" data-target="#sequencing-modal" class="journal-protocol">Sequencing</a>
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                   </p>
 
                   </p>
  
                   <p><b>Next Steps:</b> Overnight cultures in preparation for western blotting for characterisation of expression of both, <i>GFP</i>, and <i>ChrB</i> and <i>ChrBopt</i>.</p>
+
                   <p><b>Next Steps:</b> Overnight cultures in preparation for western blotting for characterisation of expression of both, GFP, and ChrB and ChrBopt.</p>
 
    
 
    
  
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             <span class="box-title">Day 7 - 02/08: Set up cultures in preparation for western blotting.</span> <!--Title of collapsible box-->
+
             <span class="box-title">02/08: Set up cultures of JM110 + pSB1C3-P<i>chr-gfp</i> (A), MC1061 + pSB1C3-P<i>chr-gfp</i> (B) and JM110 pUniprom-<i>chrB</i> and JM110 pUniprom-<i>chrB</i> (opt). in preparation for western blotting.</span> <!--Title of collapsible box-->
 
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                 <div id="collapsiblew6-7" class="collapse box-content">
  
  
                   <p><b>Aim of experiment:</b> Overnight cultures of sequence confirmed <i>ChrB</i>, <i>ChrBopt</i>, <i>pChrGFP1</i> (from 15/07), <i>pChrGFP3</i> and <i>pChrGFP4</i> (31/07) for subsequent western blotting.</p>
+
                   <p><b>Aim of experiment:</b> Overnight cultures of <i>E. coli</i> strains with sequence confirmed pUniprom-<i>chrB</i>, pUniprom-<i>chrB</i> (opt), pSB1C3-P<i>chr-gfp</i> (A), pSB1C3-P<i>chr-gfp</i> (B) - colony 3 and pSB1C3-P<i>chr-gfp</i> (B) - colony 4 for subsequent western blotting.</p>
  
 
                   <p><b>Protocols Used:</b>
 
                   <p><b>Protocols Used:</b>
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                   <p><b>Results:</b> N/A</p>
 
                   <p><b>Results:</b> N/A</p>
  
                   <p><b>Next Steps:</b> Western blotting for characterisation of expression of both, <i>GFP</i>, and <i>ChrB</i> and <i>ChrBopt</i>.</p>
+
                   <p><b>Next Steps:</b> Western blotting for characterisation of expression of both, GFP, and ChrB and ChrB (opt).</p>
  
 
                    
 
                    
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         <div class="labtitle">Week Beginning 03/08</div>
+
         <div class="labtitle">Week Beginning 03/08/2015</div>
 
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         <p class="journal-content">This week was entirely used for characterising the expression of GFP from the promoter <i>pChr</i>, and the expression of <i>ChrB</i> from the <a href="http://parts.igem.org/Part:BBa_K895000" target="_blank"><i>tat-promoter</i></a> in pUniprom.</p>
+
         <p class="journal-content">This week was entirely used for characterising the expression of GFP from the promoter P<i>chr</i>, and the expression of ChrB from the <a href="http://parts.igem.org/Part:BBa_K895000" target="_blank">P<i>tat</i></a> in pUniprom.</p>
 
         </div>
 
         </div>
 
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             <span class="box-title">Day 1 - Day 7 - 03/08 - 09/08: Determined expression of <i>ChrB</i>, <i>ChrBopt</i>, and GFP by western blotting.</span> <!--Title of collapsible box-->
+
             <span class="box-title">03/08 - 09/08: Determined expression of ChrB, ChrB (opt), and GFP by western blotting.</span> <!--Title of collapsible box-->
 
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                 <div id="collapsiblew7-1" class="collapse box-content">
  
  
                   <p><b>Aim of experiment:</b> To determine whether or not the required proteins, <i>GFP</i>, and <i>ChrB</i>, are expressed by blotting against GFP and against a 6His-tag respectively.</p>
+
                   <p><b>Aim of experiment:</b> To determine whether or not the required proteins, GFP, and ChrB, are expressed by blotting against GFP and against a 6-His Tag of ChrB and ChrB (opt) respectively.</p>
  
 
                   <p><b>Protocols Used:</b>
 
                   <p><b>Protocols Used:</b>
                     <a data-toggle="modal" data-target="#sds-modal" class="journal-protocol">SDS-PAGE and western blot</a>
+
                     <a data-toggle="modal" data-target="#sds-modal" class="journal-protocol"> SDS-PAGE and western blot</a>
 
                   </p>
 
                   </p>
  
                   <p><b>Results:</b> This procedure was repeated several times during this week in order to find appropriate concentrations and volumes to load. Figure 26 was blotted after SDS-PAGE with the following concentrations:
+
                   <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15_chr_150809_blot" class="journal-protocol"> Figure 26 </a></p>
                    <ul>
+
                      <li>GFP: Dilute pellet of 1ml overnight culture with 1ml TBS. Take 100µl of this solution and mix with 100µl Laemmli Buffer. Load 3µl on the SDS-gel.</li>
+
                      <li>HIS: Dilute pellet of 1ml overnight culture with 200µl Laemmli buffer. Load 10µl on the SDS-gel.</li>
+
                    </ul>
+
                  </p>
+
 
+
                  <p><a data-toggle="modal" data-target="#dundee15_chr_150809_blot" class="journal-protocol"> Figure 26 </a></p>
+
  
                   <p><b>Next Steps:</b>Transformation of both plasmids into MG1655 with the goal to characterise the interaction between <i>ChrB</i> and <i>pChr</i>. Furthermore the original biobrick, <a href="http://parts.igem.org/Part:BBa_K1058008" target="_blank">BBa_K1058008</a> will be tested under the same conditions for comparison.</p>
+
                   <p><b>Next Steps:</b> Transformation of both plasmids into MG1655 with the goal to characterise the interaction between ChrB and P<i>chr</i>. Furthermore the original biobrick, <a href="http://parts.igem.org/Part:BBa_K1058008" target="_blank">BBa_K1058008</a> will be tested under the same conditions for comparison.</p>
  
 
                    
 
                    
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     <div class="box">
         <div class="labtitle">Week Beginning 10/08</div>
+
         <div class="labtitle">Week Beginning 10/08/2015</div>
 
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         <p class="journal-content">This week was spent with transforming all combinations of pSB1C3-pChr-GFP and pUniprom-ChrB into <i>E. coli MG1655</i>. Tests for their interaction were also prepared.</p>
+
         <p class="journal-content">This week was spent with transforming all combinations of pSB1C3-P<i>chr-gfp</i> and pUniprom-<i>chrB</i> into <i>E. coli</i> MG1655. Tests for their interaction were also prepared.</p>
 
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         <div class="box">
             <span class="box-title">Day 1 - 10/08: Transformed both pSB1C3-pChr-GFP-plasmids, pUniprom-ChrB, and pUniprom-ChrBopt, as well as <a href="http://parts.igem.org/Part:BBa_K1058008" target="_blank">BBa_K1058008</a> into <i>E. coli MG1655</i>.</span>
+
             <span class="box-title">10/08: Transformed pSB1C3-P<i>chr-gfp</i> (A) and pSB1C3-P<i>chr-gfp</i> (B), and pUniprom-<i>chrB</i>, and pUniprom-<i>chrB</i> (opt), as well as <a href="http://parts.igem.org/Part:BBa_K1058008" target="_blank">BBa_K1058008</a> into <i>E. coli</i> MG1655.</span>
 
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                 <div id="collapsiblew8-1" class="collapse box-content">
  
  
                   <p><b>Aim of experiment:</b> To transform the following combinations of pSB1C3-pChr-GFP and pUniprom-ChrB into <i>MG1655</i>:
+
                   <p><b>Aim of experiment:</b> To transform the following combinations of pSB1C3-pChr-GFP and pUniprom-ChrB into MG1655:
 
                     <ul>
 
                     <ul>
                       <li>pUniprom-ChrB2 (29/07) - pSB1C3-pChr-GFP (31/07)</li>
+
                       <li>pUniprom-<i>chrB</i> (from colony 2 - 29/07) - pSB1C3-P<i>chr-gfp</i> (A)</li>
                       <li>pUniprom-ChrB2 (29/07) - pSB1C3-pChr-GFP (15/07)</li>
+
                       <li>pUniprom-<i>chrB</i> (from colony 2 - 29/07) - pSB1C3-P<i>chr-gfp</i> (B)</li>
                       <li>pUniprom-ChrBopt3 (29/07) - pSB1C3-pChr-GFP (31/07)</li>
+
                       <li>pUniprom-<i>chrB</i> (opt) (from colony 3 - 29/07) - pSB1C3-P<i>chr-gfp</i> (A)</li>
                       <li>pUniprom-ChrBopt3 (29/07) - pSB1C3-pChr-GFP (15/07)</li>
+
                       <li>pUniprom-<i>chrB</i> (opt) (from colony 3 - 29/07) - pSB1C3-P<i>chr-gfp</i> (B)</li>
 
                     </ul>
 
                     </ul>
                   in a stepwise fashion. Furthermore transform <a href="http://parts.igem.org/Part:BBa_K1058008" target="_blank">BBa_K1058008</a> into <i>MG1655</i>. Transformations were done in a stepwise fashion with only one of the plasmids at a time.</p>
+
                   in a stepwise fashion. Furthermore transform <a href="http://parts.igem.org/Part:BBa_K1058008" target="_blank">BBa_K1058008</a> into MG1655. Transformations were done in a stepwise fashion with only one of the plasmids at a time.</p>
  
 
                   <p><b>Protocols Used:</b>
 
                   <p><b>Protocols Used:</b>
                     <a data-toggle="modal" data-target="#miniprep-modal" class="journal-protocol">Plasmid purification, </a>
+
                     <a data-toggle="modal" data-target="#miniprep-modal" class="journal-protocol"> Plasmid purification, </a>
 
                     <a data-toggle="modal" data-target="#transformation-modal" class="journal-protocol">Transformation</a>
 
                     <a data-toggle="modal" data-target="#transformation-modal" class="journal-protocol">Transformation</a>
 
                   </p>
 
                   </p>
  
                   <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15_chr_150811_transformations" class="journal-protocol"> Figure 27 </a></p>
+
                   <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15_chr_150811_transformations" class="journal-protocol"> Figure 27 </a>Plates after transformation.</p>
  
 
                   <p><b>Next Steps:</b> Making newly transformed cells competent again in order to transform the second plasmid as well.</p>
 
                   <p><b>Next Steps:</b> Making newly transformed cells competent again in order to transform the second plasmid as well.</p>
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             <span class="box-title">Day 2 - 11/08: Set up overnight cultures of all transformed cells.</span> <!--Title of collapsible box-->
+
             <span class="box-title">11/08: Set up overnight cultures of all transformed cells.</span> <!--Title of collapsible box-->
 
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                 <div id="collapsiblew8-2" class="collapse box-content">
  
  
                   <p><b>Aim of experiment:</b> To prepare overnight cultures of yesterdays transformaions in order to make them competent to transform the second plasmid as well.</p>
+
                   <p><b>Aim of experiment:</b> To prepare overnight cultures of yesterdays transformations in order to make them competent to transform the second plasmid as well.</p>
  
 
                   <p><b>Protocols Used:</b>
 
                   <p><b>Protocols Used:</b>
                     <a data-toggle="modal" data-target="#overnightculture-modal" class="journal-protocol">Overnight culture</a>
+
                     <a data-toggle="modal" data-target="#overnightculture-modal" class="journal-protocol"> Overnight culture</a>
 
                   </p>
 
                   </p>
  
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             <span class="box-title">Day 3 - 12/08: Made competent <i>MG1655</i> with pUniprom-ChrB2, pUniprom-ChrBopt3, pSB1C3-pChr-GFP (15/07), pSB1C3-pChr-GFP (31/07).</span>
+
             <span class="box-title">12/08: Made competent MG1655 with pUniprom-ChrB2, pUniprom-ChrBopt3, pSB1C3-pChr-GFP (15/07), pSB1C3-pChr-GFP (31/07).</span>
 
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                 <div id="collapsiblew8-3" class="collapse box-content">
  
  
                   <p><b>Aim of experiment:</b>To make transformed cells competent in order to transform the second plasmid. The same plasmids that were used for the initial transformations were also used for this set of transformations.</p>
+
                   <p><b>Aim of experiment:</b> To make transformed cells competent in order to transform the second plasmid. The same plasmids that were used for the initial transformations were also used for this set of transformations.</p>
  
 
                   <p><b>Protocols Used:</b>
 
                   <p><b>Protocols Used:</b>
                     <a data-toggle="modal" data-target="#competent-modal" class="journal-protocol">Competent cells, </a>
+
                     <a data-toggle="modal" data-target="#competent-modal" class="journal-protocol"> Competent cells, </a>
 
                     <a data-toggle="modal" data-target="#transformation-modal" class="journal-protocol">Transformations </a>
 
                     <a data-toggle="modal" data-target="#transformation-modal" class="journal-protocol">Transformations </a>
 
                   </p>
 
                   </p>
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                   <p><b>Results:</b> N/A</p>
 
                   <p><b>Results:</b> N/A</p>
  
                   <p><b>Next Steps:</b> Blot against GFP in order to confirm (or refute) that <i>ChrB</i> interacts with <i>pChr</i> and hence switches off expression of GFP.</p>
+
                   <p><b>Next Steps:</b> Blot against GFP in order to confirm (or refute) that ChrB interacts with P<i>chr</i> and hence switches off expression of GFP.</p>
  
 
                    
 
                    
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             <span class="box-title">Day 4 - 13/08: Transformed pUniprom-ChrB2 and pUniprom-ChrBopt3 into <i>MG1655+pSB1C3-pChr-GFP</i>(15/07) and <i>MG1655+pChrGFP</i>(31/07)</span>
+
             <span class="box-title">13/08: Transformed pUniprom-<i>chrB</i> (from colony 2 - 29/07) and pUniprom-<i>chrB</i> (opt) (from colony 3 - 29/07) into MG1655 + pSB1C3-P<i>chr-gfp</i> (A) and pSB1C3-P<i>chr-gfp</i> (B)</span>
 
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                   <p><b>Aim of experiment:</b> To build a strain of <i>E. coli MG1655</i>, containing both plasmids required for a chromate sensor.</p>
+
                   <p><b>Aim of experiment:</b> To build a strain of <i>E. coli</i> MG1655, containing both plasmids required for a chromate sensor.</p>
  
 
                   <p><b>Protocols Used:</b>
 
                   <p><b>Protocols Used:</b>
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                   <p><b>Results:</b> No successful transformants were found.</p>
 
                   <p><b>Results:</b> No successful transformants were found.</p>
  
                   <p><b>Next Steps:</b> Repeat this experiment but transform pSB1C3-pChr-GFP (15/07) and pSB1C3-pChr-GFP (31/07) into competent <i>MG1655+pUniprom-ChrB2</i> and <i>MG1655+pUniprom-ChrBopt3</i> respectively.</p>
+
                   <p><b>Next Steps:</b> Repeat this experiment but transform pSB1C3-P<i>chr-gfp</i> (A) and pSB1C3-P<i>chr-gfp</i> (B) into competent MG1655 + pUniprom-<i>chrB</i> and MG1655 + pUniprom-<i>chrB</i> (opt) respectively.</p>
 
                    
 
                    
 
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+
         <div class="labtitle">Week Beginning 17/08/2015</div>
 
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         <p class="journal-content">During this week <i>E. coli MG1655</i> containing both plasmids for a. Western Blots were used to determine expression of <i>ChrB</i>, <i>ChrBopt</i>, and GFP.</a></p>
+
         <p class="journal-content">During this week <i>E. coli</i> MG1655 containing both plasmids for a. Western Blots were used to determine expression of ChrB and GFP.</a></p>
 
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             <span class="box-title">Day 1 - 17/08: Transformations akin to 13/08 were done, but pSB1C3-pChr-GFP(15/07) and pSB1C3-pChr-GFP(31/07) were transformed into <i>MG1655+pUniprom-ChrB2</i> and <i>MG1655+pUniprom-ChrBopt3</i>.</span>
+
             <span class="box-title">17/08: Transformations akin to 13/08 were done, but pSB1C3-P<i>chr-gfp</i> (A) and pSB1C3-P<i>chr-gfp</i> (A) were transformed into MG1655 + pUniprom-<i>chrB</i> and MG1655 + pUniprom-<i>chrB</i> (opt).</span>
 
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                   <p><b>Results:</b> Successful transformants were found on all plates.</p>
 
                   <p><b>Results:</b> Successful transformants were found on all plates.</p>
  
                   <p><b>Next Steps:</b> To determine expression of <i>ChrB</i> and <i>ChrBopt</i> (via 6His-tag) and expression of GFP.</p>
+
                   <p><b>Next Steps:</b> To determine expression of ChrB and GFP.</p>
 
                    
 
                    
 
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             <span class="box-title">Day 2 - 18/08: Preparation of samples for western blot.</span> <!--Title of collapsible box-->
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             <span class="box-title">18/08: Preparation of samples for western blot.</span> <!--Title of collapsible box-->
 
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                   <p><b>Aim of experiment:</b> To determine whether <i>ChrB</i>, <i>ChrBopt</i>, and GFP are expressed and have first estimates of differences in expression levels.</p>
+
                   <p><b>Aim of experiment:</b> To determine whether ChrB and GFP are expressed and have first estimates of differences in expression levels.</p>
  
 
                   <p><b>Protocols Used:</b>
 
                   <p><b>Protocols Used:</b>
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             <span class="box-title">Day 3-6 - 19/08 - 22/08: Repeated western blots with different concentrations to determine whether the expression of <i>ChrB</i> has an effect on the expression of GFP.</span> <!--Title of collapsible box-->
+
             <span class="box-title">19/08 - 22/08: Repeated western blots with different concentrations to determine whether the expression of ChrB has an effect on GFP levels.</span> <!--Title of collapsible box-->
 
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                   <p><b>Aim of experiment:</b> To determine whether expression of <i>ChrB</i> or <i>ChrBopt</i> has an effect on the expression of GFP.</p>
+
                   <p><b>Aim of experiment:</b> To determine whether expression of ChrB has an effect on the expression of GFP.</p>
  
 
                   <p><b>Protocols Used:</b>
 
                   <p><b>Protocols Used:</b>
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             <span class="box-title">Day 1 - Day 7 - 24/08-30/08: Design of Plate reader experiments.</span> <!--Title of collapsible box-->
+
             <span class="box-title">24/08-30/08: Design of Plate reader experiments.</span> <!--Title of collapsible box-->
 
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                   <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15_chr_150824_platereader_BBa_K1058008" class="journal-protocol"> Figure 29 </a> <i>MG1655 + <a href="http://parts.igem.org/Part:BBa_K1058008" target="_blank">BBa_K1058008</a></i></p>
+
                   <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15_chr_150824_platereader_BBa_K1058008" class="journal-protocol"> Figure 29 </a>MG1655 + <a href="http://parts.igem.org/Part:BBa_K1058008" target="_blank">BBa_K1058008</a></p>
  
                   <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15_chr_150824_platereader_pchrbgfp+chrb" class="journal-protocol"> Figure 30 </a><i>MG1655 + pSB1C3-pChr-GFP + pUniprom-ChrB</i></p>
+
                   <p><b>Results:</b><a data-toggle="modal" data-target="#icecream" class="journal-protocol"> Figure 30 </a>MG1655 + pSB1C3-P<i>chr-gfp</i> (B) + pUniprom-<i>chrB</i></p>
  
                   <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15_chr_150824_platereader_pchrbgfp+chrbopt" class="journal-protocol"> Figure 31 </a><i>MG1655 + pSB1C3-pChr-GFP + pUniprom-ChrBopt</i></p>
+
                   <p><b>Results:</b><a data-toggle="modal" data-target="#potato" class="journal-protocol"> Figure 31 </a>MG1655 + pSB1C3-P<i>chr-gfp</i> (B) + pUniprom-<i>chrB</i> (opt)</p>
  
                   <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15_chr_150824_platereader_pchrbgfp" class="journal-protocol"> Figure 32 </a><i>MG1655 + pSB1C3-pChr-GFP</i></p>
+
                   <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15_chr_150824_platereader_pchrbgfp" class="journal-protocol"> Figure 32 </a>MG1655 + pSB1C3-P<i>chr-gfp</i> (B)</p>
  
                   <p><b>Next Steps:</b> Try the system with different expression levels of <i>ChrB</i> and <i>ChrBopt</i>. Induce overexpression by exploiting the T7-promoter in pUniprom after transforming the different combinations between pSB1C3-pChr-GFP and pUniprom-ChrB/pUniprom-ChrBopt into <i>BL21(DE3)</i> and adding IPTG.</p>
+
                   <p><b>Next Steps:</b> Try the system with different expression levels of ChrB. Induce overexpression by exploiting the T7-promoter in pUniprom after transforming the different combinations between pSB1C3-P<i>chr-gfp</i> and pUniprom<i>chrB</i> / pUniprom<i>chrB</i> (opt) into <i>BL21(DE3)</i> and adding IPTG.</p>
 
                    
 
                    
 
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         <div class="labtitle">Week Beginning 31/08/2015</div>
 
          
 
          
 
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             <span class="box-title">Day 1 - 07/09: Transformation of pSB1K3 (Bielefeld) into <i>MG1655</i>.</span> <!--Title of collapsible box-->
+
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                   <p><b>Protocols Used:</b>
 
                   <p><b>Protocols Used:</b>
 
                     <a data-toggle="modal" data-target="#transformation-modal" class="journal-protocol"> Transformation, </a>
 
                     <a data-toggle="modal" data-target="#transformation-modal" class="journal-protocol"> Transformation, </a>
                     <p><a data-toggle="modal" data-target="#overnightculture-modal" class="journal-protocol"> Overnight culture</a> for new <i>E. coli MG1655</i></p>
+
                     <p><a data-toggle="modal" data-target="#overnightculture-modal" class="journal-protocol"> Overnight culture</a> for new <i>E. coli</i> MG1655</p>
 
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                   <p><b>Results:</b> No transformants were found.</p>
 
                   <p><b>Results:</b> No transformants were found.</p>
  
                   <p><b>Next Steps:</b> Retry transformation with new competent <i>E. coli MG1655</i>.</p>
+
                   <p><b>Next Steps:</b> Retry transformation with new competent <i>E. coli</i> MG1655.</p>
 
                    
 
                    
 
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             <span class="box-title">Day 2 - 08/09: Transformation of pSB1K3 into MG1655.</span> <!--Title of collapsible box-->
+
             <span class="box-title">08/09: Transformation of pSB1K3 into MG1655.</span> <!--Title of collapsible box-->
 
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                   <p><b>Aim of experiment:</b> Test transformation of BBa_K1058008 into new <i>MG1655</i> competent cells and re-transformation of pSB1K3 for subsequent miniprep.</p>
+
                   <p><b>Aim of experiment:</b> Test transformation of BBa_K1058008 into new MG1655 competent cells and re-transformation of pSB1K3 for subsequent miniprep.</p>
  
 
                   <p><b>Protocols Used:</b>
 
                   <p><b>Protocols Used:</b>
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                   </p>
 
                   </p>
  
                   <p><b>Results:</b> Successful test transformation, but no colonies on pSB1K3 plate.</p>
+
                   <p><b>Results:</b> Successful test transformation, but no colonies were found on the pSB1K3 plate.</p>
  
                   <p><b>Next Steps:</b> Use an alternative compatible plasmid for ligation of codon optimised <i>ChrB</i> (Bielefeld).</p>
+
                   <p><b>Next Steps:</b> Use an alternative compatible plasmid for ligation of the codon optimised <i>chrB</i> (Bielefeld).</p>
 
                    
 
                    
 
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             <span class="box-title">Day 3 - 09/09: Transformation of pSB1K3 into MG1655.</span> <!--Title of collapsible box-->
+
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                   <p><b>Aim of experiment:</b> Restriction digest of pT7KS with EcoRI and PstI as alternative to pSB1K3. Ligation of digested <i>ChrB</i> (Bielefeld) and transformation of pT7KS-ChrB. Also transformations of Chromate-sensor plasmids into BL21 (DE3) in the following combinations: <ul>
+
                   <p><b>Aim of experiment:</b> Restriction digest of pT7KS with EcoRI and PstI as alternative to pSB1K3. Ligation of digested <i>chrB</i> (Bielefeld) and transformation of pT7KS-<i>chrB</i>. Also transformations of Chromate-sensor plasmids into <i>BL21 (DE3)</i> in the following combinations: <ul>
                                   <li>pSB1C3-pChr-GFP + pUniprom-ChrB</li>
+
                                   <li>pSB1C3-P<i>chr-gfp</i> (B) + pUniprom-<i>chrB</i></li>
                                   <li>pSB1C3-pChr-GFP + pUniprom-ChrBopt</li>
+
                                   <li>pSB1C3-P<i>chr-gfp</i> (B) + pUniprom-<i>chrB</i> (opt)</li>
                                   <li>pUniprom-ChrB</li>
+
                                   <li>pUniprom-<i>chrB</i></li>
                                   <li>pUniprom-ChrBopt</li>
+
                                   <li>pUniprom-<i>chrB</i> (opt)</li>
 
                                 </ul>
 
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                   </p>
 
                   </p>
  
                   <p><b>Results:</b>Transformations of the plasmid with pT7KS-ChrB (Bielefeld) yielded 12 colonies, but also colonies on the vector control plate. Transformations of pSB1C3-pChr-GFP + pUniprom-ChrB, pSB1C3-pChr-GFP + pUniprom-ChrBopt, pUniprom-ChrB, and pUniprom-ChrBopt into <i>BL21(DE3)</i> were successful.</p>
+
                   <p><b>Results:</b> Transformations of the plasmid with pT7KS-<i>chrB</i> (Bielefeld) yielded 12 colonies, but also colonies on the vector control plate. Transformations of pSB1C3-P<i>chr-gfp</i> (B) + pUniprom-<i>chrB</i>, pSB1C3-P<i>chr-gfp</i> (B) + pUniprom-<i>chrB</i> (opt), pUniprom-<i>chrB</i>, and pUniprom-<i>chrB</i> (opt) into <i>BL21(DE3)</i> were successful.</p>
  
 
                   <p><b>Results:</b>
 
                   <p><b>Results:</b>
                     <p><a data-toggle="modal" data-target="#dundee15_chr_150911_chrbbielefeld" class="journal-protocol"> Figure 33 </a> pT7KS-ChrB (Bielefeld) transformations</p>
+
                     <p><a data-toggle="modal" data-target="#dundee15_chr_150911_chrbbielefeld" class="journal-protocol"> Figure 33 </a> pT7KS-<i>chrB</i> (Bielefeld) transformations</p>
 
                     <p><a data-toggle="modal" data-target="#dundee15_chr_150911_bl21" class="journal-protocol"> Figure 34 </a> Transformations into <i>BL21(DE3)</i></p>
 
                     <p><a data-toggle="modal" data-target="#dundee15_chr_150911_bl21" class="journal-protocol"> Figure 34 </a> Transformations into <i>BL21(DE3)</i></p>
 
                   </p>
 
                   </p>
  
                   <p><b>Next Steps:</b> Confirmation of colonies with pT7KS-ChrB by colony PCR. Preparation of recombinant <i>BL21(DE3)</i> for plate reader experiments.</p>
+
                   <p><b>Next Steps:</b> Confirmation of colonies with pT7KS-<i>chrB</i> (Bielefeld) by colony PCR. Preparation of recombinant <i>BL21(DE3)</i> for plate reader experiments.</p>
 
                    
 
                    
 
                 </div>
 
                 </div>
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       <div class="border-day">
 
       <div class="border-day">
 
         <div class="box">
 
         <div class="box">
             <span class="box-title">Day 4 - 10/09: Colony PCR of pT7KS-ChrB (Bielefeld).</span> <!--Title of collapsible box-->
+
             <span class="box-title">10/09: Colony PCR of pT7KS-<i>chrB</i> (Bielefeld).</span> <!--Title of collapsible box-->
 
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                 <div id="collapsiblew12-4" class="collapse box-content">
  
  
                   <p><b>Aim of experiment:</b> Check if any of the colonies after ligation of ChrB (Bielefeld) into pT7KS were successful despite colonies on the control plate.</p>
+
                   <p><b>Aim of experiment:</b> Check if any of the colonies after ligation of <i>chrB</i> (Bielefeld) into pT7KS were successful despite colonies on the control plate.</p>
  
 
                   <p><b>Protocols Used:</b>
 
                   <p><b>Protocols Used:</b>
Line 1,448: Line 1,440:
 
                   </p>
 
                   </p>
  
                   <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15_chr_150911_igem050" class="journal-protocol"> Figure 34 </a></p>
+
                   <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15_chr_150911_igem047" class="journal-protocol"> Figure 35 </a></p>
  
                   <p><b>Next Steps:</b> Prepare transformed <i>BL21(DE3)</i> for plate reader experiments. Attempt new transformation of pT7KS-ChrB (Bielefeld).</p>
+
                   <p><b>Next Steps:</b> Prepare transformed <i>BL21(DE3)</i> for plate reader experiments. Attempt new transformation of pT7KS-<i>chrB</i> (Bielefeld).</p>
 
                    
 
                    
 
                 </div>
 
                 </div>
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       <div class="border-day">
 
         <div class="box">
 
         <div class="box">
             <span class="box-title">Day 5 - 11/09: Preparation for Plate reader experiment with <i>BL21(DE3)</i></span>
+
             <span class="box-title">11/09: Preparation for Plate reader experiment with <i>BL21(DE3)</i></span>
 
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       <div class="border-day">
 
         <div class="box">
 
         <div class="box">
             <span class="box-title">Day 6 - 12/09: Set up plate reader.</span> <!--Title of collapsible box-->
+
             <span class="box-title">12/09: Set up plate reader.</span> <!--Title of collapsible box-->
 
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                   </p>
 
                   </p>
  
                   <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15_chr_platereaderinduced" class="journal-protocol"> Figure 35 </a></p>
+
                   <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15_chr_platereaderinduced" class="journal-protocol"> Figure 36 </a></p>
  
                   <p><b>Next Steps:</b>Repeat experiments with added chromate substances.</p>
+
                   <p><b>Next Steps:</b> Repeat experiments with added chromate substances.</p>
 
                    
 
                    
 
                 </div>
 
                 </div>
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     <div class="border-week">
 
     <div class="box">
 
     <div class="box">
         <div class="labtitle">Week Beginning 14/09</div>
+
         <div class="labtitle">Week Beginning 14/09/2015</div>
 
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           <div class="box-content">
 
           <div class="box-content">
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         <p class="journal-content">During this week the samples from the previous plate reader were retrieved and prepared for blotting against both GFP, and the 6His-Tag of <i>ChrB</i> and <i>ChrBopt</i>.</a></p>
+
         <p class="journal-content">During this week the samples from the previous plate reader were retrieved and prepared for blotting against both GFP, and the 6-His - Tag of ChrB.</a></p>
 
         </div>
 
         </div>
 
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         <div id="collapsiblew13" class="collapse week-content">
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       <div class="border-day">
 
       <div class="border-day">
 
         <div class="box">
 
         <div class="box">
             <span class="box-title">Day 1 - 14/09: Western blot of plate reader samples <i>MG1655 + pSB1C3-pChr-GFP + pUniprom-ChrB</i>, and  <i>MG1655 + pSB1C3-pChr-GFP + pUniprom-ChrBopt</i>.</span> <!--Title of collapsible box-->
+
             <span class="box-title">14/09: Western blot of plate reader samples MG1655 + pSB1C3-P<i>chr-gfp</i> + pUniprom-<i>chrB</i>, and MG1655 + pSB1C3-P<i>chr-gfp</i> + pUniprom-<i>chrB</i> (opt).</span> <!--Title of collapsible box-->
 
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                 <div id="collapsiblew13-1" class="collapse box-content">
  
  
                   <p><b>Aim of experiment:</b> To identify whether or not there is any detectable difference in both, the level of GFP, and the level of ChrB or ChrBopt, before and after induction with IPTG.</p>
+
                   <p><b>Aim of experiment:</b> To identify whether or not there is any detectable difference in both, the level of GFP, and the level of ChrB, before and after induction with IPTG.</p>
  
 
                   <p><b>Protocols Used:</b>
 
                   <p><b>Protocols Used:</b>
Line 1,545: Line 1,537:
 
                   </p>
 
                   </p>
  
                   <p><b>Results:</b> Insert blot image here.</p>
+
                   <p><b>Results:</b> Even though the blot was very blurred, expression of GFP could be confirmed in all samples. There seemed to be no noteable difference between induced and uninduced.</p>
  
                   <p><b>Next Steps:</b> Repeat the assays of the previous weeks by subcloning ChrB, ChrBopt, and ChrB (Bielefeld) into vectors with different expression levels and see how it influences the level of GFP expression. Then add different chromate salts and also probe the system with other substances, e.g. other heavy-metal salts.</p>
+
                   <p><b>Next Steps:</b> Repeat the assays of the previous weeks by subcloning <i>chrB</i>, <i>chrB</i> (opt), and <i>chrB</i> (Bielefeld) into vectors with different expression levels and see how it influences the level of GFP expression. Then add different chromate salts and also probe the system with other substances, e.g. other heavy-metal salts.</p>
 
                    
 
                    
 
                 </div>
 
                 </div>
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       <ol>
 
       <ol>
         <li>A 5ml overnight culture of the desired strain was grown at 37<sup>o</sup>C.</li>
+
         <li>A 5 ml overnight culture of the desired strain was grown at 37 <sup>o</sup>C.</li>
 
         <li>Equalise OD<sub>600</sub> if required. Dilute culture 1:1000.</li>
 
         <li>Equalise OD<sub>600</sub> if required. Dilute culture 1:1000.</li>
         <li>Aliquot 198µl into the required amount of wells in a 96-well plate.</li>
+
         <li>Aliquot 198 µl into the required amount of wells in a 96-well plate.</li>
 
         <li>Add additional substances and controls as required.</li>
 
         <li>Add additional substances and controls as required.</li>
 
         <li>Preheat platereader to required temperature and start experiment.</li>
 
         <li>Preheat platereader to required temperature and start experiment.</li>
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       <ol>
 
       <ol>
         <li>A 5ml overnight culture of the desired strain was grown at 37<sup>o</sup>C.</li>
+
         <li>A 5 ml overnight culture of the desired strain was grown at 37<sup>o</sup>C.</li>
         <li>Subsample <sup>1</sup>/<sub>10</sub> of the volume of the subculture. I.e. 1ml of the overnight culture into 10ml fresh LB.</li>
+
         <li>Subsample <sup>1</sup>/<sub>10</sub> of the volume of the subculture. I.e. 1 ml of the overnight culture into 10 ml fresh LB.</li>
         <li>Grow subculture at at 37<sup>o</sup>C for 2h.</li>
+
         <li>Grow subculture at at 37 <sup>o</sup>C for 2 h.</li>
         <li>Spin down subculture at 4000rpm at 4<sup>o</sup>C for 10min.</li>
+
         <li>Spin down subculture at 4000 rpm at 4<sup>o</sup>C for 10 min.</li>
 
         <li>Resuspend the pellet in ice cold transformation buffer. Use <sup>1</sup>/<sub>10</sub> of the initial volume of the subculture.</li>
 
         <li>Resuspend the pellet in ice cold transformation buffer. Use <sup>1</sup>/<sub>10</sub> of the initial volume of the subculture.</li>
         <li>Use 100µl per transformation.</li>
+
         <li>Use 100 µl per transformation.</li>
 
       </ol>
 
       </ol>
  
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       <ul>
 
       <ul>
 
         <li>Volume of sample equivalent to 550ng DNA</li>
 
         <li>Volume of sample equivalent to 550ng DNA</li>
         <li>2µl of 3mM primer</li>
+
         <li>2 µl of 3 mM primer</li>
         <li>Remaining volume of water for a total volume of 30µl</li>
+
         <li>Remaining volume of water for a total volume of 30 µl</li>
 
       </ul>
 
       </ul>
 
     </p>
 
     </p>
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         <li>Agar plates with required antibiotic were taken out of 4<sup>o</sup>C to warm to room temperature.</li>
 
         <li>Agar plates with required antibiotic were taken out of 4<sup>o</sup>C to warm to room temperature.</li>
 
         <li>Cells or colonies were picked up from the source with a toothpick under sterile conditions.</li>
 
         <li>Cells or colonies were picked up from the source with a toothpick under sterile conditions.</li>
         <li>5ml of LB medium, supplemented with the required antibiotic, were inoculated with the toothpick.</li>
+
         <li>5 ml of LB medium, supplemented with the required antibiotic, were inoculated with the toothpick.</li>
 
         <li>Tubes were incubated at 37<sup>o</sup>C in a rotary incubator at 200rpm over night.</li>
 
         <li>Tubes were incubated at 37<sup>o</sup>C in a rotary incubator at 200rpm over night.</li>
 
       </ol>
 
       </ol>
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       <li>A numbered grid according to the desired number of PCR reactions is drawn on the back of the plate.</li>
 
       <li>A numbered grid according to the desired number of PCR reactions is drawn on the back of the plate.</li>
 
       <li>Cells or colonies were picked up from the source with a toothpick under sterile conditions.</li>
 
       <li>Cells or colonies were picked up from the source with a toothpick under sterile conditions.</li>
       <li>Each colony is transferred to a 30µl aliquot of sterile water.</li>
+
       <li>Each colony is transferred to a 30 µl aliquot of sterile water.</li>
 
       <li>With the same toothpick the grid with the respctive number on the plate was inoculated.</li>
 
       <li>With the same toothpick the grid with the respctive number on the plate was inoculated.</li>
 
       <li><ul>
 
       <li><ul>
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</table>
 
</table>
  
<p>15µl of the reaction mixture was aliquoted into separate PCR tubes. 5µl of DNA fraction was added to each respective tube in order to have a total reaction volume of 20µl per tube.</p>
+
<p>15 µl of the reaction mixture was aliquoted into separate PCR tubes. 5 µl of DNA fraction was added to each respective tube in order to have a total reaction volume of 20 µl per tube.</p>
  
 
<p>
 
<p>
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       <ol>
 
       <ol>
         <li>5ml bacterial overnight culture was pelleted by stepwise centrifugation in 1.5ml microcentrifuge tubes at 13300rpm for 3 minutes at room temperature.</li>
+
         <li>5 ml bacterial overnight culture was pelleted by stepwise centrifugation in 1.5 ml microcentrifuge tubes at 13300rpm for 3 minutes at room temperature.</li>
         <li>Pelleted bacterial cells were resuspended in 250µl of Buffer P1.</li>
+
         <li>Pelleted bacterial cells were resuspended in 250 µl of Buffer P1.</li>
         <li>250µl of Buffer P2 was added and mixed thoroughly by inverting the tube 4-6 times until solution became clear.</li>
+
         <li>250 µl of Buffer P2 was added and mixed thoroughly by inverting the tube 4-6 times until solution became clear.</li>
         <li>350µl of Buffer N3 was added and immediately and thoroughly mixed by inverting the tube 4-6 times.</li>
+
         <li>350 µl of Buffer N3 was added and immediately and thoroughly mixed by inverting the tube 4-6 times.</li>
 
         <li>The mixture was centrifuged for 10 minutes at 13300rpm in a microcentrifuge.</li>
 
         <li>The mixture was centrifuged for 10 minutes at 13300rpm in a microcentrifuge.</li>
 
         <li>The supernatant was applied to a QIAprep spin column by pipetting.</li>
 
         <li>The supernatant was applied to a QIAprep spin column by pipetting.</li>
         <li>500µl of Buffer PB was added to the QIAprep spin column and centrifuged for 1 minute. The flow-through was discarded.</li>
+
         <li>500 µl of Buffer PB was added to the QIAprep spin column and centrifuged for 1 minute. The flow-through was discarded.</li>
         <li>750µl of Buffer PE was added to the QIAprep spin column and centrifuged for 1 minute. The flow-through was discarded.</li>
+
         <li>750 µl of Buffer PE was added to the QIAprep spin column and centrifuged for 1 minute. The flow-through was discarded.</li>
 
         <li>The QIAprep spin column was spun for an additional minute to remove residual wash buffer.</li>
 
         <li>The QIAprep spin column was spun for an additional minute to remove residual wash buffer.</li>
         <li>The QIAprep spin column was placed in a clean 1.5ml microcentrifuge and the DNA eluted by adding 30µl of H<sub>2</sub>O to the QIAprep spin column and centrifuging for 1 minute. </li>
+
         <li>The QIAprep spin column was placed in a clean 1.5 ml microcentrifuge and the DNA eluted by adding 30 µl of H<sub>2</sub>O to the QIAprep spin column and centrifuging for 1 minute. </li>
 
       </ol>
 
       </ol>
  
Line 2,055: Line 2,047:
 
       <ol>
 
       <ol>
 
         <li>The desired DNA fragment was excised from the agarose gel with a clean, sharp scalpel.</li>
 
         <li>The desired DNA fragment was excised from the agarose gel with a clean, sharp scalpel.</li>
         <li>The gel slice was transferred into a microcentrifuge tube. 700µl of Buffer QG was added.</li>
+
         <li>The gel slice was transferred into a microcentrifuge tube. 700 µl of Buffer QG was added.</li>
 
         <li>The gel slice was incubated in a water bath at 50°C for 10 min (or until the gel slice has completely dissolved). The tube was vortexed every 2–3 min to help dissolve gel.</li>
 
         <li>The gel slice was incubated in a water bath at 50°C for 10 min (or until the gel slice has completely dissolved). The tube was vortexed every 2–3 min to help dissolve gel.</li>
 
         <li>After the gel slice had been dissolved completely, the color of the mixture was checked that it was yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture was orange or violet, 10 μl 3 M sodium acetate, pH 5.0 would be added, and mixed. The color of the mixture would turn yellow.</li>
 
         <li>After the gel slice had been dissolved completely, the color of the mixture was checked that it was yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture was orange or violet, 10 μl 3 M sodium acetate, pH 5.0 would be added, and mixed. The color of the mixture would turn yellow.</li>
         <li>230µl of isopropanol was added to the sample and mixed.</li>
+
         <li>230 µl of isopropanol was added to the sample and mixed.</li>
         <li>A QIAquick spin column was placed in a 2ml collection tube. The sample was applied to the QIAquick spin column and centrifuged for 1 minute. The flow-through was discarded.</li>
+
         <li>A QIAquick spin column was placed in a 2 ml collection tube. The sample was applied to the QIAquick spin column and centrifuged for 1 minute. The flow-through was discarded.</li>
         <li>500µl of QG buffer was added to the QIAquick spin column and centrifuged for 1 minute. The flow-through was discarded.</li>
+
         <li>500 µl of QG buffer was added to the QIAquick spin column and centrifuged for 1 minute. The flow-through was discarded.</li>
         <li>750µl of Buffer PE was added to the QIAquick spin column and centrifuged for 1 minute. The flow-through was discarded.</li>
+
         <li>750 µl of Buffer PE was added to the QIAquick spin column and centrifuged for 1 minute. The flow-through was discarded.</li>
 
         <li>The QIAprep spin column was spun for an additional minute to remove residual wash buffer.</li>
 
         <li>The QIAprep spin column was spun for an additional minute to remove residual wash buffer.</li>
         <li>The QIAprep spin column was placed in a clean 1.5ml microcentrifuge and the DNA eluted by adding 30µl of H2O to the QIAprep spin column and centrifuging for 1 minute.</li>
+
         <li>The QIAprep spin column was placed in a clean 1.5 ml microcentrifuge and the DNA eluted by adding 30 µl of H2O to the QIAprep spin column and centrifuging for 1 minute.</li>
 
       </ol>
 
       </ol>
  
Line 2,121: Line 2,113:
 
             <td width="151" valign="top">
 
             <td width="151" valign="top">
 
                 <p>
 
                 <p>
                     50µl
+
                     50 µl
 
                 </p>
 
                 </p>
 
             </td>
 
             </td>
Line 2,246: Line 2,238:
 
     <ul>
 
     <ul>
 
       <li>The digests were incubated at 37<sup>o</sup>C for 3 hours.</li>
 
       <li>The digests were incubated at 37<sup>o</sup>C for 3 hours.</li>
       <li>Note that when performing plasmid digestions, 2.5µl of alkaline phosphatase and 2.5µl of its buffer were added at the 2 hour and 2.5 hour mark.</li>
+
       <li>Note that when performing plasmid digestions, 2.5 µl of alkaline phosphatase and 2.5 µl of its buffer were added at the 2 hour and 2.5 hour mark.</li>
 
     </ul>
 
     </ul>
 
      
 
      
Line 2,461: Line 2,453:
  
 
       <ol>
 
       <ol>
         <li>100µl of competent cells were defrosted. Thaw on ice.</li>
+
         <li>100 µl of competent cells were defrosted. Thaw on ice.</li>
 
         <li>Agar plates were taken out of 4<sup>o</sup>C to warm to room temperature.</li>
 
         <li>Agar plates were taken out of 4<sup>o</sup>C to warm to room temperature.</li>
         <li>1-5µl of DNA was added to cells.</li>
+
         <li>1-5 µl of DNA was added to cells.</li>
 
         <li>Cell and DNA mixture was left on ice for 20 minutes.</li>
 
         <li>Cell and DNA mixture was left on ice for 20 minutes.</li>
 
         <li>Mixture was placed in 42<sup>o</sup>C water bath for 90 seconds.</li>
 
         <li>Mixture was placed in 42<sup>o</sup>C water bath for 90 seconds.</li>
 
         <li>Mixture was put back on ice for 2 minutes.</li>
 
         <li>Mixture was put back on ice for 2 minutes.</li>
         <li>1ml of LB (without antibiotic) was added to cells.</li>
+
         <li>1 ml of LB (without antibiotic) was added to cells.</li>
 
         <li>Cells were allowed to grow for 1 hour at 37<sup>o</sup>C in a shaking incubator.</li>
 
         <li>Cells were allowed to grow for 1 hour at 37<sup>o</sup>C in a shaking incubator.</li>
 
         <li>Cells were then spun down and the supernatant discarded.</li>
 
         <li>Cells were then spun down and the supernatant discarded.</li>
         <li>The pellet was resuspended in the 100µl.</li>
+
         <li>The pellet was resuspended in the 100 µl.</li>
 
         <li>Cells were plated on agar plates with appropriate antibiotic.</li>
 
         <li>Cells were plated on agar plates with appropriate antibiotic.</li>
 
       </ol>
 
       </ol>
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       <ol>
 
       <ol>
         <li>50µl of an overnight culture was used to inoculate 5ml of fresh LB containing the appropriate antibiotics and left to grow at 37<sup>o</sup>C in a shaking incubator.</li>
+
         <li>50 µl of an overnight culture was used to inoculate 5 ml of fresh LB containing the appropriate antibiotics and left to grow at 37<sup>o</sup>C in a shaking incubator.</li>
         <li>Once the OD<sub>600</sub>=0.6-0.8, 1mM of IPTG was added to the culture and left to grow for a further 3 hours.</li>
+
         <li>Once the OD<sub>600</sub>=0.6-0.8, 1 mM of IPTG was added to the culture and left to grow for a further 3 hours.</li>
         <li>1ml of each culture was taken and spun down in a microcentrifuge for 3 minutes.</li>
+
         <li>1 ml of each culture was taken and spun down in a microcentrifuge for 3 minutes.</li>
         <li>Meanwhile, 950µl of 2x Laemmli sample buffer was added to 50µl of B-mercaptoethanol.</li>
+
         <li>Meanwhile, 950 µl of 2x Laemmli sample buffer was added to 50 µl of B-mercaptoethanol.</li>
         <li>The supernatant was discarded from the samples and the pellet resuspended in 200µl of the above solution.</li>
+
         <li>The supernatant was discarded from the samples and the pellet resuspended in 200 µl of the above solution.</li>
 
         <li>The samples were then boiled at 90<sup>o</sup>C for 10 minutes.</li>
 
         <li>The samples were then boiled at 90<sup>o</sup>C for 10 minutes.</li>
 
         <li>Afterwards, they were spun down for 1 minute in a microcentrifuge.</li>
 
         <li>Afterwards, they were spun down for 1 minute in a microcentrifuge.</li>
Line 2,508: Line 2,500:
 
         <li>One gel was stained using Instant Blue and the other was used to transfer the proteins onto a membrane.</li>
 
         <li>One gel was stained using Instant Blue and the other was used to transfer the proteins onto a membrane.</li>
 
         <li>After the proteins had been transferred onto the membrane, the membrane was blocked in 5% Marvel milk overnight at 4<sup>o</sup>C.</li>
 
         <li>After the proteins had been transferred onto the membrane, the membrane was blocked in 5% Marvel milk overnight at 4<sup>o</sup>C.</li>
         <li>The next day, the membrane was washed 3 times in 1xTBST buffer and then suspended in 10ml of 1xTBS buffer to which the primary anti-His antibody was added and left for one hour. The protein ladder was cut off before adding the primary antibody.</li>
+
         <li>The next day, the membrane was washed 3 times in 1xTBST buffer and then suspended in 10 ml of 1xTBS buffer to which the primary anti-His antibody was added and left for one hour. The protein ladder was cut off before adding the primary antibody.</li>
         <li>The membrane was then washed 3 times in 1xTBST buffer for 5 minutes and then suspended in 10ml of 1xTBS buffer, to which the secondary antibody was added and left for one hour.</li>
+
         <li>The membrane was then washed 3 times in 1xTBST buffer for 5 minutes and then suspended in 10 ml of 1xTBS buffer, to which the secondary antibody was added and left for one hour.</li>
         <li>The membrane was then washed in 3x10ml 1xTBST buffer for 5 minutes.</li>
+
         <li>The membrane was then washed in 3x10 ml 1xTBST buffer for 5 minutes.</li>
 
         <li>The blot was then soaked in developing solution for 5 minutes before being wrapped in a plastic cover aligned with the protein ladder that was cut off at step 12.</li>
 
         <li>The blot was then soaked in developing solution for 5 minutes before being wrapped in a plastic cover aligned with the protein ladder that was cut off at step 12.</li>
 
         <li>Once in the developing room, the film was exposed to the blot for 30 seconds, 2 minutes and 5 minutes and then placed in the film developer.</li>
 
         <li>Once in the developing room, the film was exposed to the blot for 30 seconds, 2 minutes and 5 minutes and then placed in the film developer.</li>
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       <div class="modal-header">
 
       <div class="modal-header">
 
         <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">&times;</span></button>
 
         <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">&times;</span></button>
         <div class="modal-title" id="myModalLabel">Figure 2: 1% agarose gel after PCR of <a href="http://parts.igem.org/Part:BBa_K1058008" target="_blank">BBa_K1058008</a> with primers specific to the promoter region, pChr, and the repressor region, ChrB. Left: pChr (expected size 169bp); Middle: 1kb+ ladder, Right: ChrB (expected size 939bp). Amplification of ChrB was not successful. pChr was further processed by restriction digest with EcoRI and PstI
+
         <div class="modal-title" id="myModalLabel">Figure 2: 1% agarose gel after PCR of <a href="http://parts.igem.org/Part:BBa_K1058008" target="_blank">BBa_K1058008</a> with primers specific to the promoter region, P<i>chr</i>, and the repressor region, <i>chrB</i>. Left: P<i>chr</i> (expected size 169bp); Middle: 1kb+ ladder, Right: <i>chrB</i> (expected size 939bp). Amplification of <i>chrB</i> was not successful. P<i>chr</i> was further processed by restriction digest with EcoRI and PstI.
 
         </div>
 
         </div>
 
       </div>
 
       </div>
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       <div class="modal-header">
 
       <div class="modal-header">
 
         <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">&times;</span></button>
 
         <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">&times;</span></button>
         <div class="modal-title" id="myModalLabel">Figure 3: 1% agarose gel after PCR of <a href="http://parts.igem.org/Part:BBa_K1058008" target="_blank">BBa_K1058008</a> with primers specific to the repressor region, ChrB. Left: 1.5µl DMSO; Middle: 1kb+ ladder, Right: 2.5µl DMSO. Expected size for each: 939bp. The annealing temperature for the PCR amplification was lowered to 50°C in order to increase the chances of accomodating a primer that is not 100% complementary.
+
         <div class="modal-title" id="myModalLabel">Figure 3: 1% agarose gel after PCR of <a href="http://parts.igem.org/Part:BBa_K1058008" target="_blank">BBa_K1058008</a> with primers specific to the repressor region, <i>chrB</i>. Left: 1.5 µl DMSO; Middle: 1kb+ ladder, Right: 2.5 µl DMSO. Expected size for each: 939bp. The annealing temperature for the PCR amplification was lowered to 50°C in order to increase the chances of accomodating a primer that is not 100% complementary.
 
         </div>
 
         </div>
 
       </div>
 
       </div>
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       <div class="modal-header">
 
       <div class="modal-header">
 
         <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">&times;</span></button>
 
         <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">&times;</span></button>
         <div class="modal-title" id="myModalLabel">Figure 4: 1% agarose gel after PCR of GFPmut2 with corresponding primers primers. Left: GFPmut2 (expected size 717bp); Middle: 1kb+ ladder, Right: PCR product of ChrB, using primers to add a BamHI restriction site in front of it and a His-Tag and a HindIII restriction site at the end. (expected size of ChrB: 939bp). Since amplification of ChrB was unsuccessful, it will be repeated.
+
         <div class="modal-title" id="myModalLabel">Figure 4: 1% agarose gel after PCR of <i>gfp</i> with corresponding primers. Left: <i>GFP</i> (expected size 717bp); Middle: 1kb+ ladder, Right: PCR product of <i>chrB</i>, using primers to add a BamHI restriction site in front of it and a 6-His-Tag and a HindIII restriction site at the end. (expected size of <i>chrB</i>: 939bp). Since amplification of <i>chrB</i> was unsuccessful, it will be repeated.
 
         </div>
 
         </div>
 
       </div>
 
       </div>
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       <div class="modal-header">
 
         <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">&times;</span></button>
 
         <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">&times;</span></button>
         <div class="modal-title" id="myModalLabel">Figure 5: 1% agarose gel after restriction digest of pChr-pSB1C3, ChrBs-pSB1C3, and ChrBw-pSB1C3 for confirmation of size. Ladder: 1kb+. Top: 1-4: pChr 1-4; ChrBs 1-2. Bottom: ChrBs 3-4; ChrBw 1-4. Expected sizes: pChr: 169bp; ChrB: 939bp.
+
         <div class="modal-title" id="myModalLabel">Figure 5: 1% agarose gel after restriction digest of pSB1C3-P<i>chr</i>, pSB1C3-<i>chrB</i> (s), and pSB1C3-<i>chrB</i> (w) with EcoRI and PstI for confirmation of size. Ladder: 1kb+. Top: 1-4: P<i>chr</i> 1-4; <i>chrB</i> (s) 1-2. Bottom: <i>chrB</i> (s) 3-4; <i>chrB</i> (w) 1-4. Expected sizes: P<i>chr</i>: 169bp; <i>chrB</i>: 939bp.
 
         </div>
 
         </div>
 
       </div>
 
       </div>
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       <div class="modal-header">
 
       <div class="modal-header">
 
         <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">&times;</span></button>
 
         <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">&times;</span></button>
         <div class="modal-title" id="myModalLabel">Figure 6: 1% agarose gel after PCR of GFPmut2 with corresponding primers primers. Left: GFPmut2 (expected size 717bp); Middle: 1kb+ ladder, Right: PCR product of ChrB, using primers to add a BamHI restriction site in front of it and a His-Tag and a HindIII restriction site at the end. (expected size of ChrB: 939bp). Colours: Green: Prefix and Suffix; Red: In frame stop codons.
+
         <div class="modal-title" id="myModalLabel">Figure 6: Alignment of P<i>chr</i> sequence retrieved from <a href="http://parts.igem.org/Part:BBa_K1058008" target="_blank">BBa_K1058008</a> with the sequencing result of this region. Colour code: Green = Prefix/Suffix
 
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         </div>
 
       </div>
 
       </div>
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       <div class="modal-header">
 
       <div class="modal-header">
 
         <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">&times;</span></button>
 
         <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">&times;</span></button>
         <div class="modal-title" id="myModalLabel">Figure 7: 1% agarose gel after PCR of GFPmut2 with corresponding primers primers. Left: GFPmut2 (expected size 717bp); Middle: 1kb+ ladder, Right: PCR product of ChrB, using primers to add a BamHI restriction site in front of it and a His-Tag and a HindIII restriction site at the end. (expected size of ChrB: 939bp). Colours: Green: Prefix and Suffix; Red: In frame stop codons; Blue: First 15 codons optimised for expression in E. coli.
+
         <div class="modal-title" id="myModalLabel">Figure 7: Alignment of <i>chrB</i> (opt) sequence retrieved from <a href="http://parts.igem.org/Part:BBa_K1058008" target="_blank">BBa_K1058008</a> with the sequencing result of this region. Colour code: Green: Prefix and Suffix; Red: In frame stop codons; Blue: First 15 codons optimised for expression in E. coli.
 
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         </div>
 
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       </div>
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       <div class="modal-header">
 
       <div class="modal-header">
 
         <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">&times;</span></button>
 
         <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">&times;</span></button>
         <div class="modal-title" id="myModalLabel">Figure 10: 1% agarose gel after PCR of ChrBw4 (Expected size 939bp).
+
         <div class="modal-title" id="myModalLabel">Figure 11: 1% agarose gel after restriction digest of pSB1C3 with EcoRI and SpeI (Expected size 2070bp).
 
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         </div>
 
       </div>
 
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       <div class="modal-header">
 
       <div class="modal-header">
 
         <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">&times;</span></button>
 
         <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">&times;</span></button>
         <div class="modal-title" id="myModalLabel">Figure 10: 1% agarose gel after PCR of optimised ChrB. (Expected size 939bp).
+
         <div class="modal-title" id="myModalLabel">Figure 9: 1% agarose gel after PCR of <i>chrB</i> (opt). (Expected size 939bp).
 
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         </div>
 
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       </div>
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         <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">&times;</span></button>
 
         <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">&times;</span></button>
         <div class="modal-title" id="myModalLabel">Figure 11: Plates after transformation of ChrB-pUniprom, optimised ChrB-pUniprom and pChr-GFPmut2-pSB1C3 into JM110.
+
         <div class="modal-title" id="myModalLabel">Figure 10: Plates after transformation of Top: pUniprom<i>chrB</i>, Middle: pUniprom<i>chrB</i> (opt) and Bottom: pSB1C3-P<i>chr-gfp</i> into JM110.
 
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         </div>
 
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       <div class="modal-header">
 
         <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">&times;</span></button>
 
         <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">&times;</span></button>
         <div class="modal-title" id="myModalLabel">Figure 12: 1% agarose gel after restriction digest of pUniprom-ChrB, pUniprom-ChrB optimised and pSB1C3-pChr-GFP for confirmation of size. Ladder: 1kb+. Top: 1-4: pSB1C3-pChr-GFP 1-4; pUniprom-ChrB-opt 1-2. Bottom: pUniprom-ChrBopt 3-4; pUniprom-ChrB 1-4. Expected sizes: pChr-GFP: 909bp; ChrB and Chrb-opt: 939bp.
+
         <div class="modal-title" id="myModalLabel">Figure 12: 1% agarose gel after restriction digest of pUniprom<i>chrB</i>, pUniprom<i>chrB</i> (opt) and pSB1C3-P<i>chr-gfp</i> for confirmation of size. Ladder: 1kb+. Top: 1-4: pSB1C3-P<i>chr-gfp</i> 1-4; pUniprom<i>chrB</i> (opt) 1-2. Bottom: pUniprom<i>chrB</i> (opt) 3-4; pUniprom<i>chrB</i> 1-4. Expected sizes: P<i>chr-gfp</i>: 909bp; <i>chrB</i> and <i>chrB</i> (opt): 939bp.
 
         </div>
 
         </div>
 
       </div>
 
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       <div class="modal-header">
 
       <div class="modal-header">
 
         <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">&times;</span></button>
 
         <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">&times;</span></button>
         <div class="modal-title" id="myModalLabel">Figure 13: 1% agarose gel after colony PCR of pUniprom-ChrB-opt. Top: colonies 1-11; Bottom: colonies 12-22. Ladder: 1kb+ Expected size: 939bp.
+
         <div class="modal-title" id="myModalLabel">Figure 13: 1% agarose gel after colony PCR of pUniprom<i>chrB</i> (opt). Top: colonies 1-11; Bottom: colonies 12-22. Colonies 2, 4, 9, and 12 were selected for sequencing. Ladder: 1kb+ Expected size of <i>chrB</i> (opt): 939bp.
 
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       <div class="modal-header">
 
         <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">&times;</span></button>
 
         <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">&times;</span></button>
         <div class="modal-title" id="myModalLabel">Figure 14: Sequencing result of pSB1C3-pChr-GFP after double ligation. Even though the sequence is as expected, it is ligated exactly in reverse. Colours: Green: Prefix and partial Suffix; Blue: SpeI/XbaI-scar; Cyan: Ribosomal binding site, Red: In frame stop codons.
+
         <div class="modal-title" id="myModalLabel">Figure 14: Sequencing result of pSB1C3-P<i>chr-gfp</i> after double ligation. Even though the sequence is as expected, it is ligated in reverse. It was hence called pSB1C3-P<i>chr-gfp</i> (A) The Result for pSB1C3-P<i>chr-gfp</i> (colony 2) was not as expected, albeit in the correct orientation. Colours: Green: Prefix and partial Suffix; Blue: SpeI/XbaI-scar; Cyan: Ribosomal binding site, Red: In frame stop codons.
 
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         <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">&times;</span></button>
 
         <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">&times;</span></button>
         <div class="modal-title" id="myModalLabel">Figure 15: Plates with pSB1C3-pChr-GFP in JM110 after double ligation und UV light. Numbered colonies were selected for initial size confirmation and sequencing. However, other colonies seemed to express GFP as well, hence a second attempt was made with different colonies from the same plate.
+
         <div class="modal-title" id="myModalLabel">Figure 15: Plates with pSB1C3-P<i>chr-gfp</i> in JM110 after double ligation under UV light. Numbered colonies were selected for initial size confirmation and sequencing. However, other colonies seemed to express GFP as well, hence a second attempt was made with different colonies from the same plate.
 
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       <div class="modal-header">
 
       <div class="modal-header">
 
         <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">&times;</span></button>
 
         <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">&times;</span></button>
         <div class="modal-title" id="myModalLabel">Figure 16: Alignment of sequencing results of pSB1C3-pChr-GFP colonies 1, 5, 6, and 7 with GFPmut2 and pChr sequence. Colony 1 remained consistent, the other tested colonies were without success. Colours: Green: Prefix and partial Suffix; Blue: SpeI/XbaI-scar; Cyan: Ribosomal binding site, Red: In frame stop codons.
+
         <div class="modal-title" id="myModalLabel">Figure 16: Alignment of sequencing results of pSB1C3-P<i>chr-gfp</i> - colonies 1, 5, 6, and 7 with sequences of <i>gfp</i> (mut2) and P<i>chr</i> sequence. Colony 1 remained consistent, the other tested colonies were without success. Colours: Green: Prefix and partial Suffix; Blue: SpeI/XbaI-scar; Cyan: Ribosomal binding site, Red: In frame stop codons.
 
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       <div class="modal-header">
 
         <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">&times;</span></button>
 
         <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">&times;</span></button>
         <div class="modal-title" id="myModalLabel">Figure 18: Plates after transformation of pChr into pSB1C3. Top: ratio insert:vector 2:1; Bottom: ratio insert:vector 3:1.
+
         <div class="modal-title" id="myModalLabel">Figure 18: Plates after transformation of P<i>chr</i> into pSB1C3. Top: ratio insert:vector 2:1; Bottom: ratio insert:vector 3:1.
 
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       <div class="modal-header">
 
         <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">&times;</span></button>
 
         <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">&times;</span></button>
         <div class="modal-title" id="myModalLabel">Figure 19: Plates after transformation of pChr into pSB1C3. Top: ratio insert:vector 2:1; Bottom: ratio insert:vector 3:1.
+
         <div class="modal-title" id="myModalLabel">Figure 19: Alignment of P<i>chr</i> sequence retrieved from <a href="http://parts.igem.org/Part:BBa_K1058008" target="_blank">BBa_K1058008</a> with the sequencing result of this region. Colour code: Green = Prefix/Suffix
 
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         <div class="modal-title" id="myModalLabel">Figure 20: Plates after transformation of, Top: ChrB and into pUniprom; Bottom: optimised ChrB into pUniprom. Ratio insert:vector left: 3:1; Right: 4:1.
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         <div class="modal-title" id="myModalLabel">Figure 20: Plates after transformation of, Top: <i>chrB</i> into pUniprom; Bottom: <i>chrB</i> (opt) into pUniprom. Ratio insert:vector for ligations: left: 3:1; Right: 4:1.
 
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         <div class="modal-title" id="myModalLabel">Figure 21: Gel after restriction digest of Left: pUniprom-ChrB, and Right: pUniprom-ChrBopt with BamHI/PstI for confirmation of insert size. Expected size 939bp. ChrB2 and ChrBopt3 were selected for sequencing.
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         <div class="modal-title" id="myModalLabel">Figure 21: Gel after restriction digest of Left: pUniprom-<i>chrB</i>, and Right: pUniprom-<i>chrB</i> (opt) with BamHI/PstI for confirmation of insert size. Expected size 939bp. <i>chrB</i> colony 2 and <i>chrB</i> (opt) colony 3 were selected for sequencing.
 
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         <div class="modal-title" id="myModalLabel">Figure 22: Alignments of ChrB (left) and ChrBopt (right). Colours: Pink = Restriction sites (BamHI/PstI); Blue = Hexa-Histidine-Tag; Red = In-frame stop codons; Cyan = optimised codons.
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         <div class="modal-title" id="myModalLabel">Figure 22: Alignments of: Left: <i>chrB</i> and Right: <i>chrB</i> (opt). Colours: Pink = Restriction sites (BamHI/PstI); Blue = 6-His Tag; Red = In-frame stop codons; Cyan = optimised codons.
 
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         <div class="modal-title" id="myModalLabel">Figure 23: Plates after ligation of GFP into pSB1C3-pChr. Left: Ratio Insert:Vector 2:1; Right: Ration Insert:Vector 3:1.
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         <div class="modal-title" id="myModalLabel">Figure 23: Plates after ligation of <i>gfp</i> into pSB1C3-P<i>chr</i>. Left: Ratio Insert:Vector 2:1; Right: Ratio Insert:Vector 3:1.
 
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         <div class="modal-title" id="myModalLabel">Figure 24: Gel for size confirmation of pSB1C3-pChr-GFP (Top). The bottom lanes are unrelated to this project. Samples 3 and 4 were submitted for sequencing.
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         <div class="modal-title" id="myModalLabel">Figure 24: Gel for size confirmation of pSB1C3-P<i>chr-gfp</i> (Top). Bottom: Left - Haptoglobin, Right - Lanosterol Synthase. pSB1C3-P<i>chr-gfp</i> (3) and pSB1C3-P<i>chr-gfp</i> (4) were submitted for sequencing.
 
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         <div class="modal-title" id="myModalLabel">Figure 25: Gel for size confirmation of pSB1C3-pChr-GFP (Top). The bottom lanes are unrelated to this project. Samples 3 and 4 were submitted for sequencing.
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         <div class="modal-title" id="myModalLabel">Figure 25: Alignment of P<i>chr</i> sequence retrieved from <a href="http://parts.igem.org/Part:BBa_K1058008" target="_blank">BBa_K1058008</a> and <i>gfp</i> (mut2) with the forward and reverse sequencing results of P<i>chr-gfp</i> this region. The sequence was as expected, it was hence called pSB1C3-P<i>chr-gfp</i> (B). Colour code: Green = Prefix/Suffix; Blue: SpeI/XbaI-scar; Cyan: Ribosomal binding site, Red: In frame stop codons.
 
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         <div class="modal-title" id="myModalLabel">Figure 26: Western blot against GFP (left) and ChrB (right). Expected sizes: GFP ~ 27kDa, ChrB ~35kDa. Left: Lane1 - pChrGFP inverse from 15/07; Lane2 - pChrGFP3 normal from 31/07; Lane3 - pChr only, negative control; Lane4 - positive control. Right: Lane1 - ChrB2, Lane2 - ChrBopt3, Lane3 - pUniprom empty vector, negative control, Lane4 - positive control, expected size ~35kDa.
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         <div class="modal-title" id="myModalLabel">Figure 26: Top: anti-GFP: Single colonies of JM110 + pSB1C3-P<i>chr-gfp</i> (A), MC1061 + pSB1C3-P<i>chr-gfp</i> (B) were used to inoculate 5 ml of LB broth supplemented with 100 <sup>µg</sup>/<sub>ml</sub> chloramphenicol. After 16h of incubation at 37°C with agitation at 200rpm, each sample was subcultured into 5 ml of fresh, equally supplemented LB and cells were grown for 2 hours more. 1 ml of the subculture was then retrieved and pelleted. The pellet was resuspended in 1 ml TBS. 100 µl of each sample was mixed with 100 µl laemmli buffer, and boiled for 10min. 3 µl of each sample was loaded on a  SDS gel (12% acrylamide). pSB1C3 was included as a negative control, and P<i>manA-gfp</i> as a positive control.  
 +
        Bottom: anti-6-His - ChrB: Single colonies of JM110 pUniprom-<i>chrB</i> and JM110 pUniprom-<i>chrB</i> (opt) were used to inoculate 5 ml of LB broth supplemented with 100 <sup>µg</sup>/<sub>ml</sub> ampicillin. After 16h of incubation at 37°C with agitation at 200rpm, each sample was subcultured into 5 ml of fresh, equally supplemented LB and cells were grown for 2 hours more. 1 ml of the subculture was then retrieved and pelleted. The pellet was resuspended in 200 µl laemmli buffer, and the sample boiled for 10min. 10 µl of each sample was loaded on a  SDS gel (12% acrylamide). pSB1C3 was included as a negative control, and <i>hydA - 6-His</i> as a positive control.
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        Expected sizes: GFP ~ 27kDa, ChrB ~35kDa.
 
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         <div class="modal-title" id="myModalLabel">Figure 27: Transformations of single constituents of Chromate sensing system.
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         <div class="modal-title" id="myModalLabel">Figure 27: Plates after transformation of single constituents of chromate sensor into <i>E. coli</i> MG1655. From top to bottom: pUniprom-<i>chrB</i> (from colony 2 - 29/07), pUniprom-<i>chrB</i> (opt) (from colony 3 - 29/07), pSB1C3-P<i>chr-gfp</i> (B), pSB1C3-P<i>chr-gfp</i> (A), <a href="http://parts.igem.org/Part:BBa_K1058008" target="_blank">BBa_K1058008</a>.
 
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         <div class="modal-title" id="myModalLabel">Figure 28: Western Blot against GFP.
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         <div class="modal-title" id="myModalLabel">Figure 28: Western blot against GFP: Single colonies of MG1655, containing all 4 combinations, pUniprom-<i>chrB</i> - pSB1C3-P<i>chr-gfp</i> (A), pUniprom-<i>chrB</i> - pSB1C3-P<i>chr-gfp</i> (B), pUniprom-<i>chrB</i> (opt) - pSB1C3-P<i>chr-gfp</i> (A), and pUniprom-<i>chrB</i> (opt) - pSB1C3-P<i>chr-gfp</i> (B) were used to inoculate 5 ml of LB broth supplemented with 100 <sup>µg</sup>/<sub>ml</sub> chloramphenicol and 100 <sup>µg</sup>/<sub>ml</sub> ampicillin. After 16h of incubation at 37°C with agitation at 200rpm, each sample was subcultured into 5 ml of fresh, equally supplemented LB and cells were grown for 2 hours more. 1 ml of the subculture was then retrieved and pelleted. The pellet was resuspended in 1 ml PBS. 100 µl of these samples was mixed with 100 µl laemmli buffer, and the sample boiled for 10 min. The indicated volume of each sample was loaded on a SDS gel (12% acrylamide). pSB1C3, and pUniprom were included as a negative control, and P<i>manA-gfp</i> as a positive control. The blot against 6-His – ChrB was without success and is not shown. No chromate of any kind was added to the medium previous to this blot, hence no expression of GFP was expected.  
 
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K<sub>2</sub>CrO<sub>4</sub>, K<sub>2</sub>Cr<sub>2</sub>O<sub>7</sub>, CrCl<sub>3</sub>, and K<sub>2</sub>SO<sub>4</sub>
  
 
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         <div class="modal-title" id="myModalLabel">Figure 29: Results of Plate reader experiment after exposing MG1655 with BBa_K1058008 to different concentrations of chromate and control substances. One measurement was made every 10min across 16h.
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         <div class="modal-title" id="myModalLabel">Figure 29: F,OD-plot of results of plate reader experiment after exposing MG1655 + BBa_K1058008 to a range of concentrations between 10 nM and 100 µM of K<sub>2</sub>CrO<sub>4</sub>, K<sub>2</sub>Cr<sub>2</sub>O<sub>7</sub>, CrCl<sub>3</sub>, and K<sub>2</sub>SO<sub>4</sub>. One measurement was made every 10min across 16h.
 
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         <div class="modal-title" id="myModalLabel">Figure 30: Results of Plate reader experiment after exposing MG1655 with pSB1C3-pChr-GFP and pUniprom-ChrB to different concentrations of chromate and control substances. One measurement was made every 10min across 16h.
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         <div class="modal-title" id="myModalLabel">Figure 30: F,OD-plot of results of plate reader experiment after exposing MG1655 + pSB1C3-P<i>chr-gfp</i> (B) + pUniprom-<i>chrB</i> to a range of concentrations between 10 nM and 100 µM of K<sub>2</sub>CrO<sub>4</sub>, K<sub>2</sub>Cr<sub>2</sub>O<sub>7</sub>, CrCl<sub>3</sub>, and K<sub>2</sub>SO<sub>4</sub>. One measurement was made every 10min across 16h.
 
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         <div class="modal-title" id="myModalLabel">Figure 31: Results of Plate reader experiment after exposing MG1655 with pSB1C3-pChr-GFP and pUniprom-ChrBopt to different concentrations of chromate and control substances. One measurement was made every 10min across 16h.
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         <div class="modal-title" id="myModalLabel">Figure 31: F,OD-plot of results of plate reader experiment after exposing MG1655 + pSB1C3-P<i>chr-gfp</i> (B) + pUniprom-<i>chrB</i> (opt) to a range of concentrations between 10 nM and 100 µM of K<sub>2</sub>CrO<sub>4</sub>, K<sub>2</sub>Cr<sub>2</sub>O<sub>7</sub>, CrCl<sub>3</sub>, and K<sub>2</sub>SO<sub>4</sub>. One measurement was made every 10min across 16h.
 
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         <div class="modal-title" id="myModalLabel">Figure 32: Results of Plate reader experiment after exposing MG1655 with pSB1C3-pChr-GFP to different concentrations of chromate and control substances. One measurement was made every 10min across 16h.
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         <div class="modal-title" id="myModalLabel">Figure 32: F,OD-plot of results of plate reader experiment after exposing MG1655 + pSB1C3-P<i>chr-gfp</i> (B) to a range of concentrations between 10 nM and 100 µM of K<sub>2</sub>CrO<sub>4</sub>, K<sub>2</sub>Cr<sub>2</sub>O<sub>7</sub>, CrCl<sub>3</sub>, and K<sub>2</sub>SO<sub>4</sub>. One measurement was made every 10min across 16h.
 
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         <div class="modal-title" id="myModalLabel">Figure 33: Plates after transformation into pT7KS.
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         <div class="modal-title" id="myModalLabel">Figure 33: Plates after ligation of <i>chrB</i> (Bielefeld) into pT7KS. From top to bottom: Ratio insert:vector 2:1; Ratio Insert:Vector 3:1; pT7KS religation control.
 
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         <div class="modal-title" id="myModalLabel">Figure 34: Plates after transformation of chromate sensor plasmids into BL21 (DE3).
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         <div class="modal-title" id="myModalLabel">Figure 34: Plates after transformation of single constituents of chromate sensor into <i>BL21 (DE3)</i>. From top to bottom: pSB1C3-P<i>chr-gfp</i> (B) + pUniprom-<i>chrB</i>, pSB1C3-P<i>chr-gfp</i> (B) + pUniprom-<i>chrB</i> (opt), pUniprom-<i>chrB</i>, and pUniprom-<i>chrB</i> (opt).
 
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         <div class="modal-title" id="myModalLabel">Figure 35: Agarose gel after colony PCR.
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         <div class="modal-title" id="myModalLabel">Figure 35: 1% agarose gel after colony PCR of pT7KS-<i>chrB</i> (Bielefeld). Top: colonies 1-6; Bottom: colonies 7-12. Ladder: 1kb+ Expected size of <i>chrB</i> (Bielefeld): 939bp
 
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         <div class="modal-title" id="myModalLabel">Figure 35: Agarose gel after colony PCR.
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         <div class="modal-title" id="myModalLabel">Figure 36: F,OD-plot of results of plate reader experiment after inducing expression of ChrB from the T7 promoter of pUniprom <i>BL21 (DE3)</i> pSB1C3-P<i>chr-gfp</i> (B) + pUniprom-<i>chrB</i>, and pSB1C3-P<i>chr-gfp</i> (B) + pUniprom-<i>chrB</i> (opt). IPTG was added after 4 hours. One measurement was made every 10min across 22h.
 
         </div>
 
         </div>
 
       </div>
 
       </div>

Latest revision as of 21:19, 17 September 2015

Dundee iGEM 2015

LABJOURNAL

BioSpray

Our forensic toolkit aims to use synthetic biology approaches to improve on the current methods used by crime scene investigators.

Chromium Detector

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Fingerprint Aging

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Week Beginning 22/06/2015

Summary

This week was all about getting started with the newly arrived biobrick BBa_K1058008

22/06: Plated BBa_K1058008 from agar stab.

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing. The culture was plated from the agar stab and also a liquid culture set up.

Protocols Used: Plating, Overnight cultures

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

23/06: Purified BBa_K1058008 from liquid culture.

Aim of experiment: To purify BBa_K1058008 for sequence confirmation.

Protocols Used: Miniprep plasmid purification, DNA sequencing

Results Sequencing: Figure 1

Next Steps: PCR of promoter region (Pchr) and repressor region (chrB) of BBa_K1058008 for storage in individual biobricks.

Week Beginning 29/06/2015

Summary

During this week, BBa_K1058008 was disassembled into its constituent parts, Pchr, and chrB.

29/06: PCR-amplification of pChr and ChrB from BBa_K1058008.

Aim of experiment: To break down BBa_K1058008 into the promoter region pChr, and the repressor gene, ChrB, while adding standard prefix and suffix to each. Furthermore optimisation of first 15 codons of ChrB in order to increase the overall rate of transcription. Restriction digest of the respective ends and subsequent ligation of pChr and ChrB into pSB1C3 for submission to registry.

Protocols Used: PCR, Gel extraction, Restriction Digest

Results: Figure 2

Next Steps: Repeat of PCR of ChrB. Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Week Beginning 06/07/2015

Summary

During this week ligations of pChr and ChrB into pSB1C3 were finalised and the constituent parts of our newly designed chromate sensing system were prepared for ligation and transformation.

06/07: Repeated amplification of ChrB. Ligation and transformation of pChr and ChrB.

Aim of experiment: To repeat PCR of ChrB for subsequent ligation into pSB1C3. Ligation of pChr and ChrB into pSB1C3. Transformation of each into a non-pathogenic lab strain of Escherichia coli. Overnight culture of E. coli DH5-alpha, containing GFPmut2, as a fluorescent reporter for our chromate sensing system

Protocols Used: PCR-amplification, Gel extraction, Restriction digest, Ligation, Overnight cultures

Results: Figure 3

2 bands were extracted from the gel for further characterisation, the strongest band, hence called ChrBs, and the band directly above, hence called ChrBw. pChr, ChrBs, and ChrBw were ligated into pSB1C3. pSB1C3-pChr, pSB1C3-ChrBs, and pSB1C3-ChrBw were transformed into E. coli MC1061.

Next Steps: Sequence and size confirmation of pChr-pSB1C3, ChrBs-pSB1C3, and ChrBw-pSB1C3. Ligation of pChr to GFP coding sequence for construction of a reporter plasmid.

07/07: Purification of gfp and overnight cultures of pSB1C3-Pchr, pSB1C3-chrB (s), and pSB1C3-chrB (w)

Aim of experiment: Purification of GFPmut2 from overnight cultures. Overnight cultures of recombinant E. coli MC1061, containing pSB1C3-Pchr, pSB1C3-chrB (s), and pSB1C3-chrB (w) respectively for further size and sequence confirmation. Overnight culture of pUniprom for subsequent ligation of chrB

Protocols Used: Miniprep, PCR-amplification, Gel extraction, Overnight cultures

Results: Figure 4

Next Steps: Repeat of amplification of ChrB. Sequence and size confirmation of pSB1C3-Pchr, pSB1C3-chrB (s), and pSB1C3-chrB (w).

08/07: Purification of plasmids pSB1C3-Pchr, pSB1C3-chrB (s), and pSB1C3-chrB (w). Size and sequence confirmation.

Aim of experiment: To purify overnight cultures for size and sequence confirmation. Repeat of PCR amplification of pChr and ChrB. Restriction digestes of all parts in order to produce compatible sticky ends.

Protocols Used: Miniprep, PCR amplification, Restriction digest, Sequencing

Restriction digest of gel purified PCR-products of Pchr, chrB, and gfp, using EcoRI/SpeI, BamHI/HindIII, and XbaI/PstI respecitvely.

Results:

Figure 5 - Size confirmation of pChr and ChrB.

Figure 6 - Sequence confirmation of Pchr

Figure 7 - Sequence confirmation of chrB.

Next Steps: Purification and digest of pUniprom for insertion of chrB.

09/07: Digested and cleaned up pUniprom for insertion of chrB. Amplification of optimised chrB with primers specific for the insertion into pUniprom.

Aim of experiment: To prepare pUniprom for insertion of chrB by digesting it with BamHI and HindIII. To amplify chrB version with first 15 codons optimised for insertion into pUniprom.

Protocols Used:

Restriction digest and Gel extraction of pUniprom

PCR and Gel extraction of chrB (opt)

Results:

Figure 8 Gel with pUniprom

Figure 9 Gel with chrB

Next Steps: Ligation of chrB and chrB (opt) into pUniprom.

10/07: Ligations of chrB and chrB (opt) into pUniprom, and of Pchr and gfp into pSB1C3.

Aim of experiment: Ligation of chrB and optimised chrB into pUniprom via BamHI and HindIII restriction sites. Ligation of Pchr and gfp to each other via SpeI/XbaI-link and into pSB1C3 via EcoRI and PstI restriction sites.

Protocols Used: Ligations

Results: N/A

Next Steps: Transformation of abovementioned ligations.

Week Beginning 13/07/2015

Summary

During this week successful transformants were identified and propagated.

13/07: Transformation of pUniprom-chrB, pUniprom-chrB (opt), and pSB1C3-Pchr-gfp.

Aim of experiment: Transformation of pUniprom-chrB, pUniprom-chrB (opt), and pSB1C3-Pchr-gfp into E. coli JM110 chassis. Setting up a backup restriction digest of pSB1C3 with EcoRI and SpeI in case the double ligation pSB1C3-Pchr-gfp is unsuccessful.

Protocols Used: Transformations, Restriction digest

Results:

Figure 10 Plates after transformation

Figure 11 Gel after digest of pSB1C3

Next Steps: Purification of recombinant vectors and confirmation of size and sequence. Store digested pSB1C3 at -20oC.

14/07: Set up cultures for subsequent purification of recombinant vectors for size and sequence confirmation.

Aim of experiment: Purification of recombinant vectors, pUniprom-chrB, pUniprom-chrB (opt), and pSB1C3-Pchr-gfp, for size and sequence confirmation. Patch plating of the same colonies for storage and retrieval once size and sequence have been confirmed.

Protocols Used: Overnight cultures, Patch plating

Results: N/A

Next Steps: Purification of the recombinant plasmids for confirmation of size and sequence.

15/07: Purified pUniprom-chrB, pUniprom-chrB (opt), and pSB1C3-Pchr-gfp from overnight cultures

Aim of experiment: Purification of pUniprom-chrB, pUniprom-chrB (opt), and pSB1C3-Pchr-gfp from overnight for confirmation of size and sequence. Restriction digest of pUniprom-chrB, pUniprom-chrB (opt) with BamHI and HindIII and of pSB1C3-Pchr-gfp with EcoRI/PstI for size confirmation.

Protocols Used: Miniprep, Restriction digest, Colony PCR, Sequencing

Results:

Figure 12 Size confirmation of chrB and chrB (opt).

Figure 13 Gel after colony PCR of chrB (opt).

Figure 14 Sequencing result of pSB1C3-Pchr-gfp (colony 1).

Next Steps: Confirmation of sequence of chrB (opt)

16/07: Set up overnight cultures of chrB (opt) - colonies chosen after colony-PCR for subsequent purification and sequence confirmation.

Aim of experiment: Set up overnight cultures of chrB (opt) colonies 2, 4, 9, and 12 for sequencing.

Protocols Used: Overnight cultures

Results: N/A

Next Steps: Purification of recombinant plasmids.

17/07: Purified and sequenced chosen chrB (opt) - colonies.

Aim of experiment: To purify recombinant plasmids for sequence confirmation.

Protocols Used: Miniprep, Sequencing

Results: chrB (opt) showed HindIII restriction sites and was hence cleaved into partial inserts.

Next Steps: Use alternative cloning protocol for chrB and chrB (opt).

Week Beginning 20/07/2015

Summary

During this week an alternative cloning strategy for chrB and chrB (opt) was employed. Furthermore, the pSB1C3-Pchr-gfp - construct was cloned in a stepwise fashion.

20/07: Went back to the original plate of pSB1C3-Pchr-gfp for visualising colonies that do or do not express GFP.

Aim of experiment: To compare colonies on the patch plate of pSB1C3-Pchr-gfp by differentially visualising colonies that do or do not express GFP under UV light. The background of this experiment was that if there is one colony that has both inserts in the correct orientation with respect to each other, then there is perhaps one that also has them with the correct orientation with respect to the plasmid. Colonies identified as expressing GFP were patch plated and grown in liquid cultures over night in order to confirm the sequence of the insert.

Protocols Used: Patch-plating, overnight cultures

Results: Figure 15 pSB1C3-Pchr-gfp in E. coli JM110.

Next Steps: Sequence confirmation of newly selected colonies.

21/07: Purified plasmids from new colonies and confirmed sequence. Restriction digest of pUniprom with BamHI and PstI.

Aim of experiment: Purification of newly selected pSB1C3-Pchr-gfp - colonies 1, 5, 6, and 7, for subsequent sequence confirmation. Restriction digest of pUniprom with BamHI and PstI to employ an alternative cloning strategy of chrB and chrB (opt).

Protocols Used:

Miniprep and Sequencing of Pchr-gfp - insert.

Restriction digest of pUniprom.

Results:

Figure 16 Alignment of sequencing results.

Figure 17 Gel after restriction digest of pUniprom.

Next steps: Stepwise ligation of Pchr and gfp into pSB1C3 in order to clone the inserts in the correct orientation with respect to the plasmid. Insertion of chrB and chrB (opt) into pUniprom via BamHI and PstI links.

22/07: Ligated Pchr into pSB1C3 via EcoRI and SpeI sites and cloned the recombinant plasmid into E. coli JM110.

Aim of experiment: Ligation of Pchr into pSB1C3 via EcoRI and SpeI sites. This is the first step of a 2-step cloning strategy with the aim of cloning Pchr and gfp into pSB1C3 in the correct orientation with respect to the plasmid.

Protocols Used: Ligation, Transformation

Results: Figure 18 Plates after transformation of Pchr into pSB1C3.

Next Steps: Sequence confirmation of Pchr in pSB1C3.

23/07: Set up overnight cultures of pSB1C3-Pchr colonies.

Aim of experiment: Set up overnight cultures of pSB1C3-Pchr colonies to prepare pSB1C3-Pchr for sequence confirmation

Protocols Used: Overnight culture, Patch-plating

Results: N/A

Next Steps: Purification of the recombinant vector for size and sequence confirmation.

24/07: Purified pSB1C3-Pchr for sequence confirmation.

Aim of experiment: Purification of pSB1C3-Pchr for sequence confirmation before ligating gfp into it.

Protocols Used:

Plasmid purification, Sequencing

Results: Figure 19 Alignment of sequenced pSB1C3-Pchr vectors with Pchr sequence.

Next Steps: Digest of pSB1C3-Pchr with SpeI/PstI and ligation of Pgfp

25/07: PCR-amplified chrB and chrB (opt).

Aim of experiment: Amplification of chrB and chrB (opt) for insertion into pUniprom. Restriction digest of the PCR product with BamHI and PstI for producing compatible sticky ends

Protocols Used: PCR-amplification, Restriction digest

Results: N/A

Next Steps: Ligation and Transformation of chrB and chrB (opt).

26/07: Ligated chrB and chrB (opt) into pUniprom.

Aim of experiment: Insertion of chrB and chrB (opt) into pUniprom.

Protocols Used: Ligation

Results: N/A

Next Steps: Transformation of ligated vectors.

Week Beginning 27/07/2015

Summary

Final touches were made on the chromate system. All parts were ligated into their final vectors during this week. Preparations were made for characterisation of each of the parts.

27/07: Transformed chrB and chrB (opt) into E. coli JM110

Aim of experiment: Transformation of the repressors, chrB and chrB (opt), into JM110

Protocols Used: Transformation

Results: Figure 20 Plates after transformation.

Next Steps: Purification of pUniprom-chrB and pUniprom-chrB (opt) for sequence confirmation.

28/07: Digested pSB1C3-Pchr for insertion of gfp

Aim of experiment: Digesting sequence confirmed pSB1C3-Pchr with SpeI and PstI for subsequent ligation with gfp. Overnight cultures of chrB and chrB (opt) for subsequent plasmid purification, size confirmation, and sequence confirmation.

Protocols Used:

Restriction digest, Ligation of gfp into pSB1C3-Pchr.

Overnight culture of of chrB and chrB (opt).

Results: N/A

Next Steps: Transformation of pSB1C3-Pchr-gfp for size and sequence confirmation. Purification of chrB and chrB (opt) for sequence confirmation.

29/07: Purified chrB and chrB (opt) for size and sequence confirmation.

Aim of experiment: Purification of chrB and chrB (opt) for confirmation of size and sequence.

Protocols Used: Plasmid purification, Restriction digest, Sequencing

Results:

Figure 21 Confirmation of insert size.

Figure 22 Confirmation of sequence.

Next Steps: Confirmation of expression of chrB and chrB (opt) by western blotting.

30/07: Repeated transformation of pSB1C3-Pchr-gfp into E.coli JM110

Aim of experiment: Digest of pSB1C3-Pchr with SpeI and PstI for insertion of GFP. Repeat of unsuccessful transformation from 28/07.

Protocols Used: Restriction digest, Ligation, Transformation

Results: Figure 23 Plates after transformation

Next Steps: Overnight cultures for purification of the pSB1C3-Pchr-gfp for confirmation of insert size and sequence.

31/07: Set up overnight cultures for confirmation of pSB1C3-Pchr-gfp.

Aim of experiment: Overnight cultures for subsequent plasmid purification of pSB1C3-Pchr-gfp.

Protocols Used: Overnight culture

Results: N/A

Next Steps: Purification of the Plasmids for confirmation of insert size and sequence.

01/08: Purified pSB1C3-Pchr-gfp for size confirmation and sequencing.

Aim of experiment: Purification of pSB1C3-Pchr-gfp for confirmation of insert size and sequence.

Protocols Used: Plasmid purification, Restriction digest, Sequencing

Results:

Figure 24 Gel for size confirmation.

Figure 25 Sequence confirmation.

Next Steps: Overnight cultures in preparation for western blotting for characterisation of expression of both, GFP, and ChrB and ChrBopt.

02/08: Set up cultures of JM110 + pSB1C3-Pchr-gfp (A), MC1061 + pSB1C3-Pchr-gfp (B) and JM110 pUniprom-chrB and JM110 pUniprom-chrB (opt). in preparation for western blotting.

Aim of experiment: Overnight cultures of E. coli strains with sequence confirmed pUniprom-chrB, pUniprom-chrB (opt), pSB1C3-Pchr-gfp (A), pSB1C3-Pchr-gfp (B) - colony 3 and pSB1C3-Pchr-gfp (B) - colony 4 for subsequent western blotting.

Protocols Used: Overnight culture

Results: N/A

Next Steps: Western blotting for characterisation of expression of both, GFP, and ChrB and ChrB (opt).

Week Beginning 03/08/2015

Summary

This week was entirely used for characterising the expression of GFP from the promoter Pchr, and the expression of ChrB from the Ptat in pUniprom.

03/08 - 09/08: Determined expression of ChrB, ChrB (opt), and GFP by western blotting.

Aim of experiment: To determine whether or not the required proteins, GFP, and ChrB, are expressed by blotting against GFP and against a 6-His Tag of ChrB and ChrB (opt) respectively.

Protocols Used: SDS-PAGE and western blot

Results: Figure 26

Next Steps: Transformation of both plasmids into MG1655 with the goal to characterise the interaction between ChrB and Pchr. Furthermore the original biobrick, BBa_K1058008 will be tested under the same conditions for comparison.

Week Beginning 10/08/2015

Summary

This week was spent with transforming all combinations of pSB1C3-Pchr-gfp and pUniprom-chrB into E. coli MG1655. Tests for their interaction were also prepared.

10/08: Transformed pSB1C3-Pchr-gfp (A) and pSB1C3-Pchr-gfp (B), and pUniprom-chrB, and pUniprom-chrB (opt), as well as BBa_K1058008 into E. coli MG1655.

Aim of experiment: To transform the following combinations of pSB1C3-pChr-GFP and pUniprom-ChrB into MG1655:

  • pUniprom-chrB (from colony 2 - 29/07) - pSB1C3-Pchr-gfp (A)
  • pUniprom-chrB (from colony 2 - 29/07) - pSB1C3-Pchr-gfp (B)
  • pUniprom-chrB (opt) (from colony 3 - 29/07) - pSB1C3-Pchr-gfp (A)
  • pUniprom-chrB (opt) (from colony 3 - 29/07) - pSB1C3-Pchr-gfp (B)
in a stepwise fashion. Furthermore transform BBa_K1058008 into MG1655. Transformations were done in a stepwise fashion with only one of the plasmids at a time.

Protocols Used: Plasmid purification, Transformation

Results: Figure 27 Plates after transformation.

Next Steps: Making newly transformed cells competent again in order to transform the second plasmid as well.

11/08: Set up overnight cultures of all transformed cells.

Aim of experiment: To prepare overnight cultures of yesterdays transformations in order to make them competent to transform the second plasmid as well.

Protocols Used: Overnight culture

Results: N/A

Next Steps: Make cells of overnight culture competent to transform in second plasmid.

12/08: Made competent MG1655 with pUniprom-ChrB2, pUniprom-ChrBopt3, pSB1C3-pChr-GFP (15/07), pSB1C3-pChr-GFP (31/07).

Aim of experiment: To make transformed cells competent in order to transform the second plasmid. The same plasmids that were used for the initial transformations were also used for this set of transformations.

Protocols Used: Competent cells, Transformations

Results: N/A

Next Steps: Blot against GFP in order to confirm (or refute) that ChrB interacts with Pchr and hence switches off expression of GFP.

13/08: Transformed pUniprom-chrB (from colony 2 - 29/07) and pUniprom-chrB (opt) (from colony 3 - 29/07) into MG1655 + pSB1C3-Pchr-gfp (A) and pSB1C3-Pchr-gfp (B)

Aim of experiment: To build a strain of E. coli MG1655, containing both plasmids required for a chromate sensor.

Protocols Used: Transformation

Results: No successful transformants were found.

Next Steps: Repeat this experiment but transform pSB1C3-Pchr-gfp (A) and pSB1C3-Pchr-gfp (B) into competent MG1655 + pUniprom-chrB and MG1655 + pUniprom-chrB (opt) respectively.

Week Beginning 17/08/2015

Summary

During this week E. coli MG1655 containing both plasmids for a. Western Blots were used to determine expression of ChrB and GFP.

17/08: Transformations akin to 13/08 were done, but pSB1C3-Pchr-gfp (A) and pSB1C3-Pchr-gfp (A) were transformed into MG1655 + pUniprom-chrB and MG1655 + pUniprom-chrB (opt).

Aim of experiment: To build a strain of MG1655, containing both plasmids required for a chromate sensor.

Protocols Used: Transformation

Results: Successful transformants were found on all plates.

Next Steps: To determine expression of ChrB and GFP.

18/08: Preparation of samples for western blot.

Aim of experiment: To determine whether ChrB and GFP are expressed and have first estimates of differences in expression levels.

Protocols Used: Overnight culture

Results: N/A

Next Steps: Lyse cells and prepare samples for SDS-PAGE + Western-Blot.

19/08 - 22/08: Repeated western blots with different concentrations to determine whether the expression of ChrB has an effect on GFP levels.

Aim of experiment: To determine whether expression of ChrB has an effect on the expression of GFP.

Protocols Used: SDS-PAGE + Western Blot

Results: Figure 28

Next Steps: Check if different chromate salts have an effect on the respective expression levels.

Week Beginning 24/08/2015

Summary

Plate reader experiments were carried out in order to determine the effect of K2CrO4, K2Cr2O7, CrCl3, and K2SO4 on the expression of GFP.

24/08-30/08: Design of Plate reader experiments.

Aim of experiment: To design a series of plate reader experiments to measure OD600 and relative fluorescence of the chromate sensor as a response to different concentrations of K2CrO4, K2Cr2O7, CrCl3, and K2SO4.

Protocols Used: Plate Reader Experiments

Results: Figure 29 MG1655 + BBa_K1058008

Results: Figure 30 MG1655 + pSB1C3-Pchr-gfp (B) + pUniprom-chrB

Results: Figure 31 MG1655 + pSB1C3-Pchr-gfp (B) + pUniprom-chrB (opt)

Results: Figure 32 MG1655 + pSB1C3-Pchr-gfp (B)

Next Steps: Try the system with different expression levels of ChrB. Induce overexpression by exploiting the T7-promoter in pUniprom after transforming the different combinations between pSB1C3-Pchr-gfp and pUnipromchrB / pUnipromchrB (opt) into BL21(DE3) and adding IPTG.

Week Beginning 31/08/2015

Summary

UK Synbio Conference and UK iGEM meetup at Westminster University.

Week Beginning 07/09/2015

Summary

Parts from CeBiTec Bielefeld arrived and attempts at cloning were made. The chromate sensing system was transformed into BL21(DE3) and some of the plate reader experiments repeated.

07/09: Transformation of pSB1K3 (Bielefeld) into MG1655.

Aim of experiment: The high-copy plasmid arrived at a concentration of 7 ng/µl. Hence it was transformed in order to produce more of it before purifying and digesting it.

Protocols Used: Transformation,

Overnight culture for new E. coli MG1655

Results: No transformants were found.

Next Steps: Retry transformation with new competent E. coli MG1655.

08/09: Transformation of pSB1K3 into MG1655.

Aim of experiment: Test transformation of BBa_K1058008 into new MG1655 competent cells and re-transformation of pSB1K3 for subsequent miniprep.

Protocols Used: Transformation

Results: Successful test transformation, but no colonies were found on the pSB1K3 plate.

Next Steps: Use an alternative compatible plasmid for ligation of the codon optimised chrB (Bielefeld).

09/09: Transformation of pSB1K3 into MG1655.

Aim of experiment: Restriction digest of pT7KS with EcoRI and PstI as alternative to pSB1K3. Ligation of digested chrB (Bielefeld) and transformation of pT7KS-chrB. Also transformations of Chromate-sensor plasmids into BL21 (DE3) in the following combinations:

  • pSB1C3-Pchr-gfp (B) + pUniprom-chrB
  • pSB1C3-Pchr-gfp (B) + pUniprom-chrB (opt)
  • pUniprom-chrB
  • pUniprom-chrB (opt)

Protocols Used: Restriction Digest, Gel Extraction, Ligation

Results: Transformations of the plasmid with pT7KS-chrB (Bielefeld) yielded 12 colonies, but also colonies on the vector control plate. Transformations of pSB1C3-Pchr-gfp (B) + pUniprom-chrB, pSB1C3-Pchr-gfp (B) + pUniprom-chrB (opt), pUniprom-chrB, and pUniprom-chrB (opt) into BL21(DE3) were successful.

Results:

Figure 33 pT7KS-chrB (Bielefeld) transformations

Figure 34 Transformations into BL21(DE3)

Next Steps: Confirmation of colonies with pT7KS-chrB (Bielefeld) by colony PCR. Preparation of recombinant BL21(DE3) for plate reader experiments.

10/09: Colony PCR of pT7KS-chrB (Bielefeld).

Aim of experiment: Check if any of the colonies after ligation of chrB (Bielefeld) into pT7KS were successful despite colonies on the control plate.

Protocols Used: Colony PCR

Results: Figure 35

Next Steps: Prepare transformed BL21(DE3) for plate reader experiments. Attempt new transformation of pT7KS-chrB (Bielefeld).

11/09: Preparation for Plate reader experiment with BL21(DE3)

Aim of experiment: Set up overnight cultures for subsequent plate reader experiments.

Protocols Used: Overnight cultures

Results: N/A

Next Steps: Set up plate reader, collect and analyse data. Assess the effect of induction of higher repressor-expression on the level of GFP.

12/09: Set up plate reader.

Aim of experiment: To design a series of plate reader experiments to measure OD600 and relative fluorescence of the chromate sensor. At first no chromate was added to this plate in order to assess the effect of IPTG induction.

Protocols Used: Plate reader experiments

Results: Figure 36

Next Steps: Repeat experiments with added chromate substances.

Week Beginning 14/09/2015

Summary

During this week the samples from the previous plate reader were retrieved and prepared for blotting against both GFP, and the 6-His - Tag of ChrB.

14/09: Western blot of plate reader samples MG1655 + pSB1C3-Pchr-gfp + pUniprom-chrB, and MG1655 + pSB1C3-Pchr-gfp + pUniprom-chrB (opt).

Aim of experiment: To identify whether or not there is any detectable difference in both, the level of GFP, and the level of ChrB, before and after induction with IPTG.

Protocols Used: SDS-PAGE + Western blot

Results: Even though the blot was very blurred, expression of GFP could be confirmed in all samples. There seemed to be no noteable difference between induced and uninduced.

Next Steps: Repeat the assays of the previous weeks by subcloning chrB, chrB (opt), and chrB (Bielefeld) into vectors with different expression levels and see how it influences the level of GFP expression. Then add different chromate salts and also probe the system with other substances, e.g. other heavy-metal salts.

K2CrO4, K2Cr2O7, CrCl3, and K2SO4