<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>

             <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-->

                   <p><b>Aim of experiment:</b> To prepare <a href="http://parts.igem.org/Part:BBa_K1058008" target="_blank">BBa_K1058008</a> for sequence confirmation, storage, and further processing. The culture was plated from the agar stab and also a liquid culture set up.</p>

             <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-->

                   <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>

         <div class="labtitle">Week Beginning 29/06/2015</div>

         <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>

             <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-->

                   <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>

         <div class="labtitle">Week Beginning 06/07/2015</div>

             <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-->

                   Overnight culture of <i>E. coli</i> DH5-alpha, containing GFPmut2, as a fluorescent reporter for our chromate sensing system</p>

                   pSB1C3-pChr, pSB1C3-ChrBs, and pSB1C3-ChrBw were transformed into <i>E. coli</i> MC1061.</p>

             <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-->

                   <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>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-P<i>chr</i>, pSB1C3-<i>chrB</i> (s), and pSB1C3-<i>chrB</i> (w).</p>

             <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-->

                     <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> 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>

                   <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 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 7 </a> - Sequence confirmation of <i>chrB</i>.</p>

                   <p><b>Next Steps:</b> Purification and digest of pUniprom for insertion of <i>chrB</i>.</p>

             <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-->

                   <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><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 <i>chrB</i> (opt)</p>

                   <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 9 </a>Gel with <i>chrB</i></p></p>

                   <p><b>Next Steps:</b> Ligation of <i>chrB</i> and <i>chrB</i> (opt) into pUniprom.</p>

             <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-->

                   <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>

                     <a data-toggle="modal" data-target="#ligation-modal" class="journal-protocol"> Ligations</a>

         <div class="labtitle">Week Beginning 13/07/2015</div>

             <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>  

                   <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>

                     <a data-toggle="modal" data-target="#transformation-modal" class="journal-protocol"> Transformations, </a>

                   <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>

             <span class="box-title">14/07: Set up cultures for subsequent purification of recombinant vectors for size and sequence confirmation.</span>

                   <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>

             <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-->

                   <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>

                     <a data-toggle="modal" data-target="#miniprep-modal" class="journal-protocol"> Miniprep, </a>

                     <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 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-P<i>chr-gfp</i> (colony 1).</p>

                   <p><b>Next Steps:</b> Confirmation of sequence of <i>chrB</i> (opt)</p>

             <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-->

                   <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>Results:</b> N/A</p>

             <span class="box-title">17/07: Purified and sequenced chosen <i>chrB</i> (opt) - colonies.</span> <!--Title of collapsible box-->

                   <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>chrB</i> (opt).</p>

         <div class="labtitle">Week Beginning 20/07/2015</div>

         <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>

             <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>

                   <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>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>

             <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-->

                   <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>

                     <a data-toggle="modal" data-target="#sequencing-modal" class="journal-protocol">Sequencing </a> of P<i>chr-gfp</i> - insert.</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>                   

             <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>

                   <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>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 P<i>chr</i> in pSB1C3.</p>

             <span class="box-title">23/07: Set up overnight cultures of pSB1C3-P<i>chr</i> colonies.</span>

                   <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>

                     <a data-toggle="modal" data-target="#overnightculture-modal" class="journal-protocol"> Overnight culture, </a>

             <span class="box-title">24/07: Purified pSB1C3-P<i>chr</i> for sequence confirmation.</span> <!--Title of collapsible box-->

                   <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>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-P<i>chr</i> with SpeI/PstI and ligation of P<i>gfp</i></p>

             <span class="box-title">25/07: PCR-amplified <i>chrB</i> and <i>chrB</i> (opt).</span> <!--Title of collapsible box-->

                   <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>Next Steps:</b> Ligation and Transformation of <i>chrB</i> and <i>chrB</i> (opt).</p>

             <span class="box-title">26/07: Ligated <i>chrB</i> and <i>chrB</i> (opt) into pUniprom.</span> <!--Title of collapsible box-->

                   <p><b>Aim of experiment:</b> Insertion of <i>chrB</i> and <i>chrB</i> (opt) into pUniprom.</p>

         <div class="labtitle">Week Beginning 27/07/2015</div>

             <span class="box-title">27/07: Transformed <i>chrB</i> and <i>chrB</i> (opt) into <i>E. coli</i> JM110</span>

                   <p><b>Aim of experiment:</b> Transformation of the repressors, <i>chrB</i> and <i>chrB</i> (opt), into JM110</p>

                     <a data-toggle="modal" data-target="#transformation-modal" class="journal-protocol"> Transformation</a>  

                   <p><b>Next Steps:</b> Purification of pUniprom-<i>chrB</i> and pUniprom-<i>chrB</i> (opt) for sequence confirmation.</p>

             <span class="box-title">28/07: Digested pSB1C3-P<i>chr</i> for insertion of <i>gfp</i></span>

                   <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><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-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>chrB</i> (opt).</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>

             <span class="box-title">29/07: Purified <i>chrB</i> and <i>chrB</i> (opt) for size and sequence confirmation.</span>

                   <p><b>Aim of experiment:</b> Purification of <i>chrB</i> and <i>chrB</i> (opt) for confirmation of size and sequence.</p>

                     <a data-toggle="modal" data-target="#miniprep-modal" class="journal-protocol"> Plasmid purification, </a>

                   <p><b>Next Steps:</b> Confirmation of expression of <i>chrB</i> and <i>chrB</i> (opt) by western blotting.</p>

             <span class="box-title">30/07: Repeated transformation of pSB1C3-P<i>chr-gfp</i> into <i>E.coli JM110</i></span>

                   <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>Next Steps:</b> Overnight cultures for purification of the pSB1C3-P<i>chr-gfp</i> for confirmation of insert size and sequence.</p>

             <span class="box-title">31/07: Set up overnight cultures for confirmation of pSB1C3-P<i>chr-gfp</i>.</span> <!--Title of collapsible box-->

                   <p><b>Aim of experiment:</b> Overnight cultures for subsequent plasmid purification of pSB1C3-P<i>chr-gfp</i>.</p>

                     <a data-toggle="modal" data-target="#overnightculture-modal" class="journal-protocol"> Overnight culture</a>

             <span class="box-title">01/08: Purified pSB1C3-P<i>chr-gfp</i> for size confirmation and sequencing.</span>

                   <p><b>Aim of experiment:</b> Purification of pSB1C3-P<i>chr-gfp</i> for confirmation of insert size and sequence.</p>

                     <a data-toggle="modal" data-target="#miniprep-modal" class="journal-protocol"> Plasmid purification, </a>

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

             <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-->

                   <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>Next Steps:</b> Western blotting for characterisation of expression of both, GFP, and ChrB and ChrB (opt).</p>

         <div class="labtitle">Week Beginning 03/08/2015</div>

         <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>

             <span class="box-title">03/08 - 09/08: Determined expression of ChrB, ChrB (opt), and GFP by western blotting.</span> <!--Title of collapsible box-->

                   <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>

                     <a data-toggle="modal" data-target="#sds-modal" class="journal-protocol"> SDS-PAGE and western blot</a>

                   <p><b>Results:</b><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 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>

         <div class="labtitle">Week Beginning 10/08/2015</div>

         <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>

             <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>

                   <p><b>Aim of experiment:</b> To transform the following combinations of pSB1C3-pChr-GFP and pUniprom-ChrB into MG1655:

                       <li>pUniprom-<i>chrB</i> (from colony 2 - 29/07) - pSB1C3-P<i>chr-gfp</i> (A)</li>

                       <li>pUniprom-<i>chrB</i> (from colony 2 - 29/07) - pSB1C3-P<i>chr-gfp</i> (B)</li>

                       <li>pUniprom-<i>chrB</i> (opt) (from colony 3 - 29/07) - pSB1C3-P<i>chr-gfp</i> (A)</li>

                       <li>pUniprom-<i>chrB</i> (opt) (from colony 3 - 29/07) - pSB1C3-P<i>chr-gfp</i> (B)</li>

                     <a data-toggle="modal" data-target="#miniprep-modal" class="journal-protocol"> Plasmid purification, </a>

                   <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>

             <span class="box-title">11/08: Set up overnight cultures of all transformed cells.</span> <!--Title of collapsible box-->

                   <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>

                     <a data-toggle="modal" data-target="#overnightculture-modal" class="journal-protocol"> Overnight culture</a>

                   <p><b>Next Steps:</b> Make cells of overnight culture competent to transform in second plasmid.</p>

             <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>

                   <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>

                     <a data-toggle="modal" data-target="#competent-modal" class="journal-protocol"> Competent cells, </a>

                   <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>

             <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>

                   <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>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>

         <div class="labtitle">Week Beginning 17/08/2015</div>

         <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>

             <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>

                   <p><b>Next Steps:</b> To determine expression of ChrB and GFP.</p>

             <span class="box-title">18/08: Preparation of samples for western blot.</span> <!--Title of collapsible box-->

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

             <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-->

                   <p><b>Aim of experiment:</b> To determine whether expression of ChrB has an effect on the expression of GFP.</p>

         <div class="labtitle">Week Beginning 24/08/2015</div>

             <span class="box-title">24/08-30/08: Design of Plate reader experiments.</span> <!--Title of collapsible box-->

                   <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="#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="#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>MG1655 + pSB1C3-P<i>chr-gfp</i> (B)</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>

         <div class="labtitle">Week Beginning 31/08/2015</div>

         <div class="labtitle">Week Beginning 07/09/2015</div>

             <span class="box-title">07/09: Transformation of pSB1K3 (Bielefeld) into MG1655.</span> <!--Title of collapsible box-->

                     <p><a data-toggle="modal" data-target="#overnightculture-modal" class="journal-protocol"> Overnight culture</a> for new <i>E. coli</i> MG1655</p>

                   <p><b>Next Steps:</b> Retry transformation with new competent <i>E. coli</i> MG1655.</p>

             <span class="box-title">08/09: Transformation of pSB1K3 into MG1655.</span> <!--Title of collapsible box-->

                   <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>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 the codon optimised <i>chrB</i> (Bielefeld).</p>

             <span class="box-title">09/09: Transformation of pSB1K3 into MG1655.</span> <!--Title of collapsible box-->

                   <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-P<i>chr-gfp</i> (B) + pUniprom-<i>chrB</i></li>

                                   <li>pSB1C3-P<i>chr-gfp</i> (B) + pUniprom-<i>chrB</i> (opt)</li>

                                   <li>pUniprom-<i>chrB</i></li>

                                   <li>pUniprom-<i>chrB</i> (opt)</li>

                   <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><a data-toggle="modal" data-target="#dundee15_chr_150911_chrbbielefeld" class="journal-protocol"> Figure 33 </a> pT7KS-<i>chrB</i> (Bielefeld) transformations</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>

             <span class="box-title">10/09: Colony PCR of pT7KS-<i>chrB</i> (Bielefeld).</span> <!--Title of collapsible box-->

                   <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>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-<i>chrB</i> (Bielefeld).</p>

             <span class="box-title">11/09: Preparation for Plate reader experiment with <i>BL21(DE3)</i></span>

             <span class="box-title">12/09: Set up plate reader.</span> <!--Title of collapsible box-->

                   <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>

         <div class="labtitle">Week Beginning 14/09/2015</div>

         <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>

             <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-->

                   <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>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 <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>

         <li>A 5 ml overnight culture of the desired strain was grown at 37 ^oC.</li>

         <li>Aliquot 198 µl into the required amount of wells in a 96-well plate.</li>

         <li>A 5 ml overnight culture of the desired strain was grown at 37^oC.</li>

         <li>Subsample ¹/₁₀ 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 ^oC for 2 h.</li>

         <li>Spin down subculture at 4000 rpm at 4^oC for 10 min.</li>

         <li>Use 100 µl per transformation.</li>

         <li>2 µl of 3 mM primer</li>

         <li>Remaining volume of water for a total volume of 30 µl</li>

         <li>5 ml of LB medium, supplemented with the required antibiotic, were inoculated with the toothpick.</li>

       <li>Each colony is transferred to a 30 µl aliquot of sterile water.</li>

<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>

         <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 250 µl of Buffer P1.</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>350 µl of Buffer N3 was added and immediately and thoroughly mixed by inverting the tube 4-6 times.</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>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 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>

         <li>The gel slice was transferred into a microcentrifuge tube. 700 µl of Buffer QG was added.</li>

         <li>230 µl of isopropanol was added to the sample and mixed.</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>500 µl of QG buffer 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 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>

                     50 µl

       <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>

         <li>100 µl of competent cells were defrosted. Thaw on ice.</li>

         <li>1-5 µl of DNA was added to cells.</li>

         <li>1 ml of LB (without antibiotic) was added to cells.</li>

         <li>The pellet was resuspended in the 100 µl.</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^oC in a shaking incubator.</li>

         <li>Once the OD₆₀₀=0.6-0.8, 1 mM of IPTG was added to the culture and left to grow for a further 3 hours.</li>

         <li>1 ml of each culture was taken and spun down in a microcentrifuge for 3 minutes.</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 200 µl of the above solution.</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 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 3x10 ml 1xTBST buffer for 5 minutes.</li>

         <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 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 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 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 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

         <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.

         <div class="modal-title" id="myModalLabel">Figure 11: 1% agarose gel after restriction digest of pSB1C3 with EcoRI and SpeI (Expected size 2070bp).

         <div class="modal-title" id="myModalLabel">Figure 9: 1% agarose gel after PCR of <i>chrB</i> (opt). (Expected size 939bp).

         <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.

         <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 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.

         <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.

         <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.

         <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.

         <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.

         <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

         <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.

         <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.

         <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.