<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-->

             <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-->

                   <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>

         <div class="labtitle">Week Beginning 29/06</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, <i>pChr</i>, and <i>ChrB</i>.</p>

             <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-->

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

             <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-->

                   Overnight culture of <i>E. coli DH5-alpha</i>, 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 MC1061.</i></p>

             <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-->

                   <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>

                   <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15-chr-igem024-modal" class="journal-protocol"> Figure 5 </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>

             <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-->

                     <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>

                   <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>

                   <p><a data-toggle="modal" data-target="#dundee15-chr-pChr1-modal" class="journal-protocol"> Figure 7 </a> - Sequence confirmation of <i>pChr</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>

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

             <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-->

                   <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>

                     <a data-toggle="modal" data-target="#gelextraction-modal" class="journal-protocol"> Gel extraction </a> of optimised <i>ChrB</i></p>

                   <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>

                   <p><a data-toggle="modal" data-target="#dundee15-chr-igem032-modal" class="journal-protocol"> Figure 10 </a>Gel with ChrB</p></p>

                   <p><b>Next Steps:</b> Ligation of <i>ChrB</i> and codon optimised <i>ChrB</i> into pUniprom.</p>

             <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-->

                   <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>

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

             <span class="box-title">Day 1 - 13/07: Transformation of pUniprom-ChrB, pUniprom-ChrBopt, and pSB1C3-pChr-GFP.</span>  

                   <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>Results:</b><a data-toggle="modal" data-target="#dundee15-chr-first-transformation-modal" class="journal-protocol"> Figure 11 </a></p>

             <span class="box-title">Day 2 - 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-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>

             <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-->

                   <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><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>

                     <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>

                     </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><b>Next Steps:</b> Confirmation of sequence of <i>ChrB-opt.</i></p>

             <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-->

                   <p><b>Aim of experiment:</b> Set up overnight cultures of <i>ChrBopt</i> colonies 2, 4, 9, and 12 for sequencing.</p>

             <span class="box-title">Day 5 - 17/07: Purified and sequenced chosen <i>ChrBopt</i>-colonies.</span> <!--Title of collapsible box-->

                   <p><b>Results:</b> <i>ChrBopt</i> 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>

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

         <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>

             <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>

                   <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>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>

             <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-->

                   <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>

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

             <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>

                   <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>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>Next Steps:</b> Sequence confirmation of pChr in pSB1C3.</p>

             <span class="box-title">Day 4 - 23/07: Set up overnight cultures of pSB1C3-pChr colonies.</span>

                   <p><b>Aim of experiment:</b> Set up overnight cultures of pSB1C3-pChr colonies to prepare pSB1C3-pChr for sequence confirmation</p>

             <span class="box-title">Day 5 - 24/07: Purified pSB1C3-pChr for sequence confirmation.</span> <!--Title of collapsible box-->

                   <p><b>Aim of experiment:</b> Purification of pSB1C3-pChr 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-pChr vectors with pChr sequence.</p>

                   <p><b>Next Steps:</b> Digest of pSB1C3-pChr with SpeI/PstI and ligation of GFPmut2.</p>

             <span class="box-title">Day 6 - 25/07: PCR-amplified <i>ChrB</i> and <i>ChrBopt</i>.</span> <!--Title of collapsible box-->

                   <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>Next Steps:</b> Ligation and Transformation of <i>ChrB</i> and <i>ChrBopt</i>.</p>

             <span class="box-title">Day 7 - 26/07: Ligated <i>ChrB</i> and <i>ChrBopt</i> into pUniprom.</span> <!--Title of collapsible box-->

                   <p><b>Aim of experiment:</b> Insertion of ChrB and optimised ChrB into pUniprom vector.</p>

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

             <span class="box-title">Day 1 - 27/07: Transformed <i>ChrB</i> and <i>ChrBopt</i> into <i>E. coli JM110</i></span>

                   <p><b>Aim of experiment:</b>Transformation of the repressors, <i>ChrB</i> and <i>ChrBopt</i>, into <i>JM110</i></p>

                   <p><b>Next Steps:</b> Purification of pUniprom-ChrB and pUniprom-ChrBopt for sequence confirmation.</p>

             <span class="box-title">Day 2 - 28/07: Digested pSB1C3-pChr for insertion of <i>GFP</i></span>

                   <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>

                     <a data-toggle="modal" data-target="#ligation-modal" class="journal-protocol">Ligation</a> of <i>GFP</i> into pSB1C3-pChr.</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><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>

             <span class="box-title">Day 3 - 29/07: Purified <i>ChrB</i> and <i>ChrBopt</i> for size and sequence confirmation.</span>

                   <p><b>Aim of experiment:</b> Purification of <i>ChrB</i> and <i>ChrBopt</i> for confirmation of size and sequence.</p>

                   <p><b>Next Steps:</b> Confirmation of expression of <i>ChrB</i> and <i>ChrBopt</i> by western blotting.</p>

             <span class="box-title">Day 4 - 30/07: Repeated transformation of pSB1C3-pChr-GFP into <i>E.coli JM110</i></span>

                   <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>Next Steps:</b> Overnight cultures for purification of the pSB1C3-pChr-GFP for confirmation of insert size and sequence.</p>

             <span class="box-title">Day 5 - 31/07: Set up overnight cultures for confirmation of pSB1C3-pChr-GFP.</span> <!--Title of collapsible box-->

                   <p><b>Aim of experiment:</b> Overnight cultures for subsequent plasmid purification of pSB1C3-pChr-GFP.</p>

             <span class="box-title">Day 6 - 01/08: Purified pSB1C3-pChr-GFP for size confirmation and sequencing.</span>

                   <p><b>Aim of experiment:</b> Purification of pSB1C3-pChr-GFP for confirmation of insert size and sequence.</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>

             <span class="box-title">Day 7 - 02/08: Set up cultures in preparation for western blotting.</span> <!--Title of collapsible box-->

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

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

         <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>

             <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-->

                   <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>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><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>

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

         <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>

             <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>

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

                       <li>pUniprom-ChrB2 (29/07) - pSB1C3-pChr-GFP (31/07)</li>

                       <li>pUniprom-ChrB2 (29/07) - pSB1C3-pChr-GFP (15/07)</li>

                       <li>pUniprom-ChrBopt3 (29/07) - pSB1C3-pChr-GFP (31/07)</li>

                       <li>pUniprom-ChrBopt3 (29/07) - pSB1C3-pChr-GFP (15/07)</li>

                   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>

                   <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15_chr_150811_transformations" class="journal-protocol"> Figure 27 </a></p>

             <span class="box-title">Day 2 - 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 transformaions in order to make them competent to transform the second plasmid as well.</p>

             <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>

                   <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>

             <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>

                   <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>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>

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

         <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>

             <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>

                   <p><b>Next Steps:</b> To determine expression of <i>ChrB</i> and <i>ChrBopt</i> (via 6His-tag) and expression of GFP.</p>

             <span class="box-title">Day 2 - 18/08: Preparation of samples for western blot.</span> <!--Title of collapsible box-->

                   <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>

             <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-->

                   <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>

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

             <span class="box-title">Day 1 - Day 7 - 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><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="#icecream" 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="#flake" 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="#dundee15_chr_150824_platereader_pchrbgfp" class="journal-protocol"> Figure 32 </a><i>MG1655 + pSB1C3-pChr-GFP</i></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>

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

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

             <span class="box-title">Day 1 - 07/09: Transformation of pSB1K3 (Bielefeld) into <i>MG1655</i>.</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 MG1655</i></p>

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

             <span class="box-title">Day 2 - 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 <i>MG1655</i> competent cells and re-transformation of pSB1K3 for subsequent miniprep.</p>

                   <p><b>Results:</b> Successful test transformation, but no colonies on pSB1K3 plate.</p>

                   <p><b>Next Steps:</b> Use an alternative compatible plasmid for ligation of codon optimised <i>ChrB</i> (Bielefeld).</p>

             <span class="box-title">Day 3 - 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-ChrB. Also transformations of Chromate-sensor plasmids into BL21 (DE3) in the following combinations: <ul>

                                   <li>pSB1C3-pChr-GFP + pUniprom-ChrB</li>

                                   <li>pSB1C3-pChr-GFP + pUniprom-ChrBopt</li>

                                   <li>pUniprom-ChrB</li>

                                   <li>pUniprom-ChrBopt</li>

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

             <span class="box-title">Day 4 - 10/09: Colony PCR of pT7KS-ChrB (Bielefeld).</span> <!--Title of collapsible box-->

                   <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>Results:</b><a data-toggle="modal" data-target="#dundee15_chr_150911_igem050" class="journal-protocol"> Figure 34 </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>

             <span class="box-title">Day 5 - 11/09: Preparation for Plate reader experiment with <i>BL21(DE3)</i></span>

             <span class="box-title">Day 6 - 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 35 </a></p>

         <div class="labtitle">Week Beginning 14/09</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 6His-Tag of <i>ChrB</i> and <i>ChrBopt</i>.</a></p>

             <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-->

                   <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>Results:</b> Insert blot image here.</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>

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

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

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

         <li>Subsample ¹/₁₀ of the volume of the subculture. I.e. 1ml of the overnight culture into 10ml fresh LB.</li>

         <li>Grow subculture at at 37^oC for 2h.</li>

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

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

         <li>2µl of 3mM primer</li>

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

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

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

<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>

         <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>Pelleted bacterial cells were resuspended in 250µl 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>350µl of Buffer N3 was added and immediately and thoroughly mixed by inverting the tube 4-6 times.</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>750µl 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 1.5ml microcentrifuge and the DNA eluted by adding 30µl of H₂O to the QIAprep spin column and centrifuging for 1 minute. </li>

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

         <li>230µl 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>500µl 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>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>

                     50µl

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

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

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

         <li>The pellet was resuspended in the 100µl.</li>

         <li>50µl of an overnight culture was used to inoculate 5ml 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, 1mM 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>Meanwhile, 950µl of 2x Laemmli sample buffer was added to 50µl 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 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 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 in 3x10ml 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, 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 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 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 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 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 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 10: 1% agarose gel after PCR of ChrBw4 (Expected size 939bp).

         <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 11: Plates after transformation of ChrB-pUniprom, optimised ChrB-pUniprom and pChr-GFPmut2-pSB1C3 into JM110.

         <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 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 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 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 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 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 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 20: Plates after transformation of, Top: ChrB and into pUniprom; Bottom: optimised ChrB into pUniprom. Ratio insert:vector left: 3:1; Right: 4:1.

         <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.

         <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.

         <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.

         <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.

         <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.

         <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.

       <img src="https://static.igem.org/mediawiki/2015/f/f2/Dundee15_chr_150809_blot.png" style="width:400px;height:auto">'  

         <div class="modal-title" id="myModalLabel">Figure 27: Transformations of single constituents of Chromate sensing system.

         <div class="modal-title" id="myModalLabel">Figure 28: Western Blot against GFP.

       <img src="https://static.igem.org/mediawiki/2015/f/f9/Dundee15_chr_150822_blot.png" style="width:400px;height:auto">'