Difference between revisions of "Team:Dundee/labjournal manu"
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| + | <title>Dundee iGEM 2015</title> | ||
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| − | <div class="labtitle">22/06 | + | <div class="labtitle">Week Beginning 22/06/2015</div> |
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| − | <p class="journal-content">This week was all about getting started with the newly arrived biobrick | + | <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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| − | <div class="labtitle">29/06 | + | <div class="labtitle">Week Beginning 29/06</div> |
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| − | <div class="labtitle">06 | + | <div class="labtitle">Week Beginning 06/07</div> |
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| − | <div class="labtitle">13 | + | <div class="labtitle">Week Beginning 13/07</div> |
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| − | <div class="labtitle">20 | + | <div class="labtitle">Week Beginning 20/07</div> |
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| − | <p class="journal-content">During this week an alternative cloning strategy for ChrB and ChrB-opt was employed. Furthermore, the pChr-GFP construct was cloned | + | <p class="journal-content">During this week an alternative cloning strategy for ChrB and ChrB-opt was employed. Furthermore, the pChr-GFP construct was cloned in a stepwise fashion.</p> |
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| − | <div class="labtitle">27/07 | + | <div class="labtitle">Week Beginning 27/07</div> |
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| − | <div class="labtitle">03 | + | <div class="labtitle">Week Beginning 03/08</div> |
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| − | <div class="labtitle">10 | + | <div class="labtitle">Week Beginning 10/08</div> |
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| − | <span class="box-title">Day 3 - 12/08</span> <!--Title of collapsible box--> | + | <span class="box-title">Day 3 - 12/08: Made competent MG1655 with ChrB2, ChrBopt3, pChrGFP (15/07), pChrGFP (31/07).</span> <!--Title of collapsible box--> |
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<div class="border-day"> | <div class="border-day"> | ||
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| − | <span class="box-title">Day 4</span> <!--Title of collapsible box--> | + | <span class="box-title">Day 4 - 13/08: Transformed ChrB2 and ChrBopt3 into MG1655+pChrGFP(15/07) and MG1655+pChrGFP(31/07)</span> <!--Title of collapsible box--> |
| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="# | + | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew8-4"></span> |
| − | <div id=" | + | <div id="collapsiblew8-4" class="collapse box-content"> |
| − | <p><b>Aim of experiment:</b> To | + | <p><b>Aim of experiment:</b> To build a strain of MG1655, containing both plasmids required for a chromate sensor.</p> |
<p><b>Protocols Used:</b> | <p><b>Protocols Used:</b> | ||
| − | + | <a data-toggle="modal" data-target="#transformation-modal" class="journal-protocol"> Transformation</a> | |
| − | + | </p> | |
| − | <p><b>Results:</b> | + | <p><b>Results:</b> No successful transformants were found.</p> |
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| − | <p><b>Next Steps:</b> | + | <p><b>Next Steps:</b> Check Plates for successful transformants.</p> |
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| − | <div class="labtitle">Week Beginning | + | <div class="labtitle">Week Beginning 17/08</div> |
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<div class="box-content"> | <div class="box-content"> | ||
<p class="journal-summary-heading">Summary</p> | <p class="journal-summary-heading">Summary</p> | ||
| − | <p class="journal-content"> | + | <p class="journal-content">During this week recombinant MG1655 were made, containing both plasmids. Western Blots were used to determine expression of ChrB, ChrBopt, and GFP.</a></p> |
</div> | </div> | ||
| − | <div id=" | + | <div id="collapsiblew9" class="collapse week-content"> |
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| − | <span class="box-title">Day 1</span> <!--Title of collapsible box--> | + | <span class="box-title">Day 1 - 17/08: Transformations akin to 13/08 were done, but pChrGFP(15/07) and pChrGFP(31/07) were transformed into MG1655+ChrB2 and MG1655+ChrBopt3.</span> <!--Title of collapsible box--> |
| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="# | + | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew9-1"></span> |
| − | <div id=" | + | <div id="collapsiblew9-1" class="collapse box-content"> |
| − | <p><b>Aim of experiment:</b> To | + | <p><b>Aim of experiment:</b> To build a strain of MG1655, containing both plasmids required for a chromate sensor.</p> |
<p><b>Protocols Used:</b> | <p><b>Protocols Used:</b> | ||
| − | + | <a data-toggle="modal" data-target="#transformation-modal" class="journal-protocol"> Transformation, </a> | |
| − | + | </p> | |
| − | <p><b>Results:</b> | + | <p><b>Results:</b> Successful transformants were found on all plates.</p> |
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| − | <p><b>Next Steps:</b> | + | <p><b>Next Steps:</b> To determine expression of ChrB and ChrBopt (via His-tag) and expression of GFP.</p> |
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| − | <span class="box-title">Day 2</span> <!--Title of collapsible box--> | + | <span class="box-title">Day 2 - 18/08: Preparation of samples for western blot.</span> <!--Title of collapsible box--> |
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| − | <div id=" | + | <div id="collapsiblew9-2" class="collapse box-content"> |
| − | <p><b>Aim of experiment:</b> To | + | <p><b>Aim of experiment:</b> To determine whether ChrB, ChrBopt, and GFP are expressed and have first estimates of differences in expression levels.</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> | |
| − | + | </p> | |
<p><b>Results:</b> N/A</p> | <p><b>Results:</b> N/A</p> | ||
| − | + | <p><b>Next Steps:</b> Lyse cells and prepare samples for SDS-PAGE + Western-Blot.</p> | |
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| − | <span class="box-title">Day 3</span> <!--Title of collapsible box--> | + | <span class="box-title">Day 3-6 - 19/08 - 22/08: Repeated western blots with different concentrations to determine whether the expression of ChrB has an effect on the expression of GFP.</span> <!--Title of collapsible box--> |
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| − | <div id=" | + | <div id="collapsiblew9-3" class="collapse box-content"> |
| − | <p><b>Aim of experiment:</b> To prepare | + | <p><b>Aim of experiment:</b> To prepare whether expression of ChrB or ChrB opt has an effect on the expression of GFP.</p> |
<p><b>Protocols Used:</b> | <p><b>Protocols Used:</b> | ||
| − | + | <a data-toggle="modal" data-target="#sds-modal" class="journal-protocol"> SDS-PAGE + Western Blot</a> | |
| − | + | </p> | |
| − | <p><b>Results:</b> | + | <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15_chr_150822_blot" class="journal-protocol"> Figure 28 </a></p> |
| − | + | <p><b>Next Steps:</b> Check if different substances containing chromate have an effect on the expression.</p> | |
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| − | <p><b>Next Steps:</b> | + | |
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| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
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| − | + | <!-- WEEK 10 --> | |
| + | <div class="row"> | ||
| + | <div class="border-week"> | ||
| + | <div class="box"> | ||
| + | <div class="labtitle">Week Beginning 24/08</div> | ||
| + | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew10"></span> | ||
| + | <div class="box-content"> | ||
| + | <p class="journal-summary-heading">Summary</p> | ||
| + | |||
| − | + | <p class="journal-content">Plate reader experiments were carried out in order to determine the effect 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> on the expression of GFP.</a></p> | |
| − | + | </div> | |
| − | + | <div id="collapsiblew10" class="collapse week-content"> | |
| − | + | <!-- when copying for next week, make sure to change labtitle, span, collapsible in last line. In each separate day change span and following div--> | |
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| − | <span class="box-title">Day | + | <span class="box-title">Day 1 - Day 7 - 24/08-30/08: Design of Plate reader experiments.</span> <!--Title of collapsible box--> |
| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="# | + | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew10-1"></span> |
| − | <div id=" | + | <div id="collapsiblew10-1" class="collapse box-content"> |
| − | <p><b>Aim of experiment:</b> To | + | <p><b>Aim of experiment:</b> To design a series of plate reader experiments to measure OD<sub>600</sub> and relative fluorescence of the chromate sensor as a response to different concentrations 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>.</p> |
<p><b>Protocols Used:</b> | <p><b>Protocols Used:</b> | ||
| − | + | <a data-toggle="modal" data-target="#platereader-modal" class="journal-protocol"> Plate Reader Experiments </a> | |
| − | + | </p> | |
| − | <p><b>Results:</b> | + | <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15_chr_150824_platereader_BBa_K1058008" class="journal-protocol"> Figure 29 </a></p> |
| − | < | + | <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15_chr_150824_platereader_pchrbgfp+chrb" class="journal-protocol"> Figure 30 </a></p> |
| − | <p><b>Results:</b><a data-toggle="modal" data-target="# | + | <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15_chr_150824_platereader_pchrbgfp+chrbopt" class="journal-protocol"> Figure 31 </a></p> |
| − | + | <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15_chr_150824_platereader_pchrbgfp" class="journal-protocol"> Figure 32 </a></p> | |
| − | + | <p><b>Next Steps:</b> Try the system with different expression levels of repressor. Induce overexpression of repressor from T7-promoter in pUniprom using BL21 and IPTG.</p> | |
| − | <p><b>Next Steps:</b> | + | |
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| − | + | <!-- WEEK 11 --> | |
| − | <!-- WEEK | + | |
<div class="row"> | <div class="row"> | ||
<div class="border-week"> | <div class="border-week"> | ||
<div class="box"> | <div class="box"> | ||
| − | <div class="labtitle">Week Beginning | + | <div class="labtitle">Week Beginning 31/08</div> |
| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="# | + | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew11"></span> |
<div class="box-content"> | <div class="box-content"> | ||
<p class="journal-summary-heading">Summary</p> | <p class="journal-summary-heading">Summary</p> | ||
| − | <p class="journal-content"> | + | <p class="journal-content">UK Synbio Conference and UK iGEM meetup at Westminster University.</a></p> |
</div> | </div> | ||
| − | <div id=" | + | <div id="collapsiblew11" class="collapse week-content"> |
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| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| − | + | <!-- WEEK 12 --> | |
| − | + | <div class="row"> | |
| − | + | <div class="border-week"> | |
| − | + | <div class="box"> | |
| − | + | <div class="labtitle">Week Beginning 07/09</div> | |
| − | + | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew12"></span> | |
| + | <div class="box-content"> | ||
| + | <p class="journal-summary-heading">Summary</p> | ||
| + | |||
| + | <p class="journal-content">Parts from CeBiTec Bielefeld arrived and attempts at cloning were made. The chromate sensing system was transformed into BL21 and some of the plate reader experiments repeated.</a></p> | ||
| + | </div> | ||
| + | <div id="collapsiblew12" class="collapse week-content"> | ||
| − | + | <!-- when copying for next week, make sure to change labtitle, span, collapsible in last line. In each separate day change span and following div--> | |
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<div class="box"> | <div class="box"> | ||
| − | <span class="box-title">Day | + | <span class="box-title">Day 1 - 07/09: Transformation of pSB1K3 into MG1655.</span> <!--Title of collapsible box--> |
| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="# | + | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew12-1"></span> |
| − | <div id=" | + | <div id="collapsiblew12-1" class="collapse box-content"> |
| − | <p><b>Aim of experiment:</b> | + | <p><b>Aim of experiment:</b> The high-copy plasmid arrived at very low concentration. Hence it was transformed in order to produce more of it before digesting it.</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="#overnightculture-modal" class="journal-protocol"> Overnight culture for competent cells </a> | |
| + | </p> | ||
| − | <p><b>Results:</b> | + | <p><b>Results:</b> No transformants were found.</p> |
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| − | <p><b>Next Steps:</b> | + | <p><b>Next Steps:</b> Retry and make more competent MG1655.</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 | + | <span class="box-title">Day 2 - 08/09: Transformation of pSB1K3 into MG1655.</span> <!--Title of collapsible box--> |
| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="# | + | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew12-2"></span> |
| − | <div id=" | + | <div id="collapsiblew12-2" class="collapse box-content"> |
| − | <p><b>Aim of experiment:</b> | + | <p><b>Aim of experiment:</b> Test transformation of BBa_K1058008 into new MG1655 competent cells and transformation of pSB1K3-plasmid</p> |
<p><b>Protocols Used:</b> | <p><b>Protocols Used:</b> | ||
| − | + | <a data-toggle="modal" data-target="#transformation-modal" class="journal-protocol"> Transformation, </a> | |
| − | + | </p> | |
| − | <p><b>Results:</b> | + | <p><b>Results:</b> Successful test transformation, but no colonies on pSB1K3 plate.</p> |
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| − | <p><b>Next Steps:</b> | + | <p><b>Next Steps:</b> Use an alternative plasmid.</p> |
</div> | </div> | ||
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<div class="box"> | <div class="box"> | ||
| − | <span class="box-title">Day | + | <span class="box-title">Day 3 - 09/09: Transformation of pSB1K3 into MG1655.</span> <!--Title of collapsible box--> |
| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="# | + | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew12-3"></span> |
| − | <div id=" | + | <div id="collapsiblew12-3" class="collapse box-content"> |
| − | <p><b>Aim of experiment:</b> | + | <p><b>Aim of experiment:</b> Restriction digest of pT7KS with EcoRI and PstI as alternative to pSB1K3. Ligation of digested ChrB (Bielefeld) and transformation of this plasmid. Also transformations of Chromate-sensor plasmids into BL21 (DE3) in the following combinations: <ul> |
| + | <li>pChrGFP + ChrB</li> | ||
| + | <li>pChrGFP + ChrBopt</li> | ||
| + | <li>ChrB</li> | ||
| + | <li>ChrBopt</li> | ||
| + | </ul> | ||
| + | </p> | ||
<p><b>Protocols Used:</b> | <p><b>Protocols Used:</b> | ||
| − | + | <a data-toggle="modal" data-target="#restriction-digest-modal" class="journal-protocol"> Restriction Digest, </a> | |
| − | + | <a data-toggle="modal" data-target="#gelextraction-modal" class="journal-protocol"> Gel Extraction, </a> | |
| + | <a data-toggle="modal" data-target="#ligation-modal" class="journal-protocol"> Ligation </a> | ||
| + | </p> | ||
| − | <p><b>Results:</b> | + | <p><b>Results:</b>Transformations of the plasmid with ChrB (Bielefeld) yielded 12 colonies, but also colonies on the vector control plate. Transformations into BL21 were successful.</p> |
| − | < | + | <p><b>Results:</b> |
| + | <a data-toggle="modal" data-target="#dundee15_chr_150911_chrbbielefeld" class="journal-protocol"> Figure 33 </a> | ||
| + | <a data-toggle="modal" data-target="#dundee15_chr_150911_bl21" class="journal-protocol"> Figure 34 </a> | ||
| + | </p> | ||
| − | + | <p><b>Next Steps:</b> Confirmation of colonies with pT7KS-ChrB by colony PCR. Preparation of recombinant BL21 for plate reader experiments.</p> | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | <p><b>Next Steps:</b> | + | |
</div> | </div> | ||
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<div class="box"> | <div class="box"> | ||
| − | <span class="box-title">Day | + | <span class="box-title">Day 4 - 10/09: Colony PCR of pT7KS.</span> <!--Title of collapsible box--> |
| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="# | + | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew12-4"></span> |
| − | <div id=" | + | <div id="collapsiblew12-4" class="collapse box-content"> |
| − | <p><b>Aim of experiment:</b> | + | <p><b>Aim of experiment:</b> Check if any of the colonies after ligation of ChrB (Bielefeld) into pT7KS were successful despite an unsuccessful control plate.</p> |
<p><b>Protocols Used:</b> | <p><b>Protocols Used:</b> | ||
| − | + | <a data-toggle="modal" data-target="#colony-modal" class="journal-protocol"> Colony PCR </a> | |
| − | + | </p> | |
| − | <p><b>Results:</b> | + | <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 recombinant BL21 for plate reader experiments. Attempt new transformation of ChrB (Bielefeld).</p> | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | <p><b>Next Steps:</b> | + | |
</div> | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
| − | </div> | + | </div> |
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<div class="box"> | <div class="box"> | ||
| − | <span class="box-title">Day | + | <span class="box-title">Day 5 - 11/09: Preparation for Plate reader experiment with BL21 (DE3)</span> <!--Title of collapsible box--> |
| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="# | + | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew12-5"></span> |
| − | <div id=" | + | <div id="collapsiblew12-5" class="collapse box-content"> |
| − | <p><b>Aim of experiment:</b> | + | <p><b>Aim of experiment:</b> Set up overnight cultures for subsequent plate reader experiments.</p> |
<p><b>Protocols Used:</b> | <p><b>Protocols Used:</b> | ||
| − | + | <a data-toggle="modal" data-target="#overnightculture-modal" class="journal-protocol"> Overnight cultures </a> | |
| − | + | </p> | |
<p><b>Results:</b> N/A</p> | <p><b>Results:</b> N/A</p> | ||
| − | + | <p><b>Next Steps:</b> Set up plate reader, collect and analyse data. Assess the effect of induction of higher repressor-expression on the level of GFP.</p> | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | <p><b>Next Steps:</b> | + | |
</div> | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
| − | </div> | + | </div> |
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<div class="box"> | <div class="box"> | ||
| − | <span class="box-title">Day | + | <span class="box-title">Day 6 - 12/09: Set up plate reader.</span> <!--Title of collapsible box--> |
| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="# | + | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew12-6"></span> |
| − | <div id=" | + | <div id="collapsiblew12-6" class="collapse box-content"> |
| − | <p><b>Aim of experiment:</b> To | + | <p><b>Aim of experiment:</b> To design a series of plate reader experiments to measure OD<sub>600</sub> 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.</p> |
<p><b>Protocols Used:</b> | <p><b>Protocols Used:</b> | ||
| − | + | <a data-toggle="modal" data-target="#platereader-modal" class="journal-protocol"> Plate reader </a> | |
| − | + | </p> | |
| − | <p><b>Results:</b> | + | <p><b>Results:</b>add images once analysed.</p> |
| − | + | <p><b>Next Steps:</b>Repeat experiments with added chromate substances.</p> | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | <p><b>Next Steps:</b> | + | |
</div> | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
| − | </div> | + | </div> |
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<!-- POPUP PROTOCOL MODAL SECTION --> | <!-- POPUP PROTOCOL MODAL SECTION --> | ||
| + | |||
| + | |||
| + | <!-- Plate Reader Modal --> | ||
| + | <div class="modal fade" id="platereader-modal" tabindex="-1" role="dialog" aria-labelledby="myModalLabel"> | ||
| + | <div class="modal-dialog" role="document"> | ||
| + | <div class="modal-content"> | ||
| + | <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">×</span></button> | ||
| + | <div class="modal-title" id="myModalLabel">Plate Reader Protocol</div> | ||
| + | </div> | ||
| + | <div class="modal-body"> | ||
| + | |||
| + | <ol> | ||
| + | <li>A 5ml 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>Aliquot 198µl into the required amount of wells in a 96-well plate.</li> | ||
| + | <li>Add additional substances and controls as required.</li> | ||
| + | <li>Preheat platereader to required temperature and start experiment.</li> | ||
| + | <li>Retrieve and analyse data.</li> | ||
| + | </ol> | ||
| + | |||
| + | </div> | ||
| + | <div class="modal-footer"> | ||
| + | <button type="button" class="btn btn-primary" data-dismiss="modal">Close</button> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
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<div class="modal-body"> | <div class="modal-body"> | ||
<img src="https://static.igem.org/mediawiki/2015/b/bf/Dundee15_chr_150811_transformations.png" style="width:400px;height:auto">' | <img src="https://static.igem.org/mediawiki/2015/b/bf/Dundee15_chr_150811_transformations.png" style="width:400px;height:auto">' | ||
| + | |||
| + | <!--insert link of uploaded files here--> | ||
| + | |||
| + | </div> | ||
| + | <div class="modal-footer"> | ||
| + | <button type="button" class="btn btn-primary" data-dismiss="modal">Close</button> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | |||
| + | |||
| + | <div class="modal fade" id="dundee15_chr_150822_blot" tabindex="-1" role="dialog" aria-labelledby="myModalLabel"> | ||
| + | <div class="modal-dialog" role="document"> | ||
| + | <div class="modal-content"> | ||
| + | <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">×</span></button> | ||
| + | <div class="modal-title" id="myModalLabel">Figure 28: Western Blot against GFP. | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="modal-body"> | ||
| + | <img src="https://static.igem.org/mediawiki/2015/f/f9/Dundee15_chr_150822_blot.png" style="width:400px;height:auto">' | ||
| + | |||
| + | <!--insert link of uploaded files here--> | ||
| + | |||
| + | </div> | ||
| + | <div class="modal-footer"> | ||
| + | <button type="button" class="btn btn-primary" data-dismiss="modal">Close</button> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | |||
| + | |||
| + | <!--plate reader results--> | ||
| + | <div class="modal fade" id="dundee15_chr_150824_platereader_BBa_K1058008" tabindex="-1" role="dialog" aria-labelledby="myModalLabel"> | ||
| + | <div class="modal-dialog" role="document"> | ||
| + | <div class="modal-content"> | ||
| + | <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">×</span></button> | ||
| + | <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. | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="modal-body"> | ||
| + | <img src="https://static.igem.org/mediawiki/2015/f/fd/Dundee15_BBa_K1058008_combinedplatereaderresults.png" style="width:400px;height:auto">' | ||
| + | |||
| + | <!--insert link of uploaded files here--> | ||
| + | |||
| + | </div> | ||
| + | <div class="modal-footer"> | ||
| + | <button type="button" class="btn btn-primary" data-dismiss="modal">Close</button> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | |||
| + | |||
| + | <div class="modal fade" id="dundee15_chr_150824_platereader_pchrgfp+chrb" tabindex="-1" role="dialog" aria-labelledby="myModalLabel"> | ||
| + | <div class="modal-dialog" role="document"> | ||
| + | <div class="modal-content"> | ||
| + | <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">×</span></button> | ||
| + | <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. | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="modal-body"> | ||
| + | <img src="https://static.igem.org/mediawiki/2015/3/32/Dundee15_pChr-GFP_%2B_ChrB_combinedplatereaderresults.png" style="width:400px;height:auto">' | ||
| + | |||
| + | <!--insert link of uploaded files here--> | ||
| + | |||
| + | </div> | ||
| + | <div class="modal-footer"> | ||
| + | <button type="button" class="btn btn-primary" data-dismiss="modal">Close</button> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | |||
| + | |||
| + | <div class="modal fade" id="dundee15_chr_150824_platereader_pchrbgfp+chrbopt" tabindex="-1" role="dialog" aria-labelledby="myModalLabel"> | ||
| + | <div class="modal-dialog" role="document"> | ||
| + | <div class="modal-content"> | ||
| + | <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">×</span></button> | ||
| + | <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. | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="modal-body"> | ||
| + | <img src="https://static.igem.org/mediawiki/2015/5/58/Dundee15_pChr-GFP_%2B_ChrBopt_combinedplatereaderresults.png" style="width:400px;height:auto">' | ||
| + | |||
| + | <!--insert link of uploaded files here--> | ||
| + | |||
| + | </div> | ||
| + | <div class="modal-footer"> | ||
| + | <button type="button" class="btn btn-primary" data-dismiss="modal">Close</button> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | |||
| + | |||
| + | <div class="modal fade" id="dundee15_chr_150824_platereader_pchrbgfp" tabindex="-1" role="dialog" aria-labelledby="myModalLabel"> | ||
| + | <div class="modal-dialog" role="document"> | ||
| + | <div class="modal-content"> | ||
| + | <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">×</span></button> | ||
| + | <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. | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="modal-body"> | ||
| + | <img src="https://static.igem.org/mediawiki/2015/0/04/Dundee15_pChr-GFP_combinedplatereaderresults.png" style="width:400px;height:auto">' | ||
| + | |||
| + | <!--insert link of uploaded files here--> | ||
| + | |||
| + | </div> | ||
| + | <div class="modal-footer"> | ||
| + | <button type="button" class="btn btn-primary" data-dismiss="modal">Close</button> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | |||
| + | |||
| + | <div class="modal fade" id="dundee15_chr_150911_chrbbielefeld" tabindex="-1" role="dialog" aria-labelledby="myModalLabel"> | ||
| + | <div class="modal-dialog" role="document"> | ||
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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">×</span></button> | ||
| + | <div class="modal-title" id="myModalLabel">Figure 33: Plates after transformation into pT7KS. | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="modal-body"> | ||
| + | <img src="http2015.igem.orgwikiimagesffdDundee15_chr_150911_ChrBBielefeld.png" style="width:400px;height:auto">' | ||
| + | |||
| + | <!--insert link of uploaded files here--> | ||
| + | |||
| + | </div> | ||
| + | <div class="modal-footer"> | ||
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| + | </div> | ||
| + | |||
| + | |||
| + | <div class="modal fade" id="dundee15_chr_150911_bl21" tabindex="-1" role="dialog" aria-labelledby="myModalLabel"> | ||
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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">×</span></button> | ||
| + | <div class="modal-title" id="myModalLabel">Figure 34: Plates after transformation of chromate sensor plasmids into BL21 (DE3). | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="modal-body"> | ||
| + | <img src="https://static.igem.org/mediawiki/2015/7/77/Dundee15_chr_150911_bl21.png" style="width:400px;height:auto">' | ||
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Revision as of 21:16, 13 September 2015
LABJOURNAL
BioSpray
Our forensic toolkit aims to use synthetic biology approaches to improve on the current methods used by crime scene investigators.
Summary
This week was all about getting started with the newly arrived biobrick BBa_K1058008
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To purify BBa_K1058008 for sequence confirmation.
Protocols Used:
Click here to see our miniprep protocol.
Click here to see our sequencing protocol.
Results Sequencing: Figure 1
Next Steps: PCR of parts of BBa_K1058008.
Summary
During this week, BBa_K1058008 was disassembled into its constituent parts, pChr, and ChrB.
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:
Click here to see our PCR protocol.
Click here to see our gel extraction protocol.
Click here to see our restriction digest protocol.
Results:
Figure 2: Agarose gel after PCR of pChr and ChrB.
As can be seen on the image of the gel, PCR of ChrB was not successful. pChr was further processed by restriction digest with EcoRI and PstI
Next Steps: Repeat of PCR of ChrB. Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
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.
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 E. coli.
Overnight culture of strain, containing GFPmut2, as a fluorescent reporter for our Chromate sensing system
Protocols Used:
Click here to see our PCR protocol.
The annealing temperature was lowered in order to increase the chances of accomodating a primer that is not 100% complementary. Furthermore different concentrations of DMSO were tested.
Click here to see our gel extraction protocol.
Click here to see our restriction digest protocol.
Click here to see our ligation protocol. Ligations were made in 2:1 and 3:1 ratio.
Click here to see our Overnight culture protocol.
Results:
Figure 3: Agarose gel after PCR of ChrB.
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.
pChr-pSB1C3, ChrBs-pSB1C3, and ChrBw-pSB1C3 were transformed into E. coli MC1061.
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: Purification of GFPmut2 from overnight cultures. Overnight cultures of recombinant MC1061, containing pChr-pSB1C3, ChrBs-pSB1C3, and ChrBw-pSB1C3 respectively. Overnight culture of pUniprom for subsequent ligation of ChrB
Protocols Used:
Miniprep for plasmid purification of overnight culture of GFPmut2.
PCR for amplification of GFPmut2 and ChrB.
Gel extraction of PCR product pf GFPmut2.
Overnight culture of MC1061 containing pChr-pSB1C3, ChrBs-pSB1C3, and ChrBw-pSB1C3 respectively. Furthermore of a strain containing the vector pUniprom.
Results: Figure 5 Since amplification of ChrB was unsuccessful, it will be repeated.
Next Steps: Sequence and size confirmation of pChr-pSB1C3, ChrBs-pSB1C3, and ChrBw-pSB1C3.
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 of pChr-pSB1C3, ChrBs-pSB1C3, ChrBw-pSB1C3 and pUniprom.
PCR for amplification of pChr and ChrB.
Restriction digest for confirmation of size of pChr and ChrB.
Results: Figure 6 Agarose gel for size confirmation.
Protocols Used:
Sequencing of pChr and ChrB that showed expectes sizes.
Results: Figure 7 Confirmed sequence of pChr1.
Results: Figure 8 Confirmed sequence of ChrBw4.
Protocols Used:
Restriction digest of gel purified PCR-products of pChr, ChrB, and GFPmut2, using EcoRI/SpeI, BamHI/HindIII, and XbaI/PstI respecitvely.
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare pUniprom for insertion of ChrB.
Protocols Used:
Restriction digest of pUniprom with BamHI and HindIII.
Gel extraction of digested pUniprom
Results: Figure 9
Next Steps: Ligation of ChrB into pUniprom.
Aim of experiment: To amplify ChrB version with first 15 codons optimised for insertion into pUniprom.
Protocols Used:
PCR of optimised ChrB from the sequence confirmed ChrBw4-pSB1C3, adding restriction sites compatible to pUniprom.
Gel extraction of optimised ChrBafter PCR.
Results: Figure 10
Next Steps: Ligation of ChrB and codon optimised ChrB into pUniprom.
Aim of experiment: Ligation of ChrB and optimised ChrB into pUniprom. Ligation of pChr and GFPmut2 into pSB1C3
Protocols Used:
Ligation of ChrB and optimised ChrB into pUniprom via BamHI and HindIII restriction sites. Ligation of pChr to GFPmut2 via Spe/Xba link and ligation of pChrGFP construct into pSB1C3 EcoRI/PstI restriction sites.
Results: N/A
Next Steps:Transformation of abovementioned ligations.
Summary
During this week successful transformations were identified and propagated.
Aim of experiment: Transform recombinant vectors into E. coli chassis.
Protocols Used:
Transformation of ChrB-pUniprom, optimised ChrB-pUniprom and pChr-GFPmut2-pSB1C3 into JM110.
Results: Figure 11
Aim of experiment: Backup of digested pSB1C3 for the case that a ligation with 2 inserts does not work.
Protocols Used:
Restriction digest of pSB1C3 with EcoRI/SpeI for subsequent insertion of pChr.
Results:N/A
Next Steps: Store digested plasmid at -20oC.
Aim of experiment: Purification of recombinant vectors for sequence and size confirmation.
Protocols Used:
Overnight culture of JM110 with ChrB-pUniprom, optimised ChrB-pUniprom and pChr-GFPmut2-pSB1C3.
Patch plating of the same colonies.
Results: N/A
Next Steps: Purification of the recombinant plasmids for confirmation of size and sequence.
Aim of experiment: Purification of recombinant plasmids for confirmation of size and sequence.
Protocols Used:
Miniprep of ChrB-pUniprom, optimised ChrB-pUniprom and pChr-GFPmut2-pSB1C3.
Results:N/A
Protocols Used:
Restriction digest of ChrB-pUniprom and optimised ChrB-pUniprom with BamHI and HindIII. Restriction digest of pChr-GFPmut2-pSB1C3 with EcoRI/PstI.
Results: Figure 1
Protocols Used:
Colony PCR of pUniprom-ChrB-opt for elucidation of insert size or presence of insert.
Results: Figure 13 Gel after colony PCR. Colonies 2, 4, 9, and 12 were selected for sequencing.
Protocols Used:
Sequencing of pSB1C3-pChr-GFP.
Results: Figure 14 Sequencing result of pSB1C3-pChr-GFPmut2_1. The Result for pSB1C3-pChr-GFPmut2_2 was not as expected, albeit in the correct orientation.
Next Steps: Confirmation of insert size and sequence of ChrB-opt.
Aim of experiment: To prepare ChrB-opt colonies 2, 4, 9, and 12 for sequencing.
Protocols Used:
Click here to see our overnight culture protocol.
Results:N/A
Next Steps: Purification of recombinant plasmids.
Aim of experiment: To purify recombinant plasmids for sequence confirmation.
Protocols Used:
Miniprep of ChrB-opt 2, 4, 9, 12.
Results:N/A
Protocols Used:
Sequencing of ChrB-opt 2, 4, 9, 12.
Results: ChrB showed HindIII restriction sites and was hence cleaved into partial inserts.
Next Steps:Use alternative cloning protocol for ChrB and ChrBopt.
Summary
During this week an alternative cloning strategy for ChrB and ChrB-opt was employed. Furthermore, the pChr-GFP construct was cloned in a stepwise fashion.
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.
Results:Figure 15 pSB1C3-pChr-GFP in JM110.
Next Steps:Size confirmation and sequencing of newly selected colonies.
Aim of experiment:Purification of newly selected colonies containing pSB1C3-pChr-GFP for subsequent sequence confirmation.
Protocols Used:
Miniprep for purification of pSB1C3-pChr-GFP.
Results:N/A
Next Steps:Sequencing of pSB1C3-pChr-GFP samples 1, 5, 6, and 7.
Protocols Used:
Sequencing of pSB1C3-pChr-GFP samples 1, 5, 6, and 7.
Results: Figure 16 Alignment of sequencing results.
Next Steps:Stepwise ligation of pChr-GFP into pSB1C3.
Protocols Used:
Restriction digest of pUniprom with BamHI/PstI for subsequent insertion of ChrB and ChrB-opt.
p>Results: Figure 17 Gel imageNext Steps:Insertion of ChrB and ChrB-opt respectively via BamHI/PstI.
Aim of experiment:Ligation of pChr into pSB1C3 via Eco/Spe link.
Protocols Used:
Ligation of pChr(08/07/15) into pSB1C3(13/07/15)
Results: N/A
Aim of experiment:Transformation
Protocols Used:
Transformation of pSB1C3-pChr into JM110.
Results: Figure 18 Plates after transformation of pChr into pSB1C3.
Next Steps:Sequence confirmation of pChr in pSB1C3.
Aim of experiment: To prepare pSB1C3-pChr for sequence confirmation
Protocols Used:
Overnight culture of pSB1C3-pChr.
Patch-plating of the same colonies of pSB1C3-pChr.
Results: N/A
Next Steps: Purification of the recombinant vector for sequence confirmation.
Aim of experiment:Purification of pSB1C3-pChr for sequence confirmation.
Protocols Used:
Miniprep of pSB1C3-pChr.
Results: Insert table here
Aim of experiment:Sequence confirmation of pSB1C3-pChr.
Protocols Used:
Sequencing of samples 1 and 2 of pSB1C3-pChr.
Results: Figure 19 Alignment of sequenced pSB1C3-pChr vectors with pChr sequence.
Next Steps: Digest of pSB1C3-pChr with SpeI/PstI and ligation of GFPmut2.
Aim of experiment: Amplification of ChrB and optimised ChrB
Protocols Used:
PCR-mplification of ChrB and ChrBopt.
Results: N/A
Next Steps:Ligation and Transformation of ChrB and optimised ChrB.
Aim of experiment: Insertion of ChrB and optimised ChrB into pUniprom vector.
Protocols Used:
Ligation of ChrB and optimised ChrB into vector pUniprom.
Results: N/A
Next Steps: Transformation of ligated vectors.
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.
Aim of experiment:Transformation of the repressors, ChrB and optimised ChrB, into JM110
Protocols Used:
Transformation of ChrB and optimised ChrB into JM110
Results: Figure 1
Next Steps: Purification of the plasmids for sequence confirmation.
Aim of experiment:Processing of pSB1C3-pChr from 24/07 for insertion of GFP.
Protocols Used:
Restriction digest of pSB1C3-pChr with SpeI/PstI for insertion of GFP.
Ligation of GFPmut2, previously digested with XbaI/PstI.
Results: N/A
Next Steps: Transformation of recombinant vector for confirmation of size and sequence.
Aim of experiment:Overnight culture from plates of ChrB and optimised ChrB.
Protocols Used:
Overnight culture of of ChrB and optimised ChrB.
Results: N/A
Next Steps: Purification of plasmids for sequence confirmation.
Aim of experiment:Purification of ChrB and optimised ChrB for confirmation of size and sequence.
Protocols Used:
Miniprep of ChrB and ChrBopt.
Restriction Digest of the purified vectors for confirmation of size.
Sequencing of ChrB and ChrBopt.
Results:
Next Steps: Confirmation of expression of ChrB and ChrBopt by western blotting.
Aim of experiment:Digest of pSB1C3-pChr with SpeI/PstI for insertion of GFP. Repeat of unsuccessful transformation from 28/07.
Protocols Used:
Restriction digest of pSB1C3-pChr with SpeI/PstI for insertion of GFP.
Ligation of GFPmut2, previously digested with XbaI/PstI.
Transformation of pChrGFP into JM110.
Results: Figure 22
Next Steps: Purification of the Plasmids for confirmation of insert size and sequence.
Aim of experiment:Overnight cultures for subsequent plasmid purification of pSB1C3-pChr-GFP.
Protocols Used:
Overnight cultureof pSB1C3-pChr-GFP.
Results: N/A
Next Steps: Purification of the Plasmids for confirmation of insert size and sequence.
Aim of experiment: Purification of pSB1C3-pChr-GFP for confirmation of insert size and sequence.
Protocols Used:
Miniprep for purification of pSB1C3-pChr-GFP.
Restriction digest of purified plasmid with EcoRI/PstI for confirmation of insert size.
Results:
Figure 24 Gel for size confirmation.
Figure 24 Sequence confirmation.
Next Steps:Overnight cultures in preparation for western blotting for characterisation of expression of both, GFP, and ChrB.
Aim of experiment:Overnight cultures of sequence confirmed ChrB, ChrBopt, pChrGFP1 (from 15/07), pChrGFP3 and pChrGFP4 (31/07) for subsequent western blotting.
Protocols Used:
Overnight culture of named constituents.
Results: N/A
Next Steps: Western blotting for characterisation of expression of both, GFP, and ChrB.
Summary
This week was entirely used for characterising the expression of GFP from the promoter pChr, and the expression of ChrB from the tat-promoter in pUniprom.
Aim of experiment:To determine whether or not the required proteins, GFP, and ChrB, are expressed.
Protocols Used:
SDS-PAGE and western blot against GFP and against His.
Results: 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:
- 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.
- HIS: Dilute pellet of 1ml overnight culture with 200µl Laemmli buffer. Load 10µl on the SDS-gel.
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.
Summary
This week was spent with transforming all combinations of plasmids into MG1655. Tests for their interaction were also prepared.
Aim of experiment:To transform the following combinations of plasmids into MG1655:
- ChrB2 (29/07) - pChrGFP (31/07)
- ChrB2 (29/07) - pChrGFP (15/07)
- ChrBopt3 (29/07) - pChrGFP (31/07)
- ChrBopt3 (29/07) - pChrGFP (15/07)
Protocols Used:
Miniprep all 4 constituents. Also purify BBa_K1058008
Transform all 4 in separate aliquots of MG1655.
Results: Figure 1
Next Steps:Making newly transformed cells competent again in order to transform the second plasmid as well.
Aim of experiment: To prepare overnight cultures of the transformations in order to make them competent again.
Protocols Used:
Overnight culture of a colony of each plate.
Results: N/A
Next Steps:Make cells of overnight culture competent to transform in second plasmid.
Aim of experiment:To make transformed cells competent in order to transform the second plasmid for a full system.
Protocols Used:
Making competent cells of MG1655, containing ChrB2, ChrBopt3, pChrGFPrev, pChrGFPfor respectively.
Transform same plasmids that were used for initial transformation into newly made competent cells in order to get all 4 combinations of plasmids as outline on day 1 of this week.
Results: insert images of plates once they are round.
Next Steps:Blot against GFP in order to confirm (or refute) that ChrB interacts with pChr and hence switches off expression of GFP.
Aim of experiment: To build a strain of MG1655, containing both plasmids required for a chromate sensor.
Protocols Used: Transformation
Results: No successful transformants were found.
Next Steps: Check Plates for successful transformants.
Summary
During this week recombinant MG1655 were made, containing both plasmids. Western Blots were used to determine expression of ChrB, ChrBopt, and GFP.
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 ChrBopt (via His-tag) and expression of GFP.
Aim of experiment: To determine whether ChrB, ChrBopt, 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.
Aim of experiment: To prepare whether expression of ChrB or ChrB opt has an effect on the expression of GFP.
Protocols Used: SDS-PAGE + Western Blot
Results: Figure 28
Next Steps: Check if different substances containing chromate have an effect on the expression.
Summary
Plate reader experiments were carried out in order to determine the effect of K2CrO4, K2Cr2O7, CrCl3, and K2SO4 on the expression of GFP.
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
Results: Figure 30
Results: Figure 31
Results: Figure 32
Next Steps: Try the system with different expression levels of repressor. Induce overexpression of repressor from T7-promoter in pUniprom using BL21 and IPTG.
Summary
UK Synbio Conference and UK iGEM meetup at Westminster University.
Summary
Parts from CeBiTec Bielefeld arrived and attempts at cloning were made. The chromate sensing system was transformed into BL21 and some of the plate reader experiments repeated.
Aim of experiment: The high-copy plasmid arrived at very low concentration. Hence it was transformed in order to produce more of it before digesting it.
Protocols Used: Transformation, Overnight culture for competent cells
Results: No transformants were found.
Next Steps: Retry and make more competent MG1655.
Aim of experiment: Test transformation of BBa_K1058008 into new MG1655 competent cells and transformation of pSB1K3-plasmid
Protocols Used: Transformation,
Results: Successful test transformation, but no colonies on pSB1K3 plate.
Next Steps: Use an alternative plasmid.
Aim of experiment: Restriction digest of pT7KS with EcoRI and PstI as alternative to pSB1K3. Ligation of digested ChrB (Bielefeld) and transformation of this plasmid. Also transformations of Chromate-sensor plasmids into BL21 (DE3) in the following combinations:
- pChrGFP + ChrB
- pChrGFP + ChrBopt
- ChrB
- ChrBopt
Protocols Used: Restriction Digest, Gel Extraction, Ligation
Results:Transformations of the plasmid with ChrB (Bielefeld) yielded 12 colonies, but also colonies on the vector control plate. Transformations into BL21 were successful.
Next Steps: Confirmation of colonies with pT7KS-ChrB by colony PCR. Preparation of recombinant BL21 for plate reader experiments.
Aim of experiment: Check if any of the colonies after ligation of ChrB (Bielefeld) into pT7KS were successful despite an unsuccessful control plate.
Protocols Used: Colony PCR
Results: Figure 34
Next Steps: Prepare recombinant BL21 for plate reader experiments. Attempt new transformation of ChrB (Bielefeld).
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.
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
Results:add images once analysed.
Next Steps:Repeat experiments with added chromate substances.
Restriction digests were set up according to the table below. The appropriate reaction and restriction enzymes that were used is stated in the Journal section.
|
Post Plasmid Purification |
Post PCR |
gBlock |
|
|
DNA |
1mg |
50µl |
100ng |
|
Cutsmart Buffer (µl) |
6 |
6 |
6 |
|
Water (µl) |
As required |
1 |
As required |
|
Enzyme 1 (µl) |
1.5 |
1.5 |
1.5 |
|
Enzyme 2 (µl) |
1.5 |
1.5 |
1.5 |
|
Total Volume (µl) |
30 |
60 |
30 |
- The digests were incubated at 37oC for 3 hours.
- 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.
Ligation reactions were set up according to the table below. The vector to insert ratio is stated for each specific reaction in the Journal section.
|
Vector (µl) |
2:1 (Vector: Insert) [µl] |
3:1 (Vector: Insert) [µl] |
4:1 (Vector: Insert) [µl] |
|
|
T4 DNA Ligase |
1 |
1 |
1 |
1 |
|
Ligase Buffer |
1 |
1 |
1 |
1 |
|
Vector |
1 |
1 |
1 |
1 |
|
Insert |
- |
2 |
3 |
4 |
|
H20 |
7 |
5 |
4 |
3 |
The reactions were incubated at room temperature for at least 3 hours.
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