http://2010.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=50&target=Macbereska2010.igem.org - User contributions [en]2024-03-29T13:27:43ZFrom 2010.igem.orgMediaWiki 1.16.5http://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_sensorTeam:Edinburgh/Bacterial/Blue light sensor2010-10-27T22:30:17Z<p>Macbereska: </p>
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<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_producer">blue light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Green_light_producer">green light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Green_light_sensor">green sensor</a></li><br />
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<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Collaboration">collaboration</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Attribution">attribution</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/BRIDGE">BRIDGE</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_producer">red light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_sensor">red sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_producer">blue light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_producer">green light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_sensor">green sensor</a></li><br />
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<a name="Overview" id="Overview"></a><h2>Overview: The blue light sensor</h2><br />
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<p>The blue-light sensor, which is composed of the LovTAP hybrid protein designed by <a href="#References">Strickland et al. (2008)</a> and made available by Professor Sosnick, was BioBricked by <a href="https://2009.igem.org/Team:EPF-Lausanne">EPF-Lausanne in iGEM 2009</a>. It is based on a α-helical domain linker between the Lov2 domain (the photoactive protein) and the <i>E. coli trp</i> repressor, which acts as a conduit for allosteric signals. The effective response of the sensor is at a wavelength of 470nm (as documented by the aforementioned Lausanne iGEM team).</p><br />
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<p>The blue-light sensor (LovTAP) consists of five parts:</p><br />
<br />
<ol><br />
<li>Photoreceptor1 (the shared helix between the Lov domain and the TrpR domain)<br />
<ul><br />
<li>Dark blue when contacting the Lov domain (dark state).</li><br />
<li>Red when contacting the TrpR domain (light state).</li><br />
</ul><br />
</li><br />
<li>Photoreceptor2 (falvin monoucleotide-FMN cofactor)</li><br />
<li>Lov domain-orange (photoactive protein)</li><br />
<li>TrpR domain-grey (DNA regulator)</li><br />
<li>Operator DNA</li><br />
</ol><br />
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<center><p><br><img src="https://static.igem.org/mediawiki/2010/5/59/Ed10-Strickland.jpg" width="640" height="505" border="0"/></p><br><br />
<p><b>Figure 1:</b> The mechanism of action of the LovTAP allosteric light sensor.</p><br />
<p>Image: <a href="#References">Strickland et al. (2008)</a></p><br></center><br />
<br />
<p><a href="img src="https://static.igem.org/mediawiki/2010/5/59/Ed10-Strickland.jpg">Figure 1</a> above shows the whole process regarding how the light sensor works, from the dark state (A) to the light-activated state (B → C) and then returning to the stable state (D → A). In the dark state, the shared helix contacts the Lov2 domain, and the inactivated TrpR dissociates from the DNA; in the light state, the Lov2 domains absorb the blue light proton and form a covalent adduct between the FMN cofactor and a conserved cysteine residue, destroying the shared helix in the Lov domain and binding / populating an active formation of the TrpR domain. This in turn leads to LovTAP binding the DNA and repressing lambda-cI. However, this binding is not stable, and thus it will eventually return to the initial state.</p><br />
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<a name="Strategy" id="Strategy"></a><h2>Strategy</h2><br />
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<p>Our original plan was to obtain and revive <a href="https://2009.igem.org/Team:EPF-Lausanne">EPF-Lausanne 2009</a>'s <a href="http://partsregistry.org/Part:BBa_K191009">BBa_K191009</a>. Once we had done so, we aimed to use it to create a blue light sensing system, and transform cells with for characterisation of the system and for analysis of their compatibility with the mutated blue luciferases. Further characterization of LovTap included investigation of the tryptophan influence on the system, and therefore TrpR mutant was transformed with LovTap-reporter construct.</p><br><br />
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<a name="Problems" id="Problems"></a><h2>Problems</h2><br />
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<p>We were unable to successfully characterise the LovTAP that we initially received from Lausanne due to a frameshift mutation. Eventually, we received a new version of LovTAP from our collaborators at <a href="https://2010.igem.org/Team:UNAM-Genomics_Mexico">Mexico UNAM-Genomics</a>, so that we could perform characterisation tests on it for them. Unfortunately, we were again unable to elicit a clear response from our sensor.</p><br />
<br />
<p>Our major problem was that transformants often grew the wrong colour. LovTAP controls the production of RFP in the cells; when LovTAP is inactive (i.e. in the dark), the cells should be producing RFP and hence should be growing red. When LovTAP is active (i.e. in white or blue light), the cells stop producing RFP and hence should produce white colonies. At the moment some cells in the light are still growing red, although some are definitely growing white, and vice versa in the dark. One suggestion was that the plasmids are unstable and dividing randomly, altering the intensity of the colour in some cells. Attempts to stabilise the colours of the colonies are ongoing (see the video in the <a href="https://2010.igem.org/Team:Edinburgh/Gallery">gallery</a> which demonstrates one of the innovative methods of providing proper culture conditions).</p><br><br />
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<a name="BioBricks" id="BioBricks"></a><h2>BioBricks</h2><br />
<br><br />
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<p>As stated above, our blue light sensor is based on a modified version of <a href="https://2009.igem.org/Team:EPF-Lausanne">Lausanne 2009</a>'s LovTAP part (<a href="http://partsregistry.org/Part:BBa_K191009">BBa_K191009</a>) developed by our collaborators at <a href="https://2010.igem.org/Team:UNAM-Genomics_Mexico">Mexico UNAM-Genomics</a>: <a href="http://partsregistry.org/Part:BBa_K360121">BBa_K360121</a>. We have coupled this with a simple reporter system (RFP) in order to perform characterisation tests.</p><br />
<br />
<p><a href="http://partsregistry.org/Part:BBa_K322999">BBa_K322999</a>: LovTAP with RFP reporter system, based on <a href="https://2010.igem.org/Team:UNAM-Genomics_Mexico">Mexico UNAM-Genomics</a> <a href="http://partsregistry.org/Part:BBa_K360121">BBa_K360121</a></p><br><br />
<br><br />
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<a name="Characterisation" id="Characterisation"></a><h2>Characterisation</h2><br />
<br><br />
The protocol for LovTap characterization:<br />
<ul><br />
<li>Liquid cultures (same as for minipreps) were grown overnight in 37C with antibiotics. The next day, bottles with 3.5 ml of broth were inoculated with the 0.5 ml of appropriate cultures (0.5. ml of broth added as a control).</li><br />
<li>samples designated DARK were wrapped in the aluminium foil straight after the inoculation; the cap was not wrapped (not see through anyway)so that samples could be easily taken without unwrapping the bottles from the foil.</li><br />
<li>All the samples were then taken to the shaker in the hot room and placed in prepared spots: for the dark- anywhere on an easily accessible holder, for light- in 2 rows of holders..</li><br />
<li>Light samples were covered with the cardboard box with blue LEDs attached on the sides(470 nm wavelength; 2200 mcd/B; V=4.5 V; RS466-3548), in a way to optimize light accession to the samples. Box was securely taped to the shaker.</li><br />
<li> Measurments were taken every half an hour: 200ul of the samples were placed in the numbered cuvette and then 800ul of water was added. Readings of fluroscence (green mode) were taken in standard fluorescence units (SFU, two readings each). Then optical density was measured (upstairs in the incubator room) at 360 nm wavelength. Control was used to calibrate the machine (set ref button). Also 2 readings were taken. Samples were shaken by inversion prior to taking measurment.</li><br />
<li>Experiment was stopped after 3 hours (6 sets of reading taken). </li><br />
Result analysis<br />
<li>To generate the data for the graph:</li><br />
<li> 1. means were calculated from 2 readings of optical density and fluorescence. </li><br />
<li> 2. (mean SFU minus background SFU) was divided by (mean OD minus background OD, if different from 0).</li><br />
<li> 3. table was made for the samples at different points of time- e.g. sample 1 after 30 min, 60 min, 90 min, 120 min etc.</li><br />
<li> 4. graph was made with series named the same as the samples</li> </ul><br />
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<center><br><br><p><img src="https://static.igem.org/mediawiki/2010/9/91/8octLovtap.jpg" width="600px"></p><br><br />
<p><b>Figure 2:</b> LovTAP activation.</p><br><br></center><br />
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<p><a href="https://static.igem.org/mediawiki/2010/9/91/8octLovtap.jpg">Figure 2</a>.</p><br />
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<center><br><br><p><img src="https://static.igem.org/mediawiki/2010/3/38/8octTRPR.jpg" width="600px"></p><br><br />
<p><b>Figure 3:</b> LovTAP TrpR mutant activation.</p><br><br></center><br />
<br />
<p><a href="https://static.igem.org/mediawiki/2010/3/38/8octTRPR.jpg">Figure 3</a>.</p><br />
<ul><br />
<li> <br />
Figure 2 shows the response of two clones of LovTap under the blue light. (Controls were not included in this experiment. There is no clear difference in response (measured by fluorescence/optical density) over time. Expected results would show higher fluorescence in the dark than in the light.</li><br />
<li> Trouble shooting: there is a nedd of testing system under different conditions: temperature, different strength of promoters etc. Further experiments are being carried. </li><br />
<li> Figure 3 shows same experiment performed on TrpR mutant- to see if there is difference of LovTap activation under blue light ilumination. Again, fluorescence should be higher in the dark state- due to RFP reporter being suppressed by blue light- LovTap activation. Again, results are inconclusive. </li><br />
<li> Trouble shooting:again, the same can be said: different strength of promoter could be used, as well as different medium- we tried repeat the experiment using CMM minimum medium, but so far cells fail to grow. </li><br />
<li> for the details of the lab book on the blue light sensor please see https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_sensor </li><br />
</ul><br />
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<a name="References" id="References"></a><h2>References</h2><br />
<br><br />
<br />
<p><b>Strickland, D., Moffat, K. & Sosnick, T. R. (2008).</b> Light-activated DNA binding in a designed allosteric protein. <i>Proceedings of the National Academy of Sciences</i> <b>105</b>, 10709-10714.</p><br />
<p><b>Schüttrigkeit, T. A., Kompa, C. K., Salomon, M., Rüdiger, W. &amp; Michel-Beyerle, M. E. (2003).</b> Primary photophysics of the FMN binding LOV2 domain of the plant blue light receptor phototropin of Avena sativa. <i>Chemical Physics</i> <b>294</b>, 501-508.</p><br />
<p><b>Wu, Y. I., D. Frey, et al. (2009).</b> A genetically encoded photoactivatable Rac controls the motility of living cells. <i>Nature</i> Vol <b>461</b></p><br />
<p>EPF Lausanne 2009 team wiki, <i>https://2009.igem.org/Team:EPF-Lausanne</i>.</p><br />
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<span style="color:ivory;">Throughout this wiki there are words in <b>bold</b> that indicate a relevance to <b>human aspects</b>. It will become obvious that <b>human aspects</b> are a part of almost everything in <b>iGEM</b>.</span><br />
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</html></div>Macbereskahttp://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_sensorTeam:Edinburgh/Bacterial/Blue light sensor2010-10-27T22:28:55Z<p>Macbereska: </p>
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<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Core_repressilator">the repressilator</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_producer">red light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_sensor">red sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_producer">blue light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Green_light_producer">green light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Green_light_sensor">green sensor</a></li><br />
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<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Bacterial">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/References">references</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling" class="dir">modelling BRIDGEs</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Kappa">kappa</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Genomic">the genomic model</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Bacterial">the bacterial model</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Signalling">the signalling model</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Tools">tools</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Modelling">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/References">references</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human" class="dir">human BRIDGEs</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Aspects">human aspects</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Communication">communication of science</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Terminology">terminology research</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Wiki">wiki</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Conversations">conversations</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Identity">identity</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Collaboration">collaboration</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Branding">branding research</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/SciFi">science fiction writing</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/FutureApps">future applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/SelfReflection">self-reflection</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Epic">the epic</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Human">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/References">references</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook" class="dir">lab notes&nbsp;&nbsp;&nbsp;</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Collaboration">collaboration</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Attribution">attribution</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/BRIDGE">BRIDGE</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_producer">red light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_sensor">red sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_producer">blue light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_producer">green light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_sensor">green sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Modelling">modelling</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Safety">safety</a></li><br />
<li><a href="http://www.openwetware.org/wiki/French_Lab">protocols</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Acknowledgements" class="dir">acknowledgements</a><br />
</li><br />
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</ul><br />
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<a name="Overview" id="Overview"></a><h2>Overview: The blue light sensor</h2><br />
<br><br />
<br />
<p>The blue-light sensor, which is composed of the LovTAP hybrid protein designed by <a href="#References">Strickland et al. (2008)</a> and made available by Professor Sosnick, was BioBricked by <a href="https://2009.igem.org/Team:EPF-Lausanne">EPF-Lausanne in iGEM 2009</a>. It is based on a α-helical domain linker between the Lov2 domain (the photoactive protein) and the <i>E. coli trp</i> repressor, which acts as a conduit for allosteric signals. The effective response of the sensor is at a wavelength of 470nm (as documented by the aforementioned Lausanne iGEM team).</p><br />
<br />
<p>The blue-light sensor (LovTAP) consists of five parts:</p><br />
<br />
<ol><br />
<li>Photoreceptor1 (the shared helix between the Lov domain and the TrpR domain)<br />
<ul><br />
<li>Dark blue when contacting the Lov domain (dark state).</li><br />
<li>Red when contacting the TrpR domain (light state).</li><br />
</ul><br />
</li><br />
<li>Photoreceptor2 (falvin monoucleotide-FMN cofactor)</li><br />
<li>Lov domain-orange (photoactive protein)</li><br />
<li>TrpR domain-grey (DNA regulator)</li><br />
<li>Operator DNA</li><br />
</ol><br />
<br />
<center><p><br><img src="https://static.igem.org/mediawiki/2010/5/59/Ed10-Strickland.jpg" width="640" height="505" border="0"/></p><br><br />
<p><b>Figure 1:</b> The mechanism of action of the LovTAP allosteric light sensor.</p><br />
<p>Image: <a href="#References">Strickland et al. (2008)</a></p><br></center><br />
<br />
<p><a href="img src="https://static.igem.org/mediawiki/2010/5/59/Ed10-Strickland.jpg">Figure 1</a> above shows the whole process regarding how the light sensor works, from the dark state (A) to the light-activated state (B → C) and then returning to the stable state (D → A). In the dark state, the shared helix contacts the Lov2 domain, and the inactivated TrpR dissociates from the DNA; in the light state, the Lov2 domains absorb the blue light proton and form a covalent adduct between the FMN cofactor and a conserved cysteine residue, destroying the shared helix in the Lov domain and binding / populating an active formation of the TrpR domain. This in turn leads to LovTAP binding the DNA and repressing lambda-cI. However, this binding is not stable, and thus it will eventually return to the initial state.</p><br />
<br><br />
<br><br />
<br />
<a name="Strategy" id="Strategy"></a><h2>Strategy</h2><br />
<br><br />
<p>Our original plan was to obtain and revive <a href="https://2009.igem.org/Team:EPF-Lausanne">EPF-Lausanne 2009</a>'s <a href="http://partsregistry.org/Part:BBa_K191009">BBa_K191009</a>. Once we had done so, we aimed to use it to create a blue light sensing system, and transform cells with for characterisation of the system and for analysis of their compatibility with the mutated blue luciferases. Further characterization of LovTap included investigation of the tryptophan influence on the system, and therefore TrpR mutant was transformed with LovTap-reporter construct.</p><br><br />
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<a name="Problems" id="Problems"></a><h2>Problems</h2><br />
<br><br />
<br />
<p>We were unable to successfully characterise the LovTAP that we initially received from Lausanne due to a frameshift mutation. Eventually, we received a new version of LovTAP from our collaborators at <a href="https://2010.igem.org/Team:UNAM-Genomics_Mexico">Mexico UNAM-Genomics</a>, so that we could perform characterisation tests on it for them. Unfortunately, we were again unable to elicit a clear response from our sensor.</p><br />
<br />
<p>Our major problem was that transformants often grew the wrong colour. LovTAP controls the production of RFP in the cells; when LovTAP is inactive (i.e. in the dark), the cells should be producing RFP and hence should be growing red. When LovTAP is active (i.e. in white or blue light), the cells stop producing RFP and hence should produce white colonies. At the moment some cells in the light are still growing red, although some are definitely growing white, and vice versa in the dark. One suggestion was that the plasmids are unstable and dividing randomly, altering the intensity of the colour in some cells. Attempts to stabilise the colours of the colonies are ongoing (see the video in the <a href="https://2010.igem.org/Team:Edinburgh/Gallery">gallery</a> which demonstrates one of the innovative methods of providing proper culture conditions).</p><br><br />
<br><br />
<br />
<a name="BioBricks" id="BioBricks"></a><h2>BioBricks</h2><br />
<br><br />
<br />
<p>As stated above, our blue light sensor is based on a modified version of <a href="https://2009.igem.org/Team:EPF-Lausanne">Lausanne 2009</a>'s LovTAP part (<a href="http://partsregistry.org/Part:BBa_K191009">BBa_K191009</a>) developed by our collaborators at <a href="https://2010.igem.org/Team:UNAM-Genomics_Mexico">Mexico UNAM-Genomics</a>: <a href="http://partsregistry.org/Part:BBa_K360121">BBa_K360121</a>. We have coupled this with a simple reporter system (RFP) in order to perform characterisation tests.</p><br />
<br />
<p><a href="http://partsregistry.org/Part:BBa_K322999">BBa_K322999</a>: LovTAP with RFP reporter system, based on <a href="https://2010.igem.org/Team:UNAM-Genomics_Mexico">Mexico UNAM-Genomics</a> <a href="http://partsregistry.org/Part:BBa_K360121">BBa_K360121</a></p><br><br />
<br><br />
<br />
<a name="Characterisation" id="Characterisation"></a><h2>Characterisation</h2><br />
<br><br />
The protocol for LovTap characterization:<br />
<ul><br />
<li>Liquid cultures (same as for minipreps) were grown overnight in 37C with antibiotics. The next day, bottles with 3.5 ml of broth were inoculated with the 0.5 ml of appropriate cultures (0.5. ml of broth added as a control).</li><br />
<li>samples designated DARK were wrapped in the aluminium foil straight after the inoculation; the cap was not wrapped (not see through anyway)so that samples could be easily taken without unwrapping the bottles from the foil.</li><br />
<li>All the samples were then taken to the shaker in the hot room and placed in prepared spots: for the dark- anywhere on an easily accessible holder, for light- in 2 rows of holders..</li><br />
<li>Light samples were covered with the cardboard box with blue LEDs attached on the sides(470 nm wavelength; 2200 mcd/B; V=4.5 V; RS466-3548), in a way to optimize light accession to the samples. Box was securely taped to the shaker.</li><br />
<li> Measurments were taken every half an hour: 200ul of the samples were placed in the numbered cuvette and then 800ul of water was added. Readings of fluroscence (green mode) were taken in standard fluorescence units (SFU, two readings each). Then optical density was measured (upstairs in the incubator room) at 360 nm wavelength. Control was used to calibrate the machine (set ref button). Also 2 readings were taken. Samples were shaken by inversion prior to taking measurment.</li><br />
<li>Experiment was stopped after 3 hours (6 sets of reading taken). </li><br />
Result analysis<br />
<li>To generate the data for the graph:</li><br />
<li> 1. means were calculated from 2 readings of optical density and fluorescence. </li><br />
<li> 2. (mean SFU minus background SFU) was divided by (mean OD minus background OD, if different from 0).</li><br />
<li> 3. table was made for the samples at different points of time- e.g. sample 1 after 30 min, 60 min, 90 min, 120 min etc.</li><br />
<li> 4. graph was made with series named the same as the samples</li> </ul><br />
<br />
<br />
<center><br><br><p><img src="https://static.igem.org/mediawiki/2010/9/91/8octLovtap.jpg" width="600px"></p><br><br />
<p><b>Figure 2:</b> LovTAP activation.</p><br><br></center><br />
<br />
<p><a href="https://static.igem.org/mediawiki/2010/9/91/8octLovtap.jpg">Figure 2</a>.</p><br />
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<center><br><br><p><img src="https://static.igem.org/mediawiki/2010/3/38/8octTRPR.jpg" width="600px"></p><br><br />
<p><b>Figure 3:</b> LovTAP TrpR mutant activation.</p><br><br></center><br />
<br />
<p><a href="https://static.igem.org/mediawiki/2010/3/38/8octTRPR.jpg">Figure 3</a>.</p><br />
<ul><br />
<li> <br />
Figure 2 shows the response of two clones of LovTap under the blue light. (Controls were not included in this experiment. There is no clear difference in response (measured by fluorescence/optical density) over time. Expected results would show higher fluorescence in the dark than in the light.</li><br />
<li> Trouble shooting: there is a nedd of testing system under different conditions: temperature, different strength of promoters etc. Further experiments are being carried. </li><br />
<li> Figure 3 shows same experiment performed on TrpR mutant- to see if there is difference of LovTap activation under blue light ilumination. Again, fluorescence should be higher in the dark state- due to RFP reporter being suppressed by blue light- LovTap activation. Again, results are inconclusive. </li><br />
<li> Trouble shooting:again, the same can be said: different strength of promoter could be used, as well as different medium- we tried repeat the experiment using CMM minimum medium, but so far cells fail to grow. </li><br />
<li> for the details of the protocol please see https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_sensor </li><br />
</ul><br />
<br><br />
<br><br />
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<a name="References" id="References"></a><h2>References</h2><br />
<br><br />
<br />
<p><b>Strickland, D., Moffat, K. & Sosnick, T. R. (2008).</b> Light-activated DNA binding in a designed allosteric protein. <i>Proceedings of the National Academy of Sciences</i> <b>105</b>, 10709-10714.</p><br />
<p><b>Schüttrigkeit, T. A., Kompa, C. K., Salomon, M., Rüdiger, W. &amp; Michel-Beyerle, M. E. (2003).</b> Primary photophysics of the FMN binding LOV2 domain of the plant blue light receptor phototropin of Avena sativa. <i>Chemical Physics</i> <b>294</b>, 501-508.</p><br />
<p><b>Wu, Y. I., D. Frey, et al. (2009).</b> A genetically encoded photoactivatable Rac controls the motility of living cells. <i>Nature</i> Vol <b>461</b></p><br />
<p>EPF Lausanne 2009 team wiki, <i>https://2009.igem.org/Team:EPF-Lausanne</i>.</p><br />
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<center><a href="#top" class="dir"><img width="100" src="https://static.igem.org/mediawiki/2010/9/9f/Ed10-RTT.png"></a></center><br />
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<span style="color:ivory;">Throughout this wiki there are words in <b>bold</b> that indicate a relevance to <b>human aspects</b>. It will become obvious that <b>human aspects</b> are a part of almost everything in <b>iGEM</b>.</span><br />
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</html></div>Macbereskahttp://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_sensorTeam:Edinburgh/Notebook/Blue light sensor2010-10-27T22:11:22Z<p>Macbereska: /* Blue Light Sensor */</p>
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<li><a href="https://2010.igem.org/Team:Edinburgh/Project/Protocol">the protocol</a></li><br />
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<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial" class="dir">bacterial BRIDGEs</a><br />
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<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Core_repressilator">the repressilator</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_producer">red light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_sensor">red sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_producer">blue light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Green_light_producer">green light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Green_light_sensor">green sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/BioBricks#Bacterial">submitted parts</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Bacterial">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/References">references</a></li><br />
</ul><br />
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<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling" class="dir">modelling BRIDGEs</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Kappa">kappa</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Genomic">the genomic model</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Bacterial">the bacterial model</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Signalling">the signalling model</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Tools">tools</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Modelling">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/References">references</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human" class="dir">human BRIDGEs</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Aspects">human aspects</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Communication">communication of science</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Terminology">terminology research</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Wiki">wiki</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Conversations">conversations</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Identity">identity</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Collaboration">collaboration</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Branding">branding research</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/SciFi">science fiction writing</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/FutureApps">future applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/SelfReflection">self-reflection</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Epic">the epic</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Human">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/References">references</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook" class="dir">lab notes&nbsp;&nbsp;&nbsp;</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Collaboration">collaboration</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Attribution">attribution</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/BRIDGE">BRIDGE</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_producer">red light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_sensor">red sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_producer">blue light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_producer">green light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_sensor">green sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Modelling">modelling</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Safety">safety</a></li><br />
<li><a href="http://www.openwetware.org/wiki/French_Lab">protocols</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Acknowledgements" class="dir">acknowledgements</a><br />
</li><br />
<br />
</ul><br />
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<span style="color:ivory;">These <b>notes</b> correspond to the part of the <b>project</b> detailed <a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_sensor">here</a>.</span><br />
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<br />
==Blue Light Sensor==<br />
<br><br />
<br />
'''14/07/10'''<br />
<br />
Received parts from Lausanne:<br />
*BBa_191006 (113 ng/µL) – LovTAP alone<br />
*BBa_191004 (166 ng/µL) – Readout <br />
*BBa_191003 (230 ng/µL) – whole system<br />
*BBa_191009 (85 ng/µL) – mutant <br />
*Sosnick vector containing TrpR (393 ng/µL) – vector <br />
<br />
'''16/07/10'''<br />
<br />
Transformations using 100 µL of cells, 900 µL broth<br />
*K191004 - amp<br />
*K191003 - kanamycin<br />
*K191009 – kanamycin<br />
*Sosnick vector with TrpR – amp<br />
<br />
Note: Labeling on tubes is unclear – K191009 and K191004 got mixed up, so plating both tubes on both Kan and amp<br />
<br />
'''22/07/10'''<br />
<br />
*Minipreps: K0998010 – 5x (1 didn’t grow, but did anyway)<br />
*Blue sensors (3 biobricks and Sosnick vector) – cultures set to grow overnight<br />
<br />
'''25/07/10'''<br />
Digest of the blue light sensor minipreps was performed with EcoRI and BamHI; samples:<br />
*3- K19100'''4'''; 900ul, (3)<br />
*4- K19100'''4'''; 100ul, (2)<br />
*5-K19100'''9'''; 100ul, (1)<br />
*6- K19100'''9'''; 100ul, (6)<br />
*7- K19100'''9'''; 900ul, (4)<br />
*8- K19100'''9'''; 100ul, (2)<br />
*9- K19100'''3'''; 900ul, (2)<br />
*10- K19100'''3'''; 900ul, (5)<br />
*11- K19100'''3'''; 100ul, (4)<br />
*12- K19100'''3'''; 100ul, (1)<br />
*13- K19100'''4'''; 100ul, (3)<br />
*14- K19100'''4'''; 900ul, (5)<br />
<br />
Lanes- 1- marker, 2-9 sampels as in order, 10-empty, 11, 12- marker instead of the samples- need to run last two again<br />
<br />
[[Image:109.JPG]]<br />
<br />
[[Image:111.JPG]]<br />
LAST TWO LANES- NSAMPLES NO 13 AND 14 FROM THE ABOVE LIST<br />
<br />
'''28/07/10'''<br />
<br />
Double transformants made using a novel cowboy procedure- double transformation with 4ul of each piece od DNA. Chances of succes- very very low... But at least it will be proved that it won't work!<br />
Trans. 1: BBa_K191004+ K191003- read out + LovTap<br />
Trans. 2: BBa_K191004+ K191009- read out + mutated LovTap<br />
<br />
'''30/7/10'''<br />
set up experiments to see if the blue light sensor will activate the response system under the daylight. Both liquid cultures and plates were set up, with red and white colonies- first supposed to turn white in the daylight and stay red in the dark (under the foil), second ones- no change of the colour. The same applies to the liquid cultures. However, the experiment failed- no proper growth was observed, and if there was a growth - it was very weak. No pigment observed, apart from the small points in the centre. <br />
Hypothesis- not stable plasmids-- cells look like they are not really resistant. Recorded growth:<br />
{|border="1"<br />
|<br />
|K191004+9 control<br />
|K191004+9<br />
|K191004+3 control<br />
|K191004+3<br />
|-<br />
|DARK<br />
| ++ , white<br />
| +++ , white<br />
| ++ , white<br />
| +++ , white with red points<br />
|-<br />
| LIGHT<br />
| + , white<br />
| +++ , white with red point<br />
| + -almost no growth <br />
| ++ white<br />
|}<br />
<br />
'''2/8/10'''<br />
<br />
Run the digest of the minipreps from the white and red cultures of double transformants. No proper DNA band in the solution- repeat the procedure or not? Don't know what to do.... again. <br />
<br />
[[Image:2.08.JPG]]<br />
<br />
Collect the thoughts and ideas and start again tomorrow!<br />
<br />
'''6/8/10'''<br />
<br />
double transformants were prepared again, using non-cowboy procedure. 04 cells were first MADE COMPETENT, then transformed with 03 and 09, also 1 neg control was plated. Grow white on the intitial plate, then turned red if subcultured. <br />
<br />
'''7-8/08'''<br />
<br />
'Subbed these to duplicate fresh plates and incubated overnight at room temperature <br />
either under a fluorescent lamp or wrapped in foil - all white in both cases (and growth <br />
not great, probably because of low temperature)'. <br />
The plates were moved to 37C with the lamp /one under the light, one in the dark. so far -no results, checking in an hour/. <br />
<br />
Another idea- check for stability of the insert- we don't know if the plasmids are compatibile. Streaking from original plates to check if single colonies will be a mixture of white and red or just red- origin? no plasmid maps from Lausanne. <br />
by Chairman-->' If we can't find this information, I think our best course is to <br />
PCR out both inserts and combine them in a single vector, preferably pSB1C3.'<br />
<br />
'''9/8/2010'''<br />
<br />
to do:<br />
<br />
*primers for the parts to put them in a vector<br />
*check the lamp every hour!!<br />
<br />
go thruough Lausanne website to find the plasmid maps<br />
get back to Mexico about the parts and etc.<br />
find out the conditions for the blue light sensor assay!<br />
<br />
'''10/08/10'''<br />
* assay with dark/light liquid cultures - fluorescence measurment and OD, after 5 h in the shaker under lamp- see video and pictures<br />
*email to Mexico<br />
<br />
'''11/08/10'''<br />
<br />
*primer designed for combining LOVTap and Readout1 into 1 cell<br />
*repeated assay with the smae samples, after overngiht in RT and %5h under lamp (for the light samples) and RT without shaking<br />
*primary cultures streaked to amp/kan/IPTG plates- part 4+9 and 4+3 -three clones each<br />
<br />
'''12/08/10'''<br />
*results analysis after LOVTap experiment:check file, add graphs later.<br />
*email from Mexico about Blue promoter<br />
<br />
'''16/08/10'''<br />
* streaked single clone for 9+4 and 3+4 (clones number 5 and 2, respectively)- for tomorrow's experiment<br />
* minipreped mysterious double transformants and Sacb smples<br />
* tried out the box on the shaker- unscrewed the things. <br />
* made the graphs from the second reading<br />
* 03 and 09 LovTap taken for sequencing. <br />
<br />
to do: decide on the illumination length and think about controls and how many samples do you want to put. check Lausanne website<br />
t-test to check to importancy of the differences between the means.<br />
<br />
'''17/07/10'''<br />
* desinged experimental set up for the LovTap blue light response experiment /see next day/<br />
* BAD NEWS: there is a suspicion of 1bp deletion in LovTap!!! Chairman noticed it when he was checking the parts base by base. Should have done it much much earlier probably to save the time... <br />
* BAD NEWS: lack of the proper thinking put in doubt all the results from previous two experiments (that is why you can't find any files of it attached)- there was no IPTG added to the samples, therefore LOVTap expression was not induced- no point to do T-test right now... :( Well, at least I will learn from it and add it next time!<br />
<br />
'''18/08/10'''<br />
<br />
* tested LovTap under the blue light (blue LEDs, 470 nm wavelength, 2200 mcd/B, V=4.5 V; RS 466-3548).<br />
* conditions:<br />
Liquid cultures (same as for minipreps) were grown overnight in 37C with antibiotics. <br />
The next day, bottles with 3.5 ml of broth were inoculated with the 0.5 ml of appropriate cultures (0.5. ml of broth added as a control).<br />
*sample designation: (# refers to the clone number form the original plate). Original plates are the ones made on 25/7 and have 2 clones on each plates (clones1-3 are K191003&4 double transformants, clones 4-6 are K191009+4 double transformants). <br />
<br />
# K191003+4 (#1) + Amp + Kan + IPTG + LIGHT <br />
# K191003+4 (#1) + Amp + Kan IPTG + DARK <br />
# K191003+4 (#2) + Amp + Kan IPTG + LIGHT <br />
# K191003+4 (#2) + Amp + Kan IPTG + DARK <br />
# K191009+4 (#4) + Amp + Kan IPTG +LIGHT <br />
# K191009+4 (#4) + Amp + Kan IPTG +DARK <br />
# K191009+4 (#5) + Amp + Kan IPTG +LIGHT <br />
# K191009+4 (#5) + Amp + Kan IPTG +DARK <br />
# K191004 ONLY #2+ Amp + LIGHT <br />
# K191004 ONLY #2+ Amp + DARK <br />
# neg. control- broth, Amp + Kan + IPTG + LIGHT<br />
<br />
* samples designated DARK were wrapped in the aluminium foil straight after the inoculation; the cap was not wrapped (not see through anyway)so that samples could be easily taken without unwrapping the bottles from the foil. <br />
*All the samples were then taken to the shaker in the hot room and placed in prepared spots: for the dark- anywhere on an easily accessible holder, for light- in 2 rows of holders. <br />
*Light samples were covered with the cardboard box with blue LEDs attached on the sides, in a way to optimize light accession to the samples. Box was securely taped to the shaker. <br />
* Measurments were taken every half an hour: 200ul of the samples were placed in the numbered cuvette and then 800ul of water was added. Readings of fluroscence (green mode) were taken in standard fluorescence units (SFU, two readings each). Then optical density was measured (upstairs in the incubator room) at 360 nm wavelength. Control was used to calibrate the machine (set ref button). Also 2 readings were taken. Samples were shaken by inversion prior to taking measurment. <br />
* Experiment was stopped after 3 hours (6 sets of reading taken). <br />
<br />
*'''Result analysis'''<br />
<br />
[[Image:Lt18aug.JPG]]<br />
<br />
To generate the data for the graph:<br />
# means were calculated from 2 readings of optical density and fluorescence<br />
# (mean SFU minus background SFU) was divided by (mean OD minus background OD, if different from 0).<br />
# table was made for the samples at different points of time- e.g. sample 1 after 30 min, 60 min, 90 min, 120 min etc. <br />
# graph was made with series named the same as the samples<br />
<br />
<br />
**BAD BAD VERY BAD NEWS: after 2 hours of measurments the results of the sequencing were know and revealed that THERE IS A 1BP DELETION IN LOVTAP 03 AND 09 WHICH RESULTS IN FRAMESHIFT AND LACK OF THE FUNCTIONAL PRODUCT!!!! :(:(:( despite this information, it was decided that the experiment will be finished and analysed as initially planned.<br />
<br />
'''19/08/2010'''<br />
<br />
* Pcr- mutagenensis of LovTap- Mabel, adding missing bp. <br />
* Puryfing PCR product from the solution. <br />
<br />
<br />
'''20/8/2010'''<br />
* PCR run on the gel -correct band of ~5.5. kb size, suggest that the PCR worked well. couple of other fainter bands <br />
* Self -ligation overnight (MABEL self ligation- 12ul of H2O, 4ul of DNA, 2 ul of Ligase buffer, 1 ul of T4 DNA ligase, 1ul of T4 polynucleotide kinase. <br />
* transformation and setting up the liquid cultures for the minipreps over the weekend. <br />
<br />
[[Image:123.JPG]]<br />
<br />
'''23/08/2010'''<br />
* mini-prep DNA from the colonies after PCR- marker worn off in the shaker,so 3.2. and 9.1. got mixed up- probably sequencing necessary to distinguish the mutated version from non-mutated <br />
* digest minipreps with EcoRI, run the gel[photo]<br />
* run 5ul of the ligated 03 and 09 on the gel /also Richards samples/.<br />
<br />
[[Image:124.JPG]]<br />
<br />
'''24/08/2010'''<br />
* repeated digest of mutated LovTap samples: 3.1, 3.?, 3.3., 3.4 9.?, 9.2 with EcoRi<br />
* minipreped 6 more clones of mutated LovTap 03 and 09 from the same plate, labelled- 3.6, 3.7, 3.8, 9.4, 9.5, 9.6. Run the digest with EcoRI. Note: 3.7 sample is higly diluted as 80ul of EB was used instead of 40ul. oops. <br />
* delivered mutated 3.? and 9.2 minipreped clones for sequencing /only 2 reactions: each sample- forward/.<br />
<br />
[[Image:LT100.jpg]] <br />
<br />
'''25/9/2010'''<br />
* trpR mutant received form KEIO collection- generously privided by Lucas Black (The University of Edinburgh). <br />
'''1/10/2010'''<br />
* LOVTap-RFP in JM109<br />
<br />
'''04/10/10'''<br />
We may have a working LovTap construct + red/white (RFP) reporter system thanks to Mexico.<br />
* Cutting Mexico's LOVTap with EcoRi/PstI & transfer into pSB1C3 plasmid (Part 1)<br />
* Adding reporter- cut out with XbaI/PstI from K191004 part provided by Lausanne (PArt 2)<br />
* Ligation of Part 1 and Part 2., selection for red colonies. <br />
<br />
Minor characterisation experiment:<br><br />
-LovTap constructed with red-white RFP reporter<br><br />
-If activated, the blue light sensor transformants should produce red colonies<br><br />
-Blue light sensor containing-colonies from transformant plate streaked onto two plates<br><br />
-One plate left in the light, the other left in the dark<br><br />
-If the one in the light grows red and the one in the dark doesn't, it is a good indication that the construct is working<br><br><br />
<br />
'''5/10/2010'''<br />
* transforming trpR mutant with LOVTap- RFP plasmid. <br />
<br />
'''8/10/2010'''<br />
* repetaed LOVTap charactierzation- protocol followed as on 18th of August 2010- with TrpR mutant and WT under light/dark conidtion. <br />
* no clear response was seen. <br />
<br />
[[Image:8octLovtap.jpg]]<br />
<br />
[[Image:8octTRPR.jpg]]<br />
<br />
'''11/10/2010'''<br />
* Transforming plasmid Ptrp with RFP reporter to JM109 strian- control for further experiments.<br />
<br />
'''14/10/2010'''<br />
* PRi in trpR host- control plasmid<br />
<br />
'''22/10/2010'''<br />
* checking temperature response of LOVTap<br />
* setting up samples with CMM minum medium, enriched in thymine, trace elements and glucose--> for TrpR mutant.<br />
CELLS FAIL TO GROW!:(</div>Macbereskahttp://2010.igem.org/Team:Edinburgh/Notebook/Green_light_sensorTeam:Edinburgh/Notebook/Green light sensor2010-10-27T22:03:26Z<p>Macbereska: /* Green Light Sensor */</p>
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<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Collaboration">collaboration</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Attribution">attribution</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/BRIDGE">BRIDGE</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_producer">red light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_sensor">red sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_producer">blue light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_producer">green light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_sensor">green sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Modelling">modelling</a></li><br />
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<li><a href="http://www.openwetware.org/wiki/French_Lab">protocols</a></li><br />
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<div id="windowbox" style="border: .2em solid #660000; padding: 5px; position:fixed; top:50%; right:30px; width:8%;"><br />
<span style="color:ivory;">These <b>notes</b> correspond to the part of the <b>project</b> detailed <a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Green_light_sensor">here</a>.</span><br />
<br />
</div><br />
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</html><br />
<br />
==Green Light Sensor==<br />
<br><br />
<br />
<br />
'''8/10/2010'''<br />
* running purified PCR: marker (lane 1), PhOR (Lane 2), Ccas (Lane 3).<br />
<br />
[[Image:148.JPG]]<br />
<br />
'''19/10/20010'''<br />
* Lane 1: marker<br />
* Lane 2: PheA (very faint band or primer smear?)<br />
* Lane 3: Ccas + PhoR ligation<br />
<br />
[[Image:173.JPG]]<br />
<br />
'''22/10/2010'''<br />
* PhoA + LacZ<br />
<br />
[[Image:174.JPG]]<br />
<br />
'''23/10/2010'''<br />
* Lane 1: marker<br />
* Lane 2: phA promoter + lac Z<br />
* rest- irrelevant<br />
<br />
[[Image:175.JPG]]<br />
<br />
'''25/10/2010'''<br />
* pCB + green light sensor<br />
<br />
[[Image:176.JPG]]<br />
<br />
'''26/10/2010'''<br />
* total green light sensor + reporter<br />
<br />
[[Image:177.JPG]]<br />
<br />
'''27/10/2010'''<br />
* green light sensor ligations into PSB1C3<br />
<br />
[[Image:178.JPG]]<br />
<br />
<br></div>Macbereskahttp://2010.igem.org/File:178.JPGFile:178.JPG2010-10-27T21:56:07Z<p>Macbereska: </p>
<hr />
<div></div>Macbereskahttp://2010.igem.org/File:177.JPGFile:177.JPG2010-10-27T21:55:54Z<p>Macbereska: </p>
<hr />
<div></div>Macbereskahttp://2010.igem.org/File:176.JPGFile:176.JPG2010-10-27T21:55:34Z<p>Macbereska: </p>
<hr />
<div></div>Macbereskahttp://2010.igem.org/File:175.JPGFile:175.JPG2010-10-27T21:55:15Z<p>Macbereska: </p>
<hr />
<div></div>Macbereskahttp://2010.igem.org/File:174.JPGFile:174.JPG2010-10-27T21:54:58Z<p>Macbereska: </p>
<hr />
<div></div>Macbereskahttp://2010.igem.org/File:173.JPGFile:173.JPG2010-10-27T21:51:56Z<p>Macbereska: </p>
<hr />
<div></div>Macbereskahttp://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_sensorTeam:Edinburgh/Notebook/Blue light sensor2010-10-27T21:42:45Z<p>Macbereska: /* Blue Light Sensor */</p>
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<div id="windowbox" style="border: .2em solid #660000; padding: 5px; position:fixed; top:50%; right:30px; width:8%;"><br />
<span style="color:ivory;">These <b>notes</b> correspond to the part of the <b>project</b> detailed <a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_sensor">here</a>.</span><br />
<br />
</div><br />
<br />
</html><br />
<br />
==Blue Light Sensor==<br />
<br><br />
<br />
'''14/07/10'''<br />
<br />
Received parts from Lausanne:<br />
*BBa_191006 (113 ng/µL) – LovTAP alone<br />
*BBa_191004 (166 ng/µL) – Readout <br />
*BBa_191003 (230 ng/µL) – whole system<br />
*BBa_191009 (85 ng/µL) – mutant <br />
*Sosnick vector containing TrpR (393 ng/µL) – vector <br />
<br />
'''16/07/10'''<br />
<br />
Transformations using 100 µL of cells, 900 µL broth<br />
*K191004 - amp<br />
*K191003 - kanamycin<br />
*K191009 – kanamycin<br />
*Sosnick vector with TrpR – amp<br />
<br />
Note: Labeling on tubes is unclear – K191009 and K191004 got mixed up, so plating both tubes on both Kan and amp<br />
<br />
'''22/07/10'''<br />
<br />
*Minipreps: K0998010 – 5x (1 didn’t grow, but did anyway)<br />
*Blue sensors (3 biobricks and Sosnick vector) – cultures set to grow overnight<br />
<br />
'''25/07/10'''<br />
Digest of the blue light sensor minipreps was performed with EcoRI and BamHI; samples:<br />
*3- K19100'''4'''; 900ul, (3)<br />
*4- K19100'''4'''; 100ul, (2)<br />
*5-K19100'''9'''; 100ul, (1)<br />
*6- K19100'''9'''; 100ul, (6)<br />
*7- K19100'''9'''; 900ul, (4)<br />
*8- K19100'''9'''; 100ul, (2)<br />
*9- K19100'''3'''; 900ul, (2)<br />
*10- K19100'''3'''; 900ul, (5)<br />
*11- K19100'''3'''; 100ul, (4)<br />
*12- K19100'''3'''; 100ul, (1)<br />
*13- K19100'''4'''; 100ul, (3)<br />
*14- K19100'''4'''; 900ul, (5)<br />
<br />
Lanes- 1- marker, 2-9 sampels as in order, 10-empty, 11, 12- marker instead of the samples- need to run last two again<br />
<br />
[[Image:109.JPG]]<br />
<br />
[[Image:111.JPG]]<br />
LAST TWO LANES- NSAMPLES NO 13 AND 14 FROM THE ABOVE LIST<br />
<br />
'''28/07/10'''<br />
<br />
Double transformants made using a novel cowboy procedure- double transformation with 4ul of each piece od DNA. Chances of succes- very very low... But at least it will be proved that it won't work!<br />
Trans. 1: BBa_K191004+ K191003- read out + LovTap<br />
Trans. 2: BBa_K191004+ K191009- read out + mutated LovTap<br />
<br />
'''30/7/10'''<br />
set up experiments to see if the blue light sensor will activate the response system under the daylight. Both liquid cultures and plates were set up, with red and white colonies- first supposed to turn white in the daylight and stay red in the dark (under the foil), second ones- no change of the colour. The same applies to the liquid cultures. However, the experiment failed- no proper growth was observed, and if there was a growth - it was very weak. No pigment observed, apart from the small points in the centre. <br />
Hypothesis- not stable plasmids-- cells look like they are not really resistant. Recorded growth:<br />
{|border="1"<br />
|<br />
|K191004+9 control<br />
|K191004+9<br />
|K191004+3 control<br />
|K191004+3<br />
|-<br />
|DARK<br />
| ++ , white<br />
| +++ , white<br />
| ++ , white<br />
| +++ , white with red points<br />
|-<br />
| LIGHT<br />
| + , white<br />
| +++ , white with red point<br />
| + -almost no growth <br />
| ++ white<br />
|}<br />
<br />
'''2/8/10'''<br />
<br />
Run the digest of the minipreps from the white and red cultures of double transformants. No proper DNA band in the solution- repeat the procedure or not? Don't know what to do.... again. <br />
<br />
[[Image:2.08.JPG]]<br />
<br />
Collect the thoughts and ideas and start again tomorrow!<br />
<br />
'''6/8/10'''<br />
<br />
double transformants were prepared again, using non-cowboy procedure. 04 cells were first MADE COMPETENT, then transformed with 03 and 09, also 1 neg control was plated. Grow white on the intitial plate, then turned red if subcultured. <br />
<br />
'''7-8/08'''<br />
<br />
'Subbed these to duplicate fresh plates and incubated overnight at room temperature <br />
either under a fluorescent lamp or wrapped in foil - all white in both cases (and growth <br />
not great, probably because of low temperature)'. <br />
The plates were moved to 37C with the lamp /one under the light, one in the dark. so far -no results, checking in an hour/. <br />
<br />
Another idea- check for stability of the insert- we don't know if the plasmids are compatibile. Streaking from original plates to check if single colonies will be a mixture of white and red or just red- origin? no plasmid maps from Lausanne. <br />
by Chairman-->' If we can't find this information, I think our best course is to <br />
PCR out both inserts and combine them in a single vector, preferably pSB1C3.'<br />
<br />
'''9/8/2010'''<br />
<br />
to do:<br />
<br />
*primers for the parts to put them in a vector<br />
*check the lamp every hour!!<br />
<br />
go thruough Lausanne website to find the plasmid maps<br />
get back to Mexico about the parts and etc.<br />
find out the conditions for the blue light sensor assay!<br />
<br />
'''10/08/10'''<br />
* assay with dark/light liquid cultures - fluorescence measurment and OD, after 5 h in the shaker under lamp- see video and pictures<br />
*email to Mexico<br />
<br />
'''11/08/10'''<br />
<br />
*primer designed for combining LOVTap and Readout1 into 1 cell<br />
*repeated assay with the smae samples, after overngiht in RT and %5h under lamp (for the light samples) and RT without shaking<br />
*primary cultures streaked to amp/kan/IPTG plates- part 4+9 and 4+3 -three clones each<br />
<br />
'''12/08/10'''<br />
*results analysis after LOVTap experiment:check file, add graphs later.<br />
*email from Mexico about Blue promoter<br />
<br />
'''16/08/10'''<br />
* streaked single clone for 9+4 and 3+4 (clones number 5 and 2, respectively)- for tomorrow's experiment<br />
* minipreped mysterious double transformants and Sacb smples<br />
* tried out the box on the shaker- unscrewed the things. <br />
* made the graphs from the second reading<br />
* 03 and 09 LovTap taken for sequencing. <br />
<br />
to do: decide on the illumination length and think about controls and how many samples do you want to put. check Lausanne website<br />
t-test to check to importancy of the differences between the means.<br />
<br />
'''17/07/10'''<br />
* desinged experimental set up for the LovTap blue light response experiment /see next day/<br />
* BAD NEWS: there is a suspicion of 1bp deletion in LovTap!!! Chairman noticed it when he was checking the parts base by base. Should have done it much much earlier probably to save the time... <br />
* BAD NEWS: lack of the proper thinking put in doubt all the results from previous two experiments (that is why you can't find any files of it attached)- there was no IPTG added to the samples, therefore LOVTap expression was not induced- no point to do T-test right now... :( Well, at least I will learn from it and add it next time!<br />
<br />
'''18/08/10'''<br />
<br />
* tested LovTap under the blue light (blue LEDs, 470 nm wavelength, 2200 mcd/B, V=4.5 V; RS 466-3548).<br />
* conditions:<br />
Liquid cultures (same as for minipreps) were grown overnight in 37C with antibiotics. <br />
The next day, bottles with 3.5 ml of broth were inoculated with the 0.5 ml of appropriate cultures (0.5. ml of broth added as a control).<br />
*sample designation: (# refers to the clone number form the original plate). Original plates are the ones made on 25/7 and have 2 clones on each plates (clones1-3 are K191003&4 double transformants, clones 4-6 are K191009+4 double transformants). <br />
<br />
# K191003+4 (#1) + Amp + Kan + IPTG + LIGHT <br />
# K191003+4 (#1) + Amp + Kan IPTG + DARK <br />
# K191003+4 (#2) + Amp + Kan IPTG + LIGHT <br />
# K191003+4 (#2) + Amp + Kan IPTG + DARK <br />
# K191009+4 (#4) + Amp + Kan IPTG +LIGHT <br />
# K191009+4 (#4) + Amp + Kan IPTG +DARK <br />
# K191009+4 (#5) + Amp + Kan IPTG +LIGHT <br />
# K191009+4 (#5) + Amp + Kan IPTG +DARK <br />
# K191004 ONLY #2+ Amp + LIGHT <br />
# K191004 ONLY #2+ Amp + DARK <br />
# neg. control- broth, Amp + Kan + IPTG + LIGHT<br />
<br />
* samples designated DARK were wrapped in the aluminium foil straight after the inoculation; the cap was not wrapped (not see through anyway)so that samples could be easily taken without unwrapping the bottles from the foil. <br />
*All the samples were then taken to the shaker in the hot room and placed in prepared spots: for the dark- anywhere on an easily accessible holder, for light- in 2 rows of holders. <br />
*Light samples were covered with the cardboard box with blue LEDs attached on the sides, in a way to optimize light accession to the samples. Box was securely taped to the shaker. <br />
* Measurments were taken every half an hour: 200ul of the samples were placed in the numbered cuvette and then 800ul of water was added. Readings of fluroscence (green mode) were taken in standard fluorescence units (SFU, two readings each). Then optical density was measured (upstairs in the incubator room) at 360 nm wavelength. Control was used to calibrate the machine (set ref button). Also 2 readings were taken. Samples were shaken by inversion prior to taking measurment. <br />
* Experiment was stopped after 3 hours (6 sets of reading taken). <br />
<br />
*'''Result analysis'''<br />
<br />
[[Image:Lt18aug.JPG]]<br />
<br />
To generate the data for the graph:<br />
# means were calculated from 2 readings of optical density and fluorescence<br />
# (mean SFU minus background SFU) was divided by (mean OD minus background OD, if different from 0).<br />
# table was made for the samples at different points of time- e.g. sample 1 after 30 min, 60 min, 90 min, 120 min etc. <br />
# graph was made with series named the same as the samples<br />
<br />
<br />
**BAD BAD VERY BAD NEWS: after 2 hours of measurments the results of the sequencing were know and revealed that THERE IS A 1BP DELETION IN LOVTAP 03 AND 09 WHICH RESULTS IN FRAMESHIFT AND LACK OF THE FUNCTIONAL PRODUCT!!!! :(:(:( despite this information, it was decided that the experiment will be finished and analysed as initially planned.<br />
<br />
'''19/08/2010'''<br />
<br />
* Pcr- mutagenensis of LovTap- Mabel, adding missing bp. <br />
* Puryfing PCR product from the solution. <br />
<br />
<br />
'''20/8/2010'''<br />
* PCR run on the gel -correct band of ~5.5. kb size, suggest that the PCR worked well. couple of other fainter bands <br />
* Self -ligation overnight (MABEL self ligation- 12ul of H2O, 4ul of DNA, 2 ul of Ligase buffer, 1 ul of T4 DNA ligase, 1ul of T4 polynucleotide kinase. <br />
* transformation and setting up the liquid cultures for the minipreps over the weekend. <br />
<br />
[[Image:123.JPG]]<br />
<br />
'''23/08/2010'''<br />
* mini-prep DNA from the colonies after PCR- marker worn off in the shaker,so 3.2. and 9.1. got mixed up- probably sequencing necessary to distinguish the mutated version from non-mutated <br />
* digest minipreps with EcoRI, run the gel[photo]<br />
* run 5ul of the ligated 03 and 09 on the gel /also Richards samples/.<br />
<br />
[[Image:124.JPG]]<br />
<br />
'''24/08/2010'''<br />
* repeated digest of mutated LovTap samples: 3.1, 3.?, 3.3., 3.4 9.?, 9.2 with EcoRi<br />
* minipreped 6 more clones of mutated LovTap 03 and 09 from the same plate, labelled- 3.6, 3.7, 3.8, 9.4, 9.5, 9.6. Run the digest with EcoRI. Note: 3.7 sample is higly diluted as 80ul of EB was used instead of 40ul. oops. <br />
* delivered mutated 3.? and 9.2 minipreped clones for sequencing /only 2 reactions: each sample- forward/.<br />
<br />
[[Image:LT100.jpg]] <br />
<br />
'''25/9/2010'''<br />
* trpR mutant received form KEIO collection- generously privided by Lucas Black (The University of Edinburgh). <br />
'''1/10/2010'''<br />
* LOVTap-RFP in JM109<br />
<br />
'''04/10/10'''<br />
We may have a working LovTap construct + red/white (RFP) reporter system thanks to Mexico.<br />
* Cutting Mexico's LOVTap with EcoRi/PstI & transfer into pSB1C3 plasmid (Part 1)<br />
* Adding reporter- cut out with XbaI/PstI from K191004 part provided by Lausanne (PArt 2)<br />
* Ligation of Part 1 and Part 2., selection for red colonies. <br />
<br />
Minor characterisation experiment:<br><br />
-LovTap constructed with red-white RFP reporter<br><br />
-If activated, the blue light sensor transformants should produce red colonies<br><br />
-Blue light sensor containing-colonies from transformant plate streaked onto two plates<br><br />
-One plate left in the light, the other left in the dark<br><br />
-If the one in the light grows red and the one in the dark doesn't, it is a good indication that the construct is working<br><br><br />
<br />
'''5/10/2010'''<br />
* transforming trpR mutant with LOVTap- RFP plasmid. <br />
<br />
'''8/10/2010'''<br />
* repetaed LOVTap charactierzation- protocol followed as on 18th of August 2010- with TrpR mutant and WT under light/dark conidtion. <br />
* no clear response was seen. <br />
<br />
[[Image:8octLovtap.jpg]]<br />
<br />
[[Image:8octTRPR.jpg]]<br />
<br />
'''11/10/2010'''<br />
* Transforming plasmid Ptrp with RFP reporter to JM109 strian- control for further experiments.<br />
<br />
'''14/10/2010'''<br />
* PRi in trpR host- control plasmid<br />
<br />
'''22/10/2010'''<br />
* checking temperature response of LOVTap<br />
* setting up samples with CMM minum medium, enriched in thymine, trace elements and glucose.<br />
CELLS FAIL TO GROW!:(</div>Macbereskahttp://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_sensorTeam:Edinburgh/Bacterial/Blue light sensor2010-10-27T21:35:59Z<p>Macbereska: </p>
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<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_sensor">red sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_producer">blue light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Green_light_producer">green light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Green_light_sensor">green sensor</a></li><br />
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<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Wiki">wiki</a></li><br />
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<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook" class="dir">lab notes&nbsp;&nbsp;&nbsp;</a><br />
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<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Collaboration">collaboration</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Attribution">attribution</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/BRIDGE">BRIDGE</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_producer">red light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_sensor">red sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_producer">blue light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_producer">green light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_sensor">green sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Modelling">modelling</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Safety">safety</a></li><br />
<li><a href="http://www.openwetware.org/wiki/French_Lab">protocols</a></li><br />
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<li><a href="https://2010.igem.org/Team:Edinburgh/Acknowledgements" class="dir">acknowledgements</a><br />
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<a name="Overview" id="Overview"></a><h2>Overview: The blue light sensor</h2><br />
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<p>The blue-light sensor, which is composed of the LovTAP hybrid protein designed by <a href="#References">Strickland et al. (2008)</a> and made available by Professor Sosnick, was BioBricked by <a href="https://2009.igem.org/Team:EPF-Lausanne">EPF-Lausanne in iGEM 2009</a>. It is based on a α-helical domain linker between the Lov2 domain (the photoactive protein) and the <i>E. coli trp</i> repressor, which acts as a conduit for allosteric signals. The effective response of the sensor is at a wavelength of 470nm (as documented by the aforementioned Lausanne iGEM team).</p><br />
<br />
<p>The blue-light sensor (LovTAP) consists of five parts:</p><br />
<br />
<ol><br />
<li>Photoreceptor1 (the shared helix between the Lov domain and the TrpR domain)<br />
<ul><br />
<li>Dark blue when contacting the Lov domain (dark state).</li><br />
<li>Red when contacting the TrpR domain (light state).</li><br />
</ul><br />
</li><br />
<li>Photoreceptor2 (falvin monoucleotide-FMN cofactor)</li><br />
<li>Lov domain-orange (photoactive protein)</li><br />
<li>TrpR domain-grey (DNA regulator)</li><br />
<li>Operator DNA</li><br />
</ol><br />
<br />
<center><p><br><img src="https://static.igem.org/mediawiki/2010/5/59/Ed10-Strickland.jpg" width="640" height="505" border="0"/></p><br><br />
<p><b>Figure 1:</b> The mechanism of action of the LovTAP allosteric light sensor.</p><br />
<p>Image: <a href="#References">Strickland et al. (2008)</a></p><br></center><br />
<br />
<p><a href="img src="https://static.igem.org/mediawiki/2010/5/59/Ed10-Strickland.jpg">Figure 1</a> above shows the whole process regarding how the light sensor works, from the dark state (A) to the light-activated state (B → C) and then returning to the stable state (D → A). In the dark state, the shared helix contacts the Lov2 domain, and the inactivated TrpR dissociates from the DNA; in the light state, the Lov2 domains absorb the blue light proton and form a covalent adduct between the FMN cofactor and a conserved cysteine residue, destroying the shared helix in the Lov domain and binding / populating an active formation of the TrpR domain. This in turn leads to LovTAP binding the DNA and repressing lambda-cI. However, this binding is not stable, and thus it will eventually return to the initial state.</p><br />
<br><br />
<br><br />
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<a name="Strategy" id="Strategy"></a><h2>Strategy</h2><br />
<br><br />
<p>Our original plan was to obtain and revive <a href="https://2009.igem.org/Team:EPF-Lausanne">EPF-Lausanne 2009</a>'s <a href="http://partsregistry.org/Part:BBa_K191009">BBa_K191009</a>. Once we had done so, we aimed to use it to create a blue light sensing system, and transform cells with for characterisation of the system and for analysis of their compatibility with the mutated blue luciferases. Further characterization of LovTap included investigation of the tryptophan influence on the system, and therefore TrpR mutant was transformed with LovTap-reporter construct.</p><br><br />
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<a name="Problems" id="Problems"></a><h2>Problems</h2><br />
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<p>We were unable to successfully characterise the LovTAP that we initially received from Lausanne due to a frameshift mutation. Eventually, we received a new version of LovTAP from our collaborators at <a href="https://2010.igem.org/Team:UNAM-Genomics_Mexico">Mexico UNAM-Genomics</a>, so that we could perform characterisation tests on it for them. Unfortunately, we were again unable to elicit a clear response from our sensor.</p><br />
<br />
<p>Our major problem was that transformants often grew the wrong colour. LovTAP controls the production of RFP in the cells; when LovTAP is inactive (i.e. in the dark), the cells should be producing RFP and hence should be growing red. When LovTAP is active (i.e. in white or blue light), the cells stop producing RFP and hence should produce white colonies. At the moment some cells in the light are still growing red, although some are definitely growing white, and vice versa in the dark. One suggestion was that the plasmids are unstable and dividing randomly, altering the intensity of the colour in some cells. Attempts to stabilise the colours of the colonies are ongoing (see the video in the <a href="https://2010.igem.org/Team:Edinburgh/Gallery">gallery</a> which demonstrates one of the innovative methods of providing proper culture conditions).</p><br><br />
<br><br />
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<a name="BioBricks" id="BioBricks"></a><h2>BioBricks</h2><br />
<br><br />
<br />
<p>As stated above, our blue light sensor is based on a modified version of <a href="https://2009.igem.org/Team:EPF-Lausanne">Lausanne 2009</a>'s LovTAP part (<a href="http://partsregistry.org/Part:BBa_K191009">BBa_K191009</a>) developed by our collaborators at <a href="https://2010.igem.org/Team:UNAM-Genomics_Mexico">Mexico UNAM-Genomics</a>: <a href="http://partsregistry.org/Part:BBa_K360121">BBa_K360121</a>. We have coupled this with a simple reporter system (RFP) in order to perform characterisation tests.</p><br />
<br />
<p><a href="http://partsregistry.org/Part:BBa_K322999">BBa_K322999</a>: LovTAP with RFP reporter system, based on <a href="https://2010.igem.org/Team:UNAM-Genomics_Mexico">Mexico UNAM-Genomics</a> <a href="http://partsregistry.org/Part:BBa_K360121">BBa_K360121</a></p><br><br />
<br><br />
<br />
<a name="Characterisation" id="Characterisation"></a><h2>Characterisation</h2><br />
<br><br />
The protocol for LovTap characterization:<br />
<ul><br />
<li>Liquid cultures (same as for minipreps) were grown overnight in 37C with antibiotics. The next day, bottles with 3.5 ml of broth were inoculated with the 0.5 ml of appropriate cultures (0.5. ml of broth added as a control).</li><br />
<li>samples designated DARK were wrapped in the aluminium foil straight after the inoculation; the cap was not wrapped (not see through anyway)so that samples could be easily taken without unwrapping the bottles from the foil.</li><br />
<li>All the samples were then taken to the shaker in the hot room and placed in prepared spots: for the dark- anywhere on an easily accessible holder, for light- in 2 rows of holders..</li><br />
<li>Light samples were covered with the cardboard box with blue LEDs attached on the sides(470 nm wavelength; 2200 mcd/B; V=4.5 V; RS466-3548), in a way to optimize light accession to the samples. Box was securely taped to the shaker.</li><br />
<li> Measurments were taken every half an hour: 200ul of the samples were placed in the numbered cuvette and then 800ul of water was added. Readings of fluroscence (green mode) were taken in standard fluorescence units (SFU, two readings each). Then optical density was measured (upstairs in the incubator room) at 360 nm wavelength. Control was used to calibrate the machine (set ref button). Also 2 readings were taken. Samples were shaken by inversion prior to taking measurment.</li><br />
<li>Experiment was stopped after 3 hours (6 sets of reading taken). </li><br />
Result analysis<br />
<li>To generate the data for the graph:</li><br />
<li> 1. means were calculated from 2 readings of optical density and fluorescence. </li><br />
<li> 2. (mean SFU minus background SFU) was divided by (mean OD minus background OD, if different from 0).</li><br />
<li> 3. table was made for the samples at different points of time- e.g. sample 1 after 30 min, 60 min, 90 min, 120 min etc.</li><br />
<li> 4. graph was made with series named the same as the samples</li> </ul><br />
<br />
<br />
<center><br><br><p><img src="https://static.igem.org/mediawiki/2010/9/91/8octLovtap.jpg" width="600px"></p><br><br />
<p><b>Figure 2:</b> LovTAP activation.</p><br><br></center><br />
<br />
<p><a href="https://static.igem.org/mediawiki/2010/9/91/8octLovtap.jpg">Figure 2</a>.</p><br />
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<center><br><br><p><img src="https://static.igem.org/mediawiki/2010/3/38/8octTRPR.jpg" width="600px"></p><br><br />
<p><b>Figure 3:</b> LovTAP TrpR mutant activation.</p><br><br></center><br />
<br />
<p><a href="https://static.igem.org/mediawiki/2010/3/38/8octTRPR.jpg">Figure 3</a>.</p><br />
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<a name="References" id="References"></a><h2>References</h2><br />
<br><br />
<br />
<p><b>Strickland, D., Moffat, K. & Sosnick, T. R. (2008).</b> Light-activated DNA binding in a designed allosteric protein. <i>Proceedings of the National Academy of Sciences</i> <b>105</b>, 10709-10714.</p><br />
<p><b>Schüttrigkeit, T. A., Kompa, C. K., Salomon, M., Rüdiger, W. &amp; Michel-Beyerle, M. E. (2003).</b> Primary photophysics of the FMN binding LOV2 domain of the plant blue light receptor phototropin of Avena sativa. <i>Chemical Physics</i> <b>294</b>, 501-508.</p><br />
<p><b>Wu, Y. I., D. Frey, et al. (2009).</b> A genetically encoded photoactivatable Rac controls the motility of living cells. <i>Nature</i> Vol <b>461</b></p><br />
<p>EPF Lausanne 2009 team wiki, <i>https://2009.igem.org/Team:EPF-Lausanne</i>.</p><br />
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<span style="color:ivory;">Throughout this wiki there are words in <b>bold</b> that indicate a relevance to <b>human aspects</b>. It will become obvious that <b>human aspects</b> are a part of almost everything in <b>iGEM</b>.</span><br />
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</html></div>Macbereskahttp://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_sensorTeam:Edinburgh/Bacterial/Blue light sensor2010-10-27T21:19:13Z<p>Macbereska: </p>
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<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Core_repressilator">the repressilator</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_producer">red light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_sensor">red sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_producer">blue light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Green_light_producer">green light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Green_light_sensor">green sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/BioBricks#Bacterial">submitted parts</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Bacterial">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/References">references</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling" class="dir">modelling BRIDGEs</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Kappa">kappa</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Genomic">the genomic model</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Bacterial">the bacterial model</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Signalling">the signalling model</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Tools">tools</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Modelling">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/References">references</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human" class="dir">human BRIDGEs</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Aspects">human aspects</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Communication">communication of science</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Terminology">terminology research</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Wiki">wiki</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Conversations">conversations</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Identity">identity</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Collaboration">collaboration</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Branding">branding research</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/SciFi">science fiction writing</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/FutureApps">future applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/SelfReflection">self-reflection</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Epic">the epic</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Human">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/References">references</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook" class="dir">lab notes&nbsp;&nbsp;&nbsp;</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Collaboration">collaboration</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Attribution">attribution</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/BRIDGE">BRIDGE</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_producer">red light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_sensor">red sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_producer">blue light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_producer">green light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_sensor">green sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Modelling">modelling</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Safety">safety</a></li><br />
<li><a href="http://www.openwetware.org/wiki/French_Lab">protocols</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Acknowledgements" class="dir">acknowledgements</a><br />
</li><br />
<br />
</ul><br />
</div><br />
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<a name="Overview" id="Overview"></a><h2>Overview: The blue light sensor</h2><br />
<br><br />
<br />
<p>The blue-light sensor, which is composed of the LovTAP hybrid protein designed by <a href="#References">Strickland et al. (2008)</a> and made available by Professor Sosnick, was BioBricked by <a href="https://2009.igem.org/Team:EPF-Lausanne">EPF-Lausanne in iGEM 2009</a>. It is based on a α-helical domain linker between the Lov2 domain (the photoactive protein) and the <i>E. coli trp</i> repressor, which acts as a conduit for allosteric signals. The effective response of the sensor is at a wavelength of 470nm (as documented by the aforementioned Lausanne iGEM team).</p><br />
<br />
<p>The blue-light sensor (LovTAP) consists of five parts:</p><br />
<br />
<ol><br />
<li>Photoreceptor1 (the shared helix between the Lov domain and the TrpR domain)<br />
<ul><br />
<li>Dark blue when contacting the Lov domain (dark state).</li><br />
<li>Red when contacting the TrpR domain (light state).</li><br />
</ul><br />
</li><br />
<li>Photoreceptor2 (falvin monoucleotide-FMN cofactor)</li><br />
<li>Lov domain-orange (photoactive protein)</li><br />
<li>TrpR domain-grey (DNA regulator)</li><br />
<li>Operator DNA</li><br />
</ol><br />
<br />
<center><p><br><img src="https://static.igem.org/mediawiki/2010/5/59/Ed10-Strickland.jpg" width="640" height="505" border="0"/></p><br><br />
<p><b>Figure 1:</b> The mechanism of action of the LovTAP allosteric light sensor.</p><br />
<p>Image: <a href="#References">Strickland et al. (2008)</a></p><br></center><br />
<br />
<p><a href="img src="https://static.igem.org/mediawiki/2010/5/59/Ed10-Strickland.jpg">Figure 1</a> above shows the whole process regarding how the light sensor works, from the dark state (A) to the light-activated state (B → C) and then returning to the stable state (D → A). In the dark state, the shared helix contacts the Lov2 domain, and the inactivated TrpR dissociates from the DNA; in the light state, the Lov2 domains absorb the blue light proton and form a covalent adduct between the FMN cofactor and a conserved cysteine residue, destroying the shared helix in the Lov domain and binding / populating an active formation of the TrpR domain. This in turn leads to LovTAP binding the DNA and repressing lambda-cI. However, this binding is not stable, and thus it will eventually return to the initial state.</p><br />
<br><br />
<br><br />
<br />
<a name="Strategy" id="Strategy"></a><h2>Strategy</h2><br />
<br><br />
<p>Our original plan was to obtain and revive <a href="https://2009.igem.org/Team:EPF-Lausanne">EPF-Lausanne 2009</a>'s <a href="http://partsregistry.org/Part:BBa_K191009">BBa_K191009</a>. Once we had done so, we aimed to use it to create a blue light sensing system, and transform cells with for characterisation of the system and for analysis of their compatibility with the mutated blue luciferases. Further characterization of LovTap included investigation of the tryptophan influence on the system, and therefore TrpR mutant was transformed with LovTap-reporter construct.</p><br><br />
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<a name="Problems" id="Problems"></a><h2>Problems</h2><br />
<br><br />
<br />
<p>We were unable to successfully characterise the LovTAP that we initially received from Lausanne due to a frameshift mutation. Eventually, we received a new version of LovTAP from our collaborators at <a href="https://2010.igem.org/Team:UNAM-Genomics_Mexico">Mexico UNAM-Genomics</a>, so that we could perform characterisation tests on it for them. Unfortunately, we were again unable to elicit a clear response from our sensor.</p><br />
<br />
<p>Our major problem was that transformants often grew the wrong colour. LovTAP controls the production of RFP in the cells; when LovTAP is inactive (i.e. in the dark), the cells should be producing RFP and hence should be growing red. When LovTAP is active (i.e. in white or blue light), the cells stop producing RFP and hence should produce white colonies. At the moment some cells in the light are still growing red, although some are definitely growing white, and vice versa in the dark. One suggestion was that the plasmids are unstable and dividing randomly, altering the intensity of the colour in some cells. Attempts to stabilise the colours of the colonies are ongoing (see the video in the <a href="https://2010.igem.org/Team:Edinburgh/Gallery">gallery</a> which demonstrates one of the innovative methods of providing proper culture conditions).</p><br><br />
<br><br />
<br />
<a name="BioBricks" id="BioBricks"></a><h2>BioBricks</h2><br />
<br><br />
<br />
<p>As stated above, our blue light sensor is based on a modified version of <a href="https://2009.igem.org/Team:EPF-Lausanne">Lausanne 2009</a>'s LovTAP part (<a href="http://partsregistry.org/Part:BBa_K191009">BBa_K191009</a>) developed by our collaborators at <a href="https://2010.igem.org/Team:UNAM-Genomics_Mexico">Mexico UNAM-Genomics</a>: <a href="http://partsregistry.org/Part:BBa_K360121">BBa_K360121</a>. We have coupled this with a simple reporter system (RFP) in order to perform characterisation tests.</p><br />
<br />
<p><a href="http://partsregistry.org/Part:BBa_K322999">BBa_K322999</a>: LovTAP with RFP reporter system, based on <a href="https://2010.igem.org/Team:UNAM-Genomics_Mexico">Mexico UNAM-Genomics</a> <a href="http://partsregistry.org/Part:BBa_K360121">BBa_K360121</a></p><br><br />
<br><br />
<br />
<a name="Characterisation" id="Characterisation"></a><h2>Characterisation</h2><br />
<br><br />
<br />
<center><br><br><p><img src="https://static.igem.org/mediawiki/2010/9/91/8octLovtap.jpg" width="600px"></p><br><br />
<p><b>Figure 2:</b> LovTAP activation.</p><br><br></center><br />
<br />
<p><a href="https://static.igem.org/mediawiki/2010/9/91/8octLovtap.jpg">Figure 2</a>.</p><br />
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<br />
<center><br><br><p><img src="https://static.igem.org/mediawiki/2010/3/38/8octTRPR.jpg" width="600px"></p><br><br />
<p><b>Figure 3:</b> LovTAP TrpR mutant activation.</p><br><br></center><br />
<br />
<p><a href="https://static.igem.org/mediawiki/2010/3/38/8octTRPR.jpg">Figure 3</a>.</p><br />
<br />
<br><br />
<br><br />
<br />
<a name="References" id="References"></a><h2>References</h2><br />
<br><br />
<br />
<p><b>Strickland, D., Moffat, K. & Sosnick, T. R. (2008).</b> Light-activated DNA binding in a designed allosteric protein. <i>Proceedings of the National Academy of Sciences</i> <b>105</b>, 10709-10714.</p><br />
<p><b>Schüttrigkeit, T. A., Kompa, C. K., Salomon, M., Rüdiger, W. &amp; Michel-Beyerle, M. E. (2003).</b> Primary photophysics of the FMN binding LOV2 domain of the plant blue light receptor phototropin of Avena sativa. <i>Chemical Physics</i> <b>294</b>, 501-508.</p><br />
<p><b>Wu, Y. I., D. Frey, et al. (2009).</b> A genetically encoded photoactivatable Rac controls the motility of living cells. <i>Nature</i> Vol <b>461</b></p><br />
<p>EPF Lausanne 2009 team wiki, <i>https://2009.igem.org/Team:EPF-Lausanne</i>.</p><br />
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<center><a href="#top" class="dir"><img width="100" src="https://static.igem.org/mediawiki/2010/9/9f/Ed10-RTT.png"></a></center><br />
</div><br />
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<div id="windowbox" style="border: .2em solid #660000; padding: 5px; position:fixed; top:50%; right:30px; width:8%;"><br />
<span style="color:ivory;">Throughout this wiki there are words in <b>bold</b> that indicate a relevance to <b>human aspects</b>. It will become obvious that <b>human aspects</b> are a part of almost everything in <b>iGEM</b>.</span><br />
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</div><br />
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</body><br />
<br />
</html></div>Macbereskahttp://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_sensorTeam:Edinburgh/Notebook/Blue light sensor2010-10-27T20:57:52Z<p>Macbereska: /* Blue Light Sensor */</p>
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<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial" class="dir">bacterial BRIDGEs</a><br />
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<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Core_repressilator">the repressilator</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_producer">red light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_sensor">red sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_producer">blue light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Green_light_producer">green light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Green_light_sensor">green sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/BioBricks#Bacterial">submitted parts</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Bacterial">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/References">references</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling" class="dir">modelling BRIDGEs</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Kappa">kappa</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Genomic">the genomic model</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Bacterial">the bacterial model</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Signalling">the signalling model</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Tools">tools</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Modelling">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/References">references</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human" class="dir">human BRIDGEs</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Aspects">human aspects</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Communication">communication of science</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Terminology">terminology research</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Wiki">wiki</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Conversations">conversations</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Identity">identity</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Collaboration">collaboration</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Branding">branding research</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/SciFi">science fiction writing</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/FutureApps">future applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/SelfReflection">self-reflection</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Epic">the epic</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Human">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/References">references</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook" class="dir">lab notes&nbsp;&nbsp;&nbsp;</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Collaboration">collaboration</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Attribution">attribution</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/BRIDGE">BRIDGE</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_producer">red light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_sensor">red sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_producer">blue light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_producer">green light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_sensor">green sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Modelling">modelling</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Safety">safety</a></li><br />
<li><a href="http://www.openwetware.org/wiki/French_Lab">protocols</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Acknowledgements" class="dir">acknowledgements</a><br />
</li><br />
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</ul><br />
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<span style="color:ivory;">These <b>notes</b> correspond to the part of the <b>project</b> detailed <a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_sensor">here</a>.</span><br />
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==Blue Light Sensor==<br />
<br><br />
<br />
'''14/07/10'''<br />
<br />
Received parts from Lausanne:<br />
*BBa_191006 (113 ng/µL) – LovTAP alone<br />
*BBa_191004 (166 ng/µL) – Readout <br />
*BBa_191003 (230 ng/µL) – whole system<br />
*BBa_191009 (85 ng/µL) – mutant <br />
*Sosnick vector containing TrpR (393 ng/µL) – vector <br />
<br />
<br />
<br />
'''16/07/10'''<br />
<br />
Transformations using 100 µL of cells, 900 µL broth<br />
*K191004 - amp<br />
*K191003 - kanamycin<br />
*K191009 – kanamycin<br />
*Sosnick vector with TrpR – amp<br />
<br />
Note: Labeling on tubes is unclear – K191009 and K191004 got mixed up, so plating both tubes on both Kan and amp<br />
<br />
<br />
'''22/07/10'''<br />
<br />
*Minipreps: K0998010 – 5x (1 didn’t grow, but did anyway)<br />
*Blue sensors (3 biobricks and Sosnick vector) – cultures set to grow overnight<br />
<br />
'''25/07/10'''<br />
Digest of the blue light sensor minipreps was performed with EcoRI and BamHI; samples:<br />
*3- K19100'''4'''; 900ul, (3)<br />
*4- K19100'''4'''; 100ul, (2)<br />
*5-K19100'''9'''; 100ul, (1)<br />
*6- K19100'''9'''; 100ul, (6)<br />
*7- K19100'''9'''; 900ul, (4)<br />
*8- K19100'''9'''; 100ul, (2)<br />
*9- K19100'''3'''; 900ul, (2)<br />
*10- K19100'''3'''; 900ul, (5)<br />
*11- K19100'''3'''; 100ul, (4)<br />
*12- K19100'''3'''; 100ul, (1)<br />
*13- K19100'''4'''; 100ul, (3)<br />
*14- K19100'''4'''; 900ul, (5)<br />
<br />
Lanes- 1- marker, 2-9 sampels as in order, 10-empty, 11, 12- marker instead of the samples- need to run last two again<br />
<br />
[[Image:109.JPG]]<br />
<br />
[[Image:111.JPG]]<br />
LAST TWO LANES- NSAMPLES NO 13 AND 14 FROM THE ABOVE LIST<br />
<br />
<br />
'''28/07/10'''<br />
<br />
Double transformants made using a novel cowboy procedure- double transformation with 4ul of each piece od DNA. Chances of succes- very very low... But at least it will be proved that it won't work!<br />
Trans. 1: BBa_K191004+ K191003- read out + LovTap<br />
Trans. 2: BBa_K191004+ K191009- read out + mutated LovTap<br />
<br />
'''30/7/10'''<br />
set up experiments to see if the blue light sensor will activate the response system under the daylight. Both liquid cultures and plates were set up, with red and white colonies- first supposed to turn white in the daylight and stay red in the dark (under the foil), second ones- no change of the colour. The same applies to the liquid cultures. However, the experiment failed- no proper growth was observed, and if there was a growth - it was very weak. No pigment observed, apart from the small points in the centre. <br />
Hypothesis- not stable plasmids-- cells look like they are not really resistant. Recorded growth:<br />
{|border="1"<br />
|<br />
|K191004+9 control<br />
|K191004+9<br />
|K191004+3 control<br />
|K191004+3<br />
|-<br />
|DARK<br />
| ++ , white<br />
| +++ , white<br />
| ++ , white<br />
| +++ , white with red points<br />
|-<br />
| LIGHT<br />
| + , white<br />
| +++ , white with red point<br />
| + -almost no growth <br />
| ++ white<br />
|}<br />
<br />
<br />
<br />
'''2/8/10'''<br />
<br />
Run the digest of the minipreps from the white and red cultures of double transformants. No proper DNA band in the solution- repeat the procedure or not? Don't know what to do.... again. <br />
<br />
[[Image:2.08.JPG]]<br />
<br />
Collect the thoughts and ideas and start again tomorrow!<br />
<br />
'''6/8/10'''<br />
<br />
double transformants were prepared again, using non-cowboy procedure. 04 cells were first MADE COMPETENT, then transformed with 03 and 09, also 1 neg control was plated. Grow white on the intitial plate, then turned red if subcultured. <br />
<br />
'''7-8/08'''<br />
<br />
'Subbed these to duplicate fresh plates and incubated overnight at room temperature <br />
either under a fluorescent lamp or wrapped in foil - all white in both cases (and growth <br />
not great, probably because of low temperature)'. <br />
The plates were moved to 37C with the lamp /one under the light, one in the dark. so far -no results, checking in an hour/. <br />
<br />
Another idea- check for stability of the insert- we don't know if the plasmids are compatibile. Streaking from original plates to check if single colonies will be a mixture of white and red or just red- origin? no plasmid maps from Lausanne. <br />
by Chairman-->' If we can't find this information, I think our best course is to <br />
PCR out both inserts and combine them in a single vector, preferably pSB1C3.'<br />
<br />
<br />
'''9/8/2010'''<br />
<br />
to do:<br />
<br />
*primers for the parts to put them in a vector<br />
*check the lamp every hour!!<br />
<br />
go thruough Lausanne website to find the plasmid maps<br />
get back to Mexico about the parts and etc.<br />
find out the conditions for the blue light sensor assay!<br />
<br />
'''10/08/10'''<br />
* assay with dark/light liquid cultures - fluorescence measurment and OD, after 5 h in the shaker under lamp- see video and pictures<br />
*email to Mexico<br />
<br />
'''11/08/10'''<br />
<br />
*primer designed for combining LOVTap and Readout1 into 1 cell<br />
*repeated assay with the smae samples, after overngiht in RT and %5h under lamp (for the light samples) and RT without shaking<br />
*primary cultures streaked to amp/kan/IPTG plates- part 4+9 and 4+3 -three clones each<br />
<br />
'''12/08/10'''<br />
*results analysis after LOVTap experiment:check file, add graphs later.<br />
*email from Mexico about Blue promoter<br />
<br />
'''16/08/10'''<br />
* streaked single clone for 9+4 and 3+4 (clones number 5 and 2, respectively)- for tomorrow's experiment<br />
* minipreped mysterious double transformants and Sacb smples<br />
* tried out the box on the shaker- unscrewed the things. <br />
* made the graphs from the second reading<br />
* 03 and 09 LovTap taken for sequencing. <br />
<br />
to do: decide on the illumination length and think about controls and how many samples do you want to put. check Lausanne website<br />
t-test to check to importancy of the differences between the means.<br />
<br />
'''17/07/10'''<br />
* desinged experimental set up for the LovTap blue light response experiment /see next day/<br />
* BAD NEWS: there is a suspicion of 1bp deletion in LovTap!!! Chairman noticed it when he was checking the parts base by base. Should have done it much much earlier probably to save the time... <br />
* BAD NEWS: lack of the proper thinking put in doubt all the results from previous two experiments (that is why you can't find any files of it attached)- there was no IPTG added to the samples, therefore LOVTap expression was not induced- no point to do T-test right now... :( Well, at least I will learn from it and add it next time!<br />
<br />
<br />
'''18/08/10'''<br />
<br />
* tested LovTap under the blue light<br />
* conditions:<br />
Liquid cultures (same as for minipreps) were grown overnight in 37C with antibiotics. <br />
The next day, bottles with 3.5 ml of broth were inoculated with the 0.5 ml of appropriate cultures (0.5. ml of broth added as a control).<br />
*sample designation: (# refers to the clone number form the original plate). Original plates are the ones made on '''INSERT THE DATE LAZY''' and have 2 clones on each plates (clones1-3 are K191003&4 double transformants, clones 4-6 are K191009+4 double transformants). <br />
<br />
# K191003+4 (#1) + Amp + Kan + IPTG + LIGHT <br />
# K191003+4 (#1) + Amp + Kan IPTG + DARK <br />
# K191003+4 (#2) + Amp + Kan IPTG + LIGHT <br />
# K191003+4 (#2) + Amp + Kan IPTG + DARK <br />
# K191009+4 (#4) + Amp + Kan IPTG +LIGHT <br />
# K191009+4 (#4) + Amp + Kan IPTG +DARK <br />
# K191009+4 (#5) + Amp + Kan IPTG +LIGHT <br />
# K191009+4 (#5) + Amp + Kan IPTG +DARK <br />
# K191004 ONLY #2+ Amp + LIGHT <br />
# K191004 ONLY #2+ Amp + DARK <br />
# neg. control- broth, Amp + Kan + IPTG + LIGHT<br />
<br />
* samples designated DARK were wrapped in the aluminium foil straight after the inoculation; the cap was not wrapped (not see through anyway)so that samples could be easily taken without unwrapping the bottles from the foil. <br />
*All the samples were then taken to the shaker in the hot room and placed in prepared spots: for the dark- anywhere on an easily accessible holder, for light- in 2 rows of holders. <br />
*Light samples were covered with the cardboard box with blue LEDs attached on the sides, in a way to optimize light accession to the samples (ask Sarah for the details). Box was securely taped to the shaker. <br />
* Measurments were taken every half an hour: 200ul of the samples were placed in the numbered cuvette and then 800ul of water was added. Readings of fluroscence (green mode) were taken in standard fluorescence units (SFU, two readings each). Then optical density was measured (upstairs in the incubator room) at 360 nm wavelength. Control was used to calibrate the machine (set ref button). Also 2 readings were taken. Samples were shaken by inversion prior to taking measurment. <br />
* Experiment was stopped after 3 hours (6 sets of reading taken). <br />
<br />
*'''Result analysis'''<br />
<br />
[[Image:Lt18aug.JPG]]<br />
<br />
To generate the data for the graph:<br />
# means were calculated from 2 readings of optical density and fluorescence<br />
# (mean SFU minus background SFU) was divided by (mean OD minus background OD, if different from 0).<br />
# table was made for the samples at different points of time- e.g. sample 1 after 30 min, 60 min, 90 min, 120 min etc. <br />
# graph was made with series named the same as the samples<br />
<br />
<br />
**BAD BAD VERY BAD NEWS: after 2 hours of measurments the results of the sequencing were know and revealed that THERE IS A 1BP DELETION IN LOVTAP 03 AND 09 WHICH RESULTS IN FRAMESHIFT AND LACK OF THE FUNCTIONAL PRODUCT!!!! :(:(:( despite this information, it was decided that the experiment will be finished and analysed as initially planned.<br />
<br />
'''19/08/2010'''<br />
<br />
* Pcr- mutagenensis of LovTap- Mabel, adding missing bp. <br />
* Puryfing PCR product from the solution. <br />
<br />
<br />
'''20/8/2010'''<br />
* PCR run on the gel -correct band of ~5.5. kb size, suggest that the PCR worked well. couple of other fainter bands <br />
* Self -ligation overnight (MABEL self ligation- 12ul of H2O, 4ul of DNA, 2 ul of Ligase buffer, 1 ul of T4 DNA ligase, 1ul of T4 polynucleotide kinase. <br />
* transformation and setting up the liquid cultures for the minipreps over the weekend. <br />
<br />
[[Image:123.JPG]]<br />
<br />
'''23/08/2010'''<br />
* mini-prep DNA from the colonies after PCR- marker worn off in the shaker,so 3.2. and 9.1. got mixed up- probably sequencing necessary to distinguish the mutated version from non-mutated <br />
* digest minipreps with EcoRI, run the gel[photo]<br />
* run 5ul of the ligated 03 and 09 on the gel /also Richards samples/.<br />
<br />
[[Image:124.JPG]]<br />
<br />
'''24/08/2010'''<br />
* repeated digest of mutated LovTap samples: 3.1, 3.?, 3.3., 3.4 9.?, 9.2 with EcoRi<br />
* minipreped 6 more clones of mutated LovTap 03 and 09 from the same plate, labelled- 3.6, 3.7, 3.8, 9.4, 9.5, 9.6. Run the digest with EcoRI. Note: 3.7 sample is higly diluted as 80ul of EB was used instead of 40ul. oops. <br />
* delivered mutated 3.? and 9.2 minipreped clones for sequencing /only 2 reactions: each sample- forward/.<br />
<br />
[[Image:LT100.jpg]] <br />
<br />
'''25/9/2010'''<br />
* trpR mutant received form KEIO collection- generously privided by Lucas Black (The University of Edinburgh). <br />
'''1/10/2010'''<br />
* LOVTap-RFP in JM109<br />
<br />
'''04/10/10'''<br />
We may have a working LovTap construct + red/white (RFP) reporter system thanks to Mexico.<br />
* Cutting Mexico's LOVTap with EcoRi/PstI & transfer into pSB1C3 plasmid (Part 1)<br />
* Adding reporter- cut out with XbaI/PstI from K191004 part provided by Lausanne (PArt 2)<br />
* Ligation of Part 1 and Part 2., selection for red colonies. <br />
<br />
Minor characterisation experiment:<br><br />
-LovTap constructed with red-white RFP reporter<br><br />
-If activated, the blue light sensor transformants should produce red colonies<br><br />
-Blue light sensor containing-colonies from transformant plate streaked onto two plates<br><br />
-One plate left in the light, the other left in the dark<br><br />
-If the one in the light grows red and the one in the dark doesn't, it is a good indication that the construct is working<br><br><br />
<br />
'''5/10/2010'''<br />
* transforming trpR mutant with LOVTap- RFP plasmid. <br />
<br />
'''8/10/2010'''<br />
* repetaed LOVTap charactierzation- protocol followed as on 18th of August 2010- with TrpR mutant and WT under light/dark conidtion. <br />
* no clear response was seen. <br />
<br />
[[Image:8octLovtap.jpg]]<br />
<br />
[[Image:8octTRPR.jpg]]<br />
<br />
'''11/10/2010'''<br />
* Transforming plasmid Ptrp with RFP reporter to JM109 strian- control for further experiments.<br />
<br />
'''14/10/2010'''<br />
* PRi in trpR host- control plasmid<br />
<br />
'''22/10/2010'''<br />
* checking temperature response of LOVTap<br />
* setting up samples with CMM minum medium, enriched in thymine, trace elements and glucose.<br />
CELLS FAIL TO GROW!:(</div>Macbereskahttp://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_sensorTeam:Edinburgh/Notebook/Blue light sensor2010-10-27T16:23:00Z<p>Macbereska: /* Blue Light Sensor */</p>
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<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_sensor">red sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_producer">blue light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_producer">green light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_sensor">green sensor</a></li><br />
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<span style="color:ivory;">These <b>notes</b> correspond to the part of the <b>project</b> detailed <a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_sensor">here</a>.</span><br />
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==Blue Light Sensor==<br />
<br><br />
<br />
'''14/07/10'''<br />
<br />
Received parts from Lausanne:<br />
*BBa_191006 (113 ng/µL) – LovTAP alone<br />
*BBa_191004 (166 ng/µL) – Readout <br />
*BBa_191003 (230 ng/µL) – whole system<br />
*BBa_191009 (85 ng/µL) – mutant <br />
*Sosnick vector containing TrpR (393 ng/µL) – vector <br />
<br />
<br />
<br />
'''16/07/10'''<br />
<br />
Transformations using 100 µL of cells, 900 µL broth<br />
*K191004 - amp<br />
*K191003 - kanamycin<br />
*K191009 – kanamycin<br />
*Sosnick vector with TrpR – amp<br />
<br />
Note: Labeling on tubes is unclear – K191009 and K191004 got mixed up, so plating both tubes on both Kan and amp<br />
<br />
<br />
'''22/07/10'''<br />
<br />
*Minipreps: K0998010 – 5x (1 didn’t grow, but did anyway)<br />
*Blue sensors (3 biobricks and Sosnick vector) – cultures set to grow overnight<br />
<br />
'''25/07/10'''<br />
Digest of the blue light sensor minipreps was performed with EcoRI and BamHI; samples:<br />
*3- K19100'''4'''; 900ul, (3)<br />
*4- K19100'''4'''; 100ul, (2)<br />
*5-K19100'''9'''; 100ul, (1)<br />
*6- K19100'''9'''; 100ul, (6)<br />
*7- K19100'''9'''; 900ul, (4)<br />
*8- K19100'''9'''; 100ul, (2)<br />
*9- K19100'''3'''; 900ul, (2)<br />
*10- K19100'''3'''; 900ul, (5)<br />
*11- K19100'''3'''; 100ul, (4)<br />
*12- K19100'''3'''; 100ul, (1)<br />
*13- K19100'''4'''; 100ul, (3)<br />
*14- K19100'''4'''; 900ul, (5)<br />
<br />
Lanes- 1- marker, 2-9 sampels as in order, 10-empty, 11, 12- marker instead of the samples- need to run last two again<br />
<br />
[[Image:109.JPG]]<br />
<br />
[[Image:111.JPG]]<br />
LAST TWO LANES- NSAMPLES NO 13 AND 14 FROM THE ABOVE LIST<br />
<br />
<br />
'''28/07/10'''<br />
<br />
Double transformants made using a novel cowboy procedure- double transformation with 4ul of each piece od DNA. Chances of succes- very very low... But at least it will be proved that it won't work!<br />
Trans. 1: BBa_K191004+ K191003- read out + LovTap<br />
Trans. 2: BBa_K191004+ K191009- read out + mutated LovTap<br />
<br />
'''30/7/10'''<br />
set up experiments to see if the blue light sensor will activate the response system under the daylight. Both liquid cultures and plates were set up, with red and white colonies- first supposed to turn white in the daylight and stay red in the dark (under the foil), second ones- no change of the colour. The same applies to the liquid cultures. However, the experiment failed- no proper growth was observed, and if there was a growth - it was very weak. No pigment observed, apart from the small points in the centre. <br />
Hypothesis- not stable plasmids-- cells look like they are not really resistant. Recorded growth:<br />
{|border="1"<br />
|<br />
|K191004+9 control<br />
|K191004+9<br />
|K191004+3 control<br />
|K191004+3<br />
|-<br />
|DARK<br />
| ++ , white<br />
| +++ , white<br />
| ++ , white<br />
| +++ , white with red points<br />
|-<br />
| LIGHT<br />
| + , white<br />
| +++ , white with red point<br />
| + -almost no growth <br />
| ++ white<br />
|}<br />
<br />
<br />
<br />
'''2/8/10'''<br />
<br />
Run the digest of the minipreps from the white and red cultures of double transformants. No proper DNA band in the solution- repeat the procedure or not? Don't know what to do.... again. <br />
<br />
[[Image:2.08.JPG]]<br />
<br />
Collect the thoughts and ideas and start again tomorrow!<br />
<br />
'''6/8/10'''<br />
<br />
double transformants were prepared again, using non-cowboy procedure. 04 cells were first MADE COMPETENT, then transformed with 03 and 09, also 1 neg control was plated. Grow white on the intitial plate, then turned red if subcultured. <br />
<br />
'''7-8/08'''<br />
<br />
'Subbed these to duplicate fresh plates and incubated overnight at room temperature <br />
either under a fluorescent lamp or wrapped in foil - all white in both cases (and growth <br />
not great, probably because of low temperature)'. <br />
The plates were moved to 37C with the lamp /one under the light, one in the dark. so far -no results, checking in an hour/. <br />
<br />
Another idea- check for stability of the insert- we don't know if the plasmids are compatibile. Streaking from original plates to check if single colonies will be a mixture of white and red or just red- origin? no plasmid maps from Lausanne. <br />
by Chairman-->' If we can't find this information, I think our best course is to <br />
PCR out both inserts and combine them in a single vector, preferably pSB1C3.'<br />
<br />
<br />
'''9/8/2010'''<br />
<br />
to do:<br />
<br />
*primers for the parts to put them in a vector<br />
*check the lamp every hour!!<br />
<br />
go thruough Lausanne website to find the plasmid maps<br />
get back to Mexico about the parts and etc.<br />
find out the conditions for the blue light sensor assay!<br />
<br />
'''10/08/10'''<br />
* assay with dark/light liquid cultures - fluorescence measurment and OD, after 5 h in the shaker under lamp- see video and pictures<br />
*email to Mexico<br />
<br />
'''11/08/10'''<br />
<br />
*primer designed for combining LOVTap and Readout1 into 1 cell<br />
*repeated assay with the smae samples, after overngiht in RT and %5h under lamp (for the light samples) and RT without shaking<br />
*primary cultures streaked to amp/kan/IPTG plates- part 4+9 and 4+3 -three clones each<br />
<br />
'''12/08/10'''<br />
*results analysis after LOVTap experiment:check file, add graphs later.<br />
*email from Mexico about Blue promoter<br />
<br />
'''16/08/10'''<br />
* streaked single clone for 9+4 and 3+4 (clones number 5 and 2, respectively)- for tomorrow's experiment<br />
* minipreped mysterious double transformants and Sacb smples<br />
* tried out the box on the shaker- unscrewed the things. <br />
* made the graphs from the second reading<br />
* 03 and 09 LovTap taken for sequencing. <br />
<br />
to do: decide on the illumination length and think about controls and how many samples do you want to put. check Lausanne website<br />
t-test to check to importancy of the differences between the means.<br />
<br />
'''17/07/10'''<br />
* desinged experimental set up for the LovTap blue light response experiment /see next day/<br />
* BAD NEWS: there is a suspicion of 1bp deletion in LovTap!!! Chairman noticed it when he was checking the parts base by base. Should have done it much much earlier probably to save the time... <br />
* BAD NEWS: lack of the proper thinking put in doubt all the results from previous two experiments (that is why you can't find any files of it attached)- there was no IPTG added to the samples, therefore LOVTap expression was not induced- no point to do T-test right now... :( Well, at least I will learn from it and add it next time!<br />
<br />
<br />
'''18/08/10'''<br />
<br />
* tested LovTap under the blue light<br />
* conditions:<br />
Liquid cultures (same as for minipreps) were grown overnight in 37C with antibiotics. <br />
The next day, bottles with 3.5 ml of broth were inoculated with the 0.5 ml of appropriate cultures (0.5. ml of broth added as a control).<br />
*sample designation: (# refers to the clone number form the original plate). Original plates are the ones made on '''INSERT THE DATE LAZY''' and have 2 clones on each plates (clones1-3 are K191003&4 double transformants, clones 4-6 are K191009+4 double transformants). <br />
<br />
# K191003+4 (#1) + Amp + Kan + IPTG + LIGHT <br />
# K191003+4 (#1) + Amp + Kan IPTG + DARK <br />
# K191003+4 (#2) + Amp + Kan IPTG + LIGHT <br />
# K191003+4 (#2) + Amp + Kan IPTG + DARK <br />
# K191009+4 (#4) + Amp + Kan IPTG +LIGHT <br />
# K191009+4 (#4) + Amp + Kan IPTG +DARK <br />
# K191009+4 (#5) + Amp + Kan IPTG +LIGHT <br />
# K191009+4 (#5) + Amp + Kan IPTG +DARK <br />
# K191004 ONLY #2+ Amp + LIGHT <br />
# K191004 ONLY #2+ Amp + DARK <br />
# neg. control- broth, Amp + Kan + IPTG + LIGHT<br />
<br />
* samples designated DARK were wrapped in the aluminium foil straight after the inoculation; the cap was not wrapped (not see through anyway)so that samples could be easily taken without unwrapping the bottles from the foil. <br />
*All the samples were then taken to the shaker in the hot room and placed in prepared spots: for the dark- anywhere on an easily accessible holder, for light- in 2 rows of holders. <br />
*Light samples were covered with the cardboard box with blue LEDs attached on the sides, in a way to optimize light accession to the samples (ask Sarah for the details). Box was securely taped to the shaker. <br />
* Measurments were taken every half an hour: 200ul of the samples were placed in the numbered cuvette and then 800ul of water was added. Readings of fluroscence (green mode) were taken in standard fluorescence units (SFU, two readings each). Then optical density was measured (upstairs in the incubator room) at 360 nm wavelength. Control was used to calibrate the machine (set ref button). Also 2 readings were taken. Samples were shaken by inversion prior to taking measurment. <br />
* Experiment was stopped after 3 hours (6 sets of reading taken). <br />
<br />
*'''Result analysis'''<br />
<br />
[[Image:Lt18aug.JPG]]<br />
<br />
To generate the data for the graph:<br />
# means were calculated from 2 readings of optical density and fluorescence<br />
# (mean SFU minus background SFU) was divided by (mean OD minus background OD, if different from 0).<br />
# table was made for the samples at different points of time- e.g. sample 1 after 30 min, 60 min, 90 min, 120 min etc. <br />
# graph was made with series named the same as the samples<br />
<br />
[[File:Untitled.JPG]]<br />
<br />
**BAD BAD VERY BAD NEWS: after 2 hours of measurments the results of the sequencing were know and revealed that THERE IS A 1BP DELETION IN LOVTAP 03 AND 09 WHICH RESULTS IN FRAMESHIFT AND LACK OF THE FUNCTIONAL PRODUCT!!!! :(:(:( despite this information, it was decided that the experiment will be finished and analysed as initially planned.<br />
<br />
'''19/08/2010'''<br />
<br />
* Pcr- mutagenensis of LovTap- Mabel, adding missing bp. <br />
* Puryfing PCR product from the solution. <br />
<br />
<br />
'''20/8/2010'''<br />
* PCR run on the gel -correct band of ~5.5. kb size, suggest that the PCR worked well. couple of other fainter bands <br />
* Self -ligation overnight (MABEL self ligation- 12ul of H2O, 4ul of DNA, 2 ul of Ligase buffer, 1 ul of T4 DNA ligase, 1ul of T4 polynucleotide kinase. <br />
* transformation and setting up the liquid cultures for the minipreps over the weekend. <br />
<br />
[[Image:123.JPG]]<br />
<br />
'''23/08/2010'''<br />
* mini-prep DNA from the colonies after PCR- marker worn off in the shaker,so 3.2. and 9.1. got mixed up- probably sequencing necessary to distinguish the mutated version from non-mutated <br />
* digest minipreps with EcoRI, run the gel[photo]<br />
* run 5ul of the ligated 03 and 09 on the gel /also Richards samples/.<br />
<br />
[[Image:124.JPG]]<br />
<br />
'''24/08/2010'''<br />
* repeated digest of mutated LovTap samples: 3.1, 3.?, 3.3., 3.4 9.?, 9.2 with EcoRi<br />
* minipreped 6 more clones of mutated LovTap 03 and 09 from the same plate, labelled- 3.6, 3.7, 3.8, 9.4, 9.5, 9.6. Run the digest with EcoRI. Note: 3.7 sample is higly diluted as 80ul of EB was used instead of 40ul. oops. <br />
* delivered mutated 3.? and 9.2 minipreped clones for sequencing /only 2 reactions: each sample- forward/.<br />
<br />
[[Image:LT100.jpg]] <br />
<br />
'''25/9/2010'''<br />
* trpR mutant received form KEIO collection- generously privided by Lucas Black (The University of Edinburgh). <br />
'''1/10/2010'''<br />
* LOVTap-RFP in JM109<br />
<br />
'''04/10/10'''<br />
We may have a working LovTap construct + red/white (RFP) reporter system thanks to Mexico.<br />
* Cutting Mexico's LOVTap with EcoRi/PstI & transfer into pSB1C3 plasmid (Part 1)<br />
* Adding reporter- cut out with XbaI/PstI from K191004 part provided by Lausanne (PArt 2)<br />
* Ligation of Part 1 and Part 2., selection for red colonies. <br />
<br />
Minor characterisation experiment:<br><br />
-LovTap constructed with red-white RFP reporter<br><br />
-If activated, the blue light sensor transformants should produce red colonies<br><br />
-Blue light sensor containing-colonies from transformant plate streaked onto two plates<br><br />
-One plate left in the light, the other left in the dark<br><br />
-If the one in the light grows red and the one in the dark doesn't, it is a good indication that the construct is working<br><br><br />
<br />
'''5/10/2010'''<br />
* transforming trpR mutant with LOVTap- RFP plasmid. <br />
<br />
'''8/10/2010'''<br />
* repetaed LOVTap charactierzation- with TrpR mutant and WT under light/dark conidtion. <br />
* no clear response was seen. <br />
<br />
[[Image:8octLovtap.jpg]]<br />
<br />
[[Image:8octTRPR.jpg]]<br />
<br />
'''11/10/2010'''<br />
* Transforming plasmid Ptrp with RFP reporter to JM109 strian- control for further experiments.<br />
<br />
'''14/10/2010'''<br />
* PRi in trpR host- control plasmid<br />
<br />
'''22/10/2010'''<br />
* checking temperature response of LOVTap<br />
* setting up samples with CMM minum medium, enriched in thymine, trace elements and glucose.<br />
CELLS FAIL TO GROW!:(</div>Macbereskahttp://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_sensorTeam:Edinburgh/Notebook/Blue light sensor2010-10-27T16:13:18Z<p>Macbereska: /* Blue Light Sensor */</p>
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<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_producer">blue light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_producer">green light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_sensor">green sensor</a></li><br />
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<span style="color:ivory;">These <b>notes</b> correspond to the part of the <b>project</b> detailed <a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_sensor">here</a>.</span><br />
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==Blue Light Sensor==<br />
<br><br />
<br />
'''14/07/10'''<br />
<br />
Received parts from Lausanne:<br />
*BBa_191006 (113 ng/µL) – LovTAP alone<br />
*BBa_191004 (166 ng/µL) – Readout <br />
*BBa_191003 (230 ng/µL) – whole system<br />
*BBa_191009 (85 ng/µL) – mutant <br />
*Sosnick vector containing TrpR (393 ng/µL) – vector <br />
<br />
<br />
<br />
'''16/07/10'''<br />
<br />
Transformations using 100 µL of cells, 900 µL broth<br />
*K191004 - amp<br />
*K191003 - kanamycin<br />
*K191009 – kanamycin<br />
*Sosnick vector with TrpR – amp<br />
<br />
Note: Labeling on tubes is unclear – K191009 and K191004 got mixed up, so plating both tubes on both Kan and amp<br />
<br />
<br />
'''22/07/10'''<br />
<br />
*Minipreps: K0998010 – 5x (1 didn’t grow, but did anyway)<br />
*Blue sensors (3 biobricks and Sosnick vector) – cultures set to grow overnight<br />
<br />
'''25/07/10'''<br />
Digest of the blue light sensor minipreps was performed with EcoRI and BamHI; samples:<br />
*3- K19100'''4'''; 900ul, (3)<br />
*4- K19100'''4'''; 100ul, (2)<br />
*5-K19100'''9'''; 100ul, (1)<br />
*6- K19100'''9'''; 100ul, (6)<br />
*7- K19100'''9'''; 900ul, (4)<br />
*8- K19100'''9'''; 100ul, (2)<br />
*9- K19100'''3'''; 900ul, (2)<br />
*10- K19100'''3'''; 900ul, (5)<br />
*11- K19100'''3'''; 100ul, (4)<br />
*12- K19100'''3'''; 100ul, (1)<br />
*13- K19100'''4'''; 100ul, (3)<br />
*14- K19100'''4'''; 900ul, (5)<br />
<br />
Lanes- 1- marker, 2-9 sampels as in order, 10-empty, 11, 12- marker instead of the samples- need to run last two again<br />
<br />
[[Image:109.JPG]]<br />
<br />
[[Image:111.JPG]]<br />
LAST TWO LANES- NSAMPLES NO 13 AND 14 FROM THE ABOVE LIST<br />
<br />
<br />
'''28/07/10'''<br />
<br />
Double transformants made using a novel cowboy procedure- double transformation with 4ul of each piece od DNA. Chances of succes- very very low... But at least it will be proved that it won't work!<br />
Trans. 1: BBa_K191004+ K191003- read out + LovTap<br />
Trans. 2: BBa_K191004+ K191009- read out + mutated LovTap<br />
<br />
'''30/7/10'''<br />
set up experiments to see if the blue light sensor will activate the response system under the daylight. Both liquid cultures and plates were set up, with red and white colonies- first supposed to turn white in the daylight and stay red in the dark (under the foil), second ones- no change of the colour. The same applies to the liquid cultures. However, the experiment failed- no proper growth was observed, and if there was a growth - it was very weak. No pigment observed, apart from the small points in the centre. <br />
Hypothesis- not stable plasmids-- cells look like they are not really resistant. Recorded growth:<br />
{|border="1"<br />
|<br />
|K191004+9 control<br />
|K191004+9<br />
|K191004+3 control<br />
|K191004+3<br />
|-<br />
|DARK<br />
| ++ , white<br />
| +++ , white<br />
| ++ , white<br />
| +++ , white with red points<br />
|-<br />
| LIGHT<br />
| + , white<br />
| +++ , white with red point<br />
| + -almost no growth <br />
| ++ white<br />
|}<br />
<br />
<br />
<br />
'''2/8/10'''<br />
<br />
Run the digest of the minipreps from the white and red cultures of double transformants. No proper DNA band in the solution- repeat the procedure or not? Don't know what to do.... again. <br />
<br />
[[Image:2.08.JPG]]<br />
<br />
Collect the thoughts and ideas and start again tomorrow!<br />
<br />
'''6/8/10'''<br />
<br />
double transformants were prepared again, using non-cowboy procedure. 04 cells were first MADE COMPETENT, then transformed with 03 and 09, also 1 neg control was plated. Grow white on the intitial plate, then turned red if subcultured. <br />
<br />
'''7-8/08'''<br />
<br />
'Subbed these to duplicate fresh plates and incubated overnight at room temperature <br />
either under a fluorescent lamp or wrapped in foil - all white in both cases (and growth <br />
not great, probably because of low temperature)'. <br />
The plates were moved to 37C with the lamp /one under the light, one in the dark. so far -no results, checking in an hour/. <br />
<br />
Another idea- check for stability of the insert- we don't know if the plasmids are compatibile. Streaking from original plates to check if single colonies will be a mixture of white and red or just red- origin? no plasmid maps from Lausanne. <br />
by Chairman-->' If we can't find this information, I think our best course is to <br />
PCR out both inserts and combine them in a single vector, preferably pSB1C3.'<br />
<br />
<br />
'''9/8/2010'''<br />
<br />
to do:<br />
<br />
*primers for the parts to put them in a vector<br />
*check the lamp every hour!!<br />
<br />
go thruough Lausanne website to find the plasmid maps<br />
get back to Mexico about the parts and etc.<br />
find out the conditions for the blue light sensor assay!<br />
<br />
'''10/08/10'''<br />
* assay with dark/light liquid cultures - fluorescence measurment and OD, after 5 h in the shaker under lamp- see video and pictures<br />
*email to Mexico<br />
<br />
'''11/08/10'''<br />
<br />
*primer designed for combining LOVTap and Readout1 into 1 cell<br />
*repeated assay with the smae samples, after overngiht in RT and %5h under lamp (for the light samples) and RT without shaking<br />
*primary cultures streaked to amp/kan/IPTG plates- part 4+9 and 4+3 -three clones each<br />
<br />
'''12/08/10'''<br />
*results analysis after LOVTap experiment:check file, add graphs later.<br />
*email from Mexico about Blue promoter<br />
<br />
'''16/08/10'''<br />
* streaked single clone for 9+4 and 3+4 (clones number 5 and 2, respectively)- for tomorrow's experiment<br />
* minipreped mysterious double transformants and Sacb smples<br />
* tried out the box on the shaker- unscrewed the things. <br />
* made the graphs from the second reading<br />
* 03 and 09 LovTap taken for sequencing. <br />
<br />
to do: decide on the illumination length and think about controls and how many samples do you want to put. check Lausanne website<br />
t-test to check to importancy of the differences between the means.<br />
<br />
'''17/07/10'''<br />
* desinged experimental set up for the LovTap blue light response experiment /see next day/<br />
* BAD NEWS: there is a suspicion of 1bp deletion in LovTap!!! Chairman noticed it when he was checking the parts base by base. Should have done it much much earlier probably to save the time... <br />
* BAD NEWS: lack of the proper thinking put in doubt all the results from previous two experiments (that is why you can't find any files of it attached)- there was no IPTG added to the samples, therefore LOVTap expression was not induced- no point to do T-test right now... :( Well, at least I will learn from it and add it next time!<br />
<br />
<br />
'''18/08/10'''<br />
<br />
* tested LovTap under the blue light<br />
* conditions:<br />
Liquid cultures (same as for minipreps) were grown overnight in 37C with antibiotics. <br />
The next day, bottles with 3.5 ml of broth were inoculated with the 0.5 ml of appropriate cultures (0.5. ml of broth added as a control).<br />
*sample designation: (# refers to the clone number form the original plate). Original plates are the ones made on '''INSERT THE DATE LAZY''' and have 2 clones on each plates (clones1-3 are K191003&4 double transformants, clones 4-6 are K191009+4 double transformants). <br />
<br />
# K191003+4 (#1) + Amp + Kan + IPTG + LIGHT <br />
# K191003+4 (#1) + Amp + Kan IPTG + DARK <br />
# K191003+4 (#2) + Amp + Kan IPTG + LIGHT <br />
# K191003+4 (#2) + Amp + Kan IPTG + DARK <br />
# K191009+4 (#4) + Amp + Kan IPTG +LIGHT <br />
# K191009+4 (#4) + Amp + Kan IPTG +DARK <br />
# K191009+4 (#5) + Amp + Kan IPTG +LIGHT <br />
# K191009+4 (#5) + Amp + Kan IPTG +DARK <br />
# K191004 ONLY #2+ Amp + LIGHT <br />
# K191004 ONLY #2+ Amp + DARK <br />
# neg. control- broth, Amp + Kan + IPTG + LIGHT<br />
<br />
* samples designated DARK were wrapped in the aluminium foil straight after the inoculation; the cap was not wrapped (not see through anyway)so that samples could be easily taken without unwrapping the bottles from the foil. <br />
*All the samples were then taken to the shaker in the hot room and placed in prepared spots: for the dark- anywhere on an easily accessible holder, for light- in 2 rows of holders. <br />
*Light samples were covered with the cardboard box with blue LEDs attached on the sides, in a way to optimize light accession to the samples (ask Sarah for the details). Box was securely taped to the shaker. <br />
* Measurments were taken every half an hour: 200ul of the samples were placed in the numbered cuvette and then 800ul of water was added. Readings of fluroscence (green mode) were taken in standard fluorescence units (SFU, two readings each). Then optical density was measured (upstairs in the incubator room) at 360 nm wavelength. Control was used to calibrate the machine (set ref button). Also 2 readings were taken. Samples were shaken by inversion prior to taking measurment. <br />
* Experiment was stopped after 3 hours (6 sets of reading taken). <br />
<br />
*'''Result analysis'''<br />
<br />
[[Image:Lt18aug.JPG]]<br />
<br />
To generate the data for the graph:<br />
# means were calculated from 2 readings of optical density and fluorescence<br />
# (mean SFU minus background SFU) was divided by (mean OD minus background OD, if different from 0).<br />
# table was made for the samples at different points of time- e.g. sample 1 after 30 min, 60 min, 90 min, 120 min etc. <br />
# graph was made with series named the same as the samples<br />
<br />
[[File:Untitled.JPG]]<br />
<br />
**BAD BAD VERY BAD NEWS: after 2 hours of measurments the results of the sequencing were know and revealed that THERE IS A 1BP DELETION IN LOVTAP 03 AND 09 WHICH RESULTS IN FRAMESHIFT AND LACK OF THE FUNCTIONAL PRODUCT!!!! :(:(:( despite this information, it was decided that the experiment will be finished and analysed as initially planned.<br />
<br />
'''19/08/2010'''<br />
<br />
* Pcr- mutagenensis of LovTap- Mabel, adding missing bp. <br />
* Puryfing PCR product from the solution. <br />
<br />
<br />
'''20/8/2010'''<br />
* PCR run on the gel -correct band of ~5.5. kb size, suggest that the PCR worked well. couple of other fainter bands <br />
* Self -ligation overnight (MABEL self ligation- 12ul of H2O, 4ul of DNA, 2 ul of Ligase buffer, 1 ul of T4 DNA ligase, 1ul of T4 polynucleotide kinase. <br />
* transformation and setting up the liquid cultures for the minipreps over the weekend. <br />
<br />
[[Image:123.JPG]]<br />
<br />
'''23/08/2010'''<br />
* mini-prep DNA from the colonies after PCR- marker worn off in the shaker,so 3.2. and 9.1. got mixed up- probably sequencing necessary to distinguish the mutated version from non-mutated <br />
* digest minipreps with EcoRI, run the gel[photo]<br />
* run 5ul of the ligated 03 and 09 on the gel /also Richards samples/.<br />
<br />
[[Image:124.JPG]]<br />
<br />
'''24/08/2010'''<br />
* repeated digest of mutated LovTap samples: 3.1, 3.?, 3.3., 3.4 9.?, 9.2 with EcoRi<br />
* minipreped 6 more clones of mutated LovTap 03 and 09 from the same plate, labelled- 3.6, 3.7, 3.8, 9.4, 9.5, 9.6. Run the digest with EcoRI. Note: 3.7 sample is higly diluted as 80ul of EB was used instead of 40ul. oops. <br />
* delivered mutated 3.? and 9.2 minipreped clones for sequencing /only 2 reactions: each sample- forward/.<br />
<br />
[[Image:LT100.jpg]]<br />
<br />
'''04/10/10'''<br />
We may have a working LovTap construct + red/white (RFP) reporter system thanks to Mexico.<br />
<br />
<br />
Minor characterisation experiment:<br><br />
-LovTap constructed with red-white RFP reporter<br><br />
-If activated, the blue light sensor transformants should produce red colonies<br><br />
-Blue light sensor containing-colonies from transformant plate streaked onto two plates<br><br />
-One plate left in the light, the other left in the dark<br><br />
-If the one in the light grows red and the one in the dark doesn't, it is a good indication that the construct is working<br><br><br />
<br />
'''8/10/2010'''<br />
* repetaed LOVTap charactierzation- with TrpR mutant and WT under light/dark conidtion. <br />
* no clear response was seen. <br />
<br />
[[Image:8octLovtap.jpg]]<br />
<br />
[[Image:8octTRPR.jpg]]<br />
<br />
'''22/10/2010'''<br />
* checking temperature response of LOVTap<br />
* setting up samples with CMM minum medium, enriched in thymine, trace elements and glucose.<br />
CELLS FAIL TO GROW!:(</div>Macbereskahttp://2010.igem.org/File:2.08.JPGFile:2.08.JPG2010-10-27T16:12:51Z<p>Macbereska: </p>
<hr />
<div></div>Macbereskahttp://2010.igem.org/Team:Edinburgh/Notebook/BRIDGETeam:Edinburgh/Notebook/BRIDGE2010-10-27T15:56:52Z<p>Macbereska: /* BRIDGE */</p>
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<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Collaboration">collaboration</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Attribution">attribution</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/BRIDGE">BRIDGE</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_producer">red light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_sensor">red sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_producer">blue light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_producer">green light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_sensor">green sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Modelling">modelling</a></li><br />
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<li><a href="http://www.openwetware.org/wiki/French_Lab">protocols</a></li><br />
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<span style="color:ivory;">These <b>notes</b> correspond to the part of the <b>project</b> detailed <a href="https://2010.igem.org/Team:Edinburgh/Project">here</a>.</span><br />
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<br />
== BRIDGE ==<br />
<br><br />
<br />
'''5/7/2010'''<br />
<br />
* sacB digest with EcoRI -band around kb '''(gel 1)'''<br />
[[Image:103.JPG]]<br />
<br />
<br />
'''6/7/2010'''<br />
* sacB digest with EcoRi/PstI. Band also around 5 kb. <br />
[[Image:1005.JPG]]<br />
<br />
'''7/7/2010''' <br />
<br />
* PCR of sacB to check insert. Used sacB tube 1 and 3.<br />
* Restriction digest of PCR with EcoRI and NotI. <br />
[[Image:106.JPG]]<br />
* lane 1- marker<br />
* lane 2, 3, 4 - sacB 1, 2 & 3 cut with NotI; band size: slightly bigger than 4 kb<br />
* Lane 5- PCR of LuxAB; band size around 1.5. kb<br />
* lane 6- Vector of sacB (pSB1C3, 2072bp, aroun 3kb with RFP) cut with NotI; band size around 1.5. kb<br />
* Lane 7- PCR of sacB with gene sepcific primers; band size around 1.5. kb<br />
<br />
* Trouble shooting: sacB cut with NotI give about 4kb, whereas cutting with EcoRI gives 5.5. kb... hmhmhm.<br />
there should be 2 NotI sites on either side of the insert but we only get 1 band--> is there only 1 NotI site? Furthermore, PstI/EcoRI digests give only 1 band (same as cut only with EcoRI), so maybe there is no PstI/NotI site?<br />
<br />
* Purifying sacB from PCR product to start making BRIDGE- row F in the BOX 1.<br />
<br />
'''9/7/2010'''<br />
* Making working solutions of primers from stock: 4ul of H20; 1 ul of stock solution<br />
* Primers: forward 72.3 C, revers- 77.6 C; length of the product: 2.6-2.7 kb<br />
{|border="1"<br />
|Lane 1<br />
|Lane 2<br />
|Lane 3<br />
|Lane 4<br />
|Lane 5<br />
|Lane 6<br />
|Lane 7<br />
|Lane8<br />
|-<br />
| ladder<br />
| sacB <br />
| sacB digest wtih EcoRI-HF<br />
| sacB +<br />
| digest `5009<br />
| S248T PCR product<br />
| Pure 356K<br />
| Pure 356R<br />
|}<br />
<br />
[[Image:152.JPG]]<br />
'''to do on Monday:'''<br />
* redo sacB- catR ligation<br />
* Run 5 ul of ligation on gel to check for large bands<br />
* redo RLS part transformations<br />
<br />
<br />
'''12/7/2010''' <br />
<br />
* Transformants of luxAB-edi1 failed- probably smth wrong at the purification stage...<br />
<br />
[[Image:107.JPG]]<br />
<br />
{|border="1"<br />
|Lane 1<br />
|Lane 2<br />
|Lane 3<br />
|Lane 4<br />
|Lane 5<br />
|Lane 6<br />
|-<br />
| ladder<br />
| luxAB-ediI digest<br />
| luxAB-ediI ligation<br />
| sacB digest<br />
| catR digest<br />
| sacB- catR lgation<br />
|-<br />
| expec. no of bands<br />
| 2<br />
| 1 or 3 <br />
| 1<br />
| 1<br />
| 1 band= to 4+5<br />
|}<br />
<br />
* If all failed- purification procedure failed<br />
* If 3 and 6 failed- ligation failed<br />
* If all fine then PCR/transformation failed<br />
conclusion:...<br />
<br />
* 3 and 6 failed- ligation problem (not enough DNA, ligase).<br />
* Band 1- luxAB but no ediI (?)<br />
<br />
* Repeated digest (sacB with XbaI, catR with SpeI, LuxAB & EdiI with SpeI & SacI) and purification.<br />
* Set up ligations with 2ul ligase instead of 1 ul (1ul of H2O less). If this fails- perhaps only 2-3 h ligation ina RT (SpeI is set up in both digest- could be common cause)<br />
<br />
'''13/7/2010'''<br />
*First, run PCR of catR-sacB ligation (use DNA'''?????'''' MK1- with sacBr1 (19.6.10); MK2- sacBr1(10)<br />
* Gel electrophoresis of: digested catR, digested sacB, ligated sacb-catR, digested LuxAB-ediI and ligated luxAB-ediI along with sacB-catR PCR<br />
<br />
[[Image:108.JPG]]<br />
<br />
{|border="1"<br />
|Lane 1<br />
|Lane 2<br />
|Lane 3<br />
|Lane 4<br />
|Lane 5<br />
|Lane 6<br />
|Lane 7<br />
|-<br />
| ladder<br />
| ediI<br />
| digested LuxAB-ediI<br />
| ligated luxAB-ediI<br />
| ligated catR & sacB<br />
| PCR 1 (MK1)<br />
| PCR 2 (MK2)<br />
|}<br />
<br />
''''AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAARGH!!!!!!!!!!!!!!!1''''<br />
<br />
* Almost completely convinced it's the ligase... or me (MK). Officially taking myself off the project. xx<br />
* (Spotted 2 purified sacBs in the freezer... maybe try the original if this one fails...?)<br />
'''3/8/2010'''<br />
*minipreps: marker (lane 1& 8) & sacb 8.1-8.6( Lanes 2-7)<br />
[[Image:115.JPG]]<br />
''''9/8/2010''''<br />
<br />
Gel with :<br />
* cat + sacB (PCR product) (6)<br />
* vector + cat + sacB (7) (both done by CF)<br />
<br />
'''20/8/2010'''<br />
<br />
* Made plates variety of sucrose combinations--> 40 cml<br />
* 0%<br />
* 0.1%<br />
* 0.25%<br />
* 0.5%<br />
* 1.0%<br />
<br />
*Plated E6 miniprep, which should show some sucrose sensitivity (1plate at each concentration)<br />
*Control- E5, 1 plate at each concentration<br />
<br />
'''30/8/2010'''<br />
* Primers have arrived for up and downstream sequences of TnaA region.<br />
'''8/9/2010'''<br />
* Lane 1 &8- marker, Lane 6- cat +sacB PCR; Lane 7- vector+cat+sacB<br />
[[Image:141.JPG]]<br />
<br />
'''13/9/2010'''<br />
Plane for week begininng 13th:<br />
* Amplify RFP with primers --> RBS-F2; YFP - R2 ---> to amplify RFP<br />
* Digest UP with SpeI and B-D with XbaI<br />
* Ligate UP to BRIDGE-DOWN<br />
* Digest RFP with XbaI and SpeI<br />
* Ligate UP and RFP<br />
* Ligate DOWN and RFP<br />
<br />
* Also- registry editing for RLS (definately dodgy sequence), LovTap<br />
* Justification of reseubmission<br />
* Look up absorption/emission rates and transmission through medium for Donal<br />
<br />
'''20/9/2010'''<br />
<br />
* Achieved: cat/sacB- traA down ligation<br />
* Attempted: traA up- cat/sacB/down ligation; traA up- RFP ligation; RFP- traA down ligation/ ALL FAILED<br />
* Running: purified digest of:<br />
- cat/sacB + SpeI <br><br />
- tnaA down- XbaI<br><br />
- tnaA up + SpeI<br><br />
- cat/sacB/down + XbaI <br><br />
- RFP + XbaI<br><br />
and also running: UP- BRIDGE- DOWN ligation; UP - RFP ligation<br />
<br />
Have: PCR of cat/sacB, RFP, BRIDGE-DOWN, traA up, traA down; ligation of BRIDGE-DOWN<br />
<br />
<br />
[[Image:146.JPG]]<br />
<br />
Run gel: <br />
{| border="1"<br />
| Lane 1 <br />
| Lane 2 <br />
| Lane 3 <br />
| Lane 4 <br />
| Lane 5 <br />
| Lane 6 <br />
| Lane 7 <br />
| Lane 8<br />
|-<br />
| Ladder <br />
| Pure SacB <br />
| Pure SacB & EcoRI <br />
| sacB & catR PCR <br />
| I15009 digest <br />
| S284T PCR Product <br />
| Purified 356K <br />
| Purified 356R<br />
|}<br />
<br />
<b>25/10/10</b><br />
<br><br />
Repetitive failure of purifications with both traditional method and kits, unsure what is going wrong. Have decided to tell Chris. Off to Firbush on Wednesday.<br><br><br />
<b>01/10/10</b><br />
<br><br />
In my absence (at Firbush with the rest of the Biotechnologists) Chris has managed to construct the tnaA UP-cat-sacB-tnaA DOWN segment that we need for the first part of the BRIDGE protocol. I will continue to build the tnaA UP-RFP-tnaA DOWN segment. <br />
<br><br />
Set up ligations of RFP-DOWN and UP-RFP, left in 16C incubator overnight. <br />
<br><br><br />
<b>02/10/10</b><br />
<br><br />
Lab on a Saturday again, fun fun fun...<br />
<br><br />
Retrieved and amplified ligations and ran on gel. UP-RFP appears to be significantly shorter than RFP-DOWN and indeed shorter than it ought to be (should be about 2kb, is only around 1.5kb). <br><br />
Purified both products, then digested RFP-DOWN with XbaI. (Used 2hour incubation time to write presentation for Wednesday).<br><br />
Set up ligation of UP with RFP-DOWN and left in incubator overnight.<br><br />
<br><br />
<b>03/10/10</b><br />
<br><br />
Bus to Darwin for 11:20am; enter Darwin; lift to 8th floor; retrieve ligations from waterbath in cold room; walk down to 7th floor; put ligation in freezer; lift to ground floor; leave Darwin; catch 11:40am bus back to central.<br />
<br><br><br />
<b>04/10/10</b><br />
<br><br />
PCR of ligation of UP-RFP-DOWN. Made and ran gel along with purified PCRs from Saturday, just in case they're still not working.<br><br />
They worked! The purifications were perfect. There is a band about the size of UP-RFP-DOWN in it's lane, but it's quite faint. I will probably repeat the PCR with a lower annealing temperature (around 50) this afternoon. I suspect the UP primers do not anneal properly above 54C.<br />
<br><br><br />
<b>07/10/10</b><br />
<br><br />
Chris set up overnight cultures of DH5-alpha cells and JM109 cells, both containing the lambda-red plasmid, in 2.5ml LB with tetracycline.<br><br />
Repeated PCR of up-RFP-down ligation as well as PCR of 3 subcultures suspected to contain the cat-sacB construct, performed using the tnaA UP forward primer and the tnaA DOWN reverse primer. <br />
<br><br><br />
<b>08/10/10</b><br><br />
Took 2 5ml LB bottles:<br><br />
- Added 1.7 microlitres of tet15 and 0.3ml DH5-alpha overnight culture to one<br><br />
- Added 5 microlitres of tet15 and 0.3ml of JM109 overnight culture to the other<br><br><br />
Grew second cultures for 2 hours at 30C with shaking <br><br />
Measured optical density after 2 hours:<br><br />
- Water = 0.00<br><br />
- DH5-alpha = 0.251<br><br />
- JM109 = 0.320<br><br><br />
Added 100microlitres arabinose to both cultures and grew at 37C for one and a half hours with shaking.<br><br><br />
Prepared cells for electroporation:<br><br />
1- Added 1.5ml of liquid culture to an eppendorf<br><br />
2- Spun down cells at 11,000rpm for 30s<br><br />
3- Removed supernatant, added 1ml of cold sterile water and resuspended cells<br><br />
4- Repeated steps 2 and 3 twice more<br><br />
5- Added 40microlitres of suspended cells and 1microlitre of DNA construct (<i>up-cat-sacB-down</i>) to chilled electroporation cuvette (also prepared one cuvette of each cell culture without DNA for controls)<br><br><br />
Performed electroporation<br><br />
Suspended cells in cuvette by adding 0.5ml LB and added to eppendorf containing 1.0ml LB<br><br />
Left in 37C incubator for one hour, no sign of growth - spun cells down and no visible pellet, resuspended in 0.9ml and decided to leave overnight<br><br><br />
Will plate up cells on chloramphenicol tomorrow and they should have grown by Monday.<br><br><br />
Annoyingly, the Red/ET recombination plasmid is lost at 37C, but it was worth following the procedure to see if it actually works first time. If we need to repeat it, the plates can be grown at 30C so that there is no need to retransform with the recombination plasmid.<br><br><br />
<b>09/10/10</b><br />
<br>Cells left to grow for ~20 hours<br><br />
Plated from 100microlitres from 900microlitre suspension onto 4 chloramphenicol plates marked JM109/lambda + cat/sacB and -cat/sacB (control) and DH5-alpha/lambda + cat/sacB and - cat/sacB (control)<br><br />
Spun cells down at 7000rpm for 2 minutes - gave decent sized pellet. Removed 700microlitres of supernatant and resuspended cells<br><br />
Plated last 100microlitres onto a further 4 chloramphenicol plates <br><br />
Plates will be left in 37C incubator - cells were concentrated enough on second set of plates so that if they are cml resistant they will grow by tomorrow. <br><br><br />
<b>10/10/10</b><br><br />
Significant growth of red colonies on DH5-alpha+ (concentrated) but none on DH5-alpha- (not-concentrated). This is highly confusing as there should be no RFP in the cells. We now believe this to be a contamination.<br />
<br><br />
There is growth on both of the negative plates of the JM109 cells, but none on the positive plates. This could be a labelling error.<br><br />
There is no growth on any of the other plates.<br><br><br />
<b>11/10/10</b><br><br />
No growth on any of the plates that had not grown yesterday. <br><br />
Colonies on JM109- plates do not smell of indole. If they are E.coli then this is promising. Subcultured 4 colonies from each plate onto cml plates.<br><br><br />
Ran gels of PCRs performed on 07/10. No bands in the three lanes from subcultures, but a strong band from the PCR of the ligation.<br><br><br />
<b>12/10/10</b><br><br />
The subcultures from yesterday have not grown particularly well. The cells are white, unlike normal E.coli, and the colonies are uncharacteristically runny. Ran PCR of subcultures to determine whether they contain the insert. (with insert ~2.5kb, without ~1.5kb)<br><br />
<br />
[[Image:FromChairman1.5.JPG]]<br />
<br />
Ran this PCR on a gel. Gave no bands. First attempt at BRIDGE did not work. <br><br><br />
Alterations to be made on second attempt:<br />
<br>1- Resuspend cells in 20-50microlitres after cleaning to increase transformation efficiency<br><br />
2- Grow at 30C instead of 37C after electroporation so as not to lose recombination plasmid<br><br />
3- Use normal chloramphenicol plates rather than cml+iptg+xgal.<br />
<br />
<br><br><br />
<b>14/10/10</b><br><br />
Prepared overnight cultures:<br />
<br><br />
- DH5-alpha/lambda in 5ml LB with 1.5microlitres tet15<br><br />
- JM109/lambda in 5ml LB with 5microlitres tet15<br><br><br />
Grew cells at 30C, 200rpm<br><br><br />
<br />
[[Image:171.jpg]]<br />
<br />
* tnaA PCR, JM109, MK2+4, CF 6.<br />
<b>15/10/10</b><br><br />
Prepared secondary liquid cultures:<br />
<br><br />
- 5ml LB + 1.5microlitres tet15 inoculated with 0.3ml overnight DH5-alpha/lambda culture<br><br />
- 5ml LB + 5microlitres tet15 inoculated with 0.3ml overnight culture JM109/lambda<br><br><br />
Grew cells at 30C, 200rpm for 2 hours<br><br><br />
Transferred 1.5ml of both cultures to individual eppendorfs and spun down cells for 3 minutes at 8000rpm. Removed supernatant and added another 1.5ml. Spun down cells again and removed supernatant.<br><br />
Suspended cells in 1ml cold sterile water and spun them down for 1 minute at 10000rpm before removing supernatant.<br />
Repeated this twice more.<br />
<br><br />
Resuspended cells in 100microlitires cold sterile water.<br><br><br />
Added 40microlitres of the relevant cell culture to cold sterile electroporation cuvettes labelled J+, J-, D+ and D-.<br><br />
Added 2microlitres DNA to the J+ cuvette.<br><br />
Added 1microlitre DNA to the D+ cuvetter.<br><br />
Prepared 8 eppendorfs, 4 containing 1.5ml LB.<br><br><br />
Electroporated all 4 samples.<br><br />
The voltage decay readings for the J-, D+ and D- cells were all above 4.0, but the reading for the J+ was 3.30. This probably indicates that there is too much DNA in the sample as the charge dissapated too quickly. From now on only 1microlitre of DNA will be used.<br><br><br />
Immediately after electroporation, 500microlitres of LB was added to the cuvette from an eppendorf to resuspend the cells and transferred back to the eppendorf. The LB was mixed and 750microlitres were transferred to a clean eppendorf.<br><br><br />
For each transformation, half the cells were grown at 30C overnight and half were grown at 37C overnight.<br />
<br><br><br />
<b>16/10/10</b><br><br />
Spun down cells from each of the 8 eppendorfs at 8000rpm for 3 minutes and removed 600microlitres of supernatant. <br><br />
Spread transformants onto chloramphenicol (one plate each) and grew at relevant temperatures for 48 hours. <br><br><br />
<b>18/10/10</b><br><br />
Only growth on plates appears to be non-E.coli. Major contamination has only occurred on 30C plates. Whatever it is, it seems to grow better at 30C. <br><br />
Since this is the second time we have seen this type of contamination the next attempt at the protocol will be combined with taking samples of cells at each step.<br><br><br />
<b>19/10/10</b><br><br />
Made more tet15 plates, as will need about 40 this week.<br><br />
Prepared overnight cultures of DH5-alpha/lambda and JM109/lamda, both grown at 30C.<br><br><br />
<b>20/10/10</b><br><br />
Took a sample of each overnight culture and spread it onto two tetracycline plates (1), one grown at 30C the other grown at 37C. The reasoning here is that any contaminants at this stage must be tetracycline resistant.<br><br><br />
Added 0.3ml overnight culture to fresh LB with tetracyline. Grew for 2 hours at 30C with shaking.<br />
<br><br><br />
Sampled each culture again, this time plating to both chloramphenicol and tetracyline plates. (2)<br><br />
NB: JM109 is not growing as well as DH5alpha in liquid culture. We may need to change the concentration of tet in one or both slightly.<br><br><br />
Something of a mini catastrophe...I knocked over my DH5alpha liquid culture and lost half the contents. Have added LB and a proportionate amount of tet15 and left both in the 30C shaker for another hour. This should recover enough cells to continue, however it also means the LB may contaminate the DH5alpha...if it is the LB, then only DH5alpha will be contaminated.<br><br><br />
In the mean time, am making 20-26 plates of cml.<br><br><br />
Added 100microlitres of 20% L-arabinose to each culture and left for one hour at 37C with shaking. Prepared cold water, cuvettes and eppendorfs with LB.<br><br><br />
Sampled cultures again, plating to chloramphenicol only. (3)<br><br />
Washed cells with cold water.<br><br />
Sampled cells again and plated to chloramphenicol. (4)<br><br><br />
<br><br><br />
Electroporated two sets with the cat/sacB construct and and two without. Transferred to LB in eppendorfs for recovery and grew each set of four at both 30C and 37C for an hour.<br><br><br />
Sampled cells again and plated to chloramphenicol. (5)<br><br />
Innoculated 5ml liquid cultures with 5microlitres of cml and 600microlitres of the transformed sample and grew overnight at 30C and 37C with shaking.<br><br><br />
<b>21/10/10</b><br />
<br><br />
No growth on any of the chloramphenicol plates from yesterdays samples. This suggests the usual contaminant has not yet reached the cultures. There is growth on each tetracycline plate, which is expected as the cells still contain the Red/ET plasmid at this point.<br><br><br />
Unfortunately there is no growth in the positive chloramphenicol liquid cultures. There is growth in the D- at 37C culture which presumably is one of our contaminants. I will keep an eye on the D- 37C plate and possibly plate out the liquid culture to examine the colonies. This contamination must have occurred during the final stage of the protocol from yesterday and must be human error as it is an isolated incident. <br><br><br />
<b>22/10/10</b><br><br />
Set up secondary liquid cultures in tet15 (3.5microlitres for JM109 as it seems to be struggling to grow) and left to grow in 30C shaker for 3 hours.<br />
<br><br />
Plated out D- 37C cell culture that survived chloramphenicol to determine strain.<br><br />
<br><br />
Checked chloramphenicol sample plates again:<br><br />
Sample 2- White colony growth on both JM109 plates but none on DH5alpha<br><br />
Sample 3- No growth (contaminants have disappeared, killed by arabinose?)<br><br />
Sample 4- White colony growth 30C plates only, two micrococcal colonies on J 37C<br><br />
Sample 5- A few very small white colonies on 30C plates D-, D+ and J-, no growth on D- at 37C<br><br><br />
The bulk of the contamination appears to occur at the washing step, but whatever they are, they don't seem to be able to survive cml40 in a liquid culture. Confusingly, whatever has contaminated the D- culture at 37C doesn't seem to be on the plates. This probably means it is an isolated event. I don't know if the same goes for the contaminants in sample 2. These are more confusing as they disappear after the arabinose is added. It is possible that they do not survive either in arabinose or at 37C in liquid.<br><br><br />
Next step to remove contaminants:<br><br />
Previously the washing step had been performed in the lab away from the clean hood. From now on, any stage where the cells are exposed will take place under the clean hood.<br><br />
A control will be set up at this step to confirm that contamination is occurring here and not previously. A second set of eppendorfs will be washed repeatedly with water and then 100microlitres of the water will be plated along with the actual samples. <br><br />
Growth on the negative control will indicate that either the water or the eppendorfs is contaminated. <br><br />
No growth on either will indicate that the use of the clean hood makes a difference.<br><br><br />
Next step in improving the efficacy of BRIDGE:<br><br />
The cat gene we're using is not very strongly induced. Couple this with the fact that DH5alpha and JM109 both struggle to grow at regular concentrations of antibiotics and we can assume that any recombinants we do get won't be able to grow in cml40. This time I will grow them overnight in a lower concentration of chloramphenicol. 3microlitres for JM109, 2microlitres for DH5alpha. <br><br><br />
This might be the last time I will be able to attempt BRIDGE before the wiki freeze. Fingers crossed.<br><br><br />
Maximised biomass this time by growing JM109 in 3.5microlitres tet15 and DH5alpha in 1.75microlitres as usual, for 2 and a half hours after adding 0.5ml overnight culture rather than 0.3ml. <br><br><br />
After the induction step spun down whole cell culture and kept cells under clean hood whenever they were exposed. <br>Also took negative controls at washing step of sterile water and LB, both kept in eppendorfs for 10minutes.<br>If there is growth on:<br />
<br><br />
- Sterile water only - sterile water is contaminated<br><br />
- LB only - LB is contaminated<br><br />
- Both - eppendorfs are contaminated<br>Also took samples of actual cell cultures at this step.<br><br><br />
Electroporated cells as normal. Grew at recovery step for 2 hours.<br><br><br />
Grew all 8 samples in cml40: JM109 in 3.5microlitres, DH5alpha in 1.75microlitres.<br><br />
Grew positive transformants in tet15 (normal concentrations) to determine if the cells were still alive. <br><br />
If the positive cells grow in:<br><br />
- cml and tet - recombinants<br><br />
- cml but not tet - contaminant<br><br />
- tet but not cml - non-recombinant<br><br />
- neither - cells have died<br><br><br />
<br />
Transformed JM109 cells with pSB1A2+cat to characterise cat and Edinbrick as a negative pSB1A2 control. Plated cells to ampicillin. <br><br><br />
<b>23/10/10</b><br><br />
The negative controls from the washing step have not grown, but then neither have the samples, so no contamination on anything. <br><br><br />
There is growth in 3 of the 4 tetracyline cultures from yesterday (JM109 has not grown at 37C, possibly because of inability of plasmid to replicate). This means the cells are not, in 3 cases, being killed by the electrporation. <br>There is no growth in any of the negative controls, so no indication of contamination. <br><br />
No growth in any of the cml positive cultures which indicates that the recombinase genes are not being induced properly. <br><br />
<br>The D- 37C contamination from Wednesday turned out to have RFP in it. This is quite confusing but, as stated earlier, all contaminations have been removed from the procedure.<br><br><br />
Good growth of cat and Edinbrick transformants on ampicillin. Have subcultured 4 of each, again to ampicillin.<br><br><br />
<b>24/10/10</b><br />
<br><br />
Subcultured the cat and Edinbrick transformants from ampicillin to cml40. <br><br><br />
<b>25/10/10</b><br><br />
No significant growth on subcultures.<br><br><br />
Set up titre of cml resistance using positive 30C transformants and negative controls. All grown in tetracycline and in increasing concentrations of chloramphenicol. Grown overnight at 30C and 200rpm.<br><br><br />
<b>26/10/10</b><br><br />
Growth on Edinbrick on cml40 but not on cat. Could be mislabelling. Have re-attempted subculture from transformant plates but if initial plates are labelled wrong then the results will be the same.<br><br><br />
Results from titre. Conclusion: recombination isn't working as cat has been known to show resistance to cml10-40.<br><br><br />
<br />
<br />
{|border="1" style="text-align: center"<br />
|-<br />
!<br />
! colspan="2" | Ju109 (tet15 / 5ul)<br />
! colspan="2" | DH5a (tet3.75 / 1.75ul)<br />
|-<br />
! Cml<br />
! + cat/sacB<br />
! control<br />
! + cat/sacB<br />
! control<br />
|-<br />
| 0 / 0 ul || + || + || + || +<br />
|-<br />
| 4 / 0.5 ul || - || - || - || -<br />
|-<br />
| 8 / 1 ul || - || - || - || -<br />
|-<br />
| 12 / 1.5 ul || - || - || - || -<br />
|-<br />
| 16 / 2 ul || - || - || - || -<br />
|-<br />
|| 20 / 2.5 ul || - || - || - || -<br />
|-<br />
| 24 / 3 ul || - || - || - || -<br />
|}</div>Macbereskahttp://2010.igem.org/File:FromChairman1.5.JPGFile:FromChairman1.5.JPG2010-10-27T15:56:25Z<p>Macbereska: </p>
<hr />
<div></div>Macbereskahttp://2010.igem.org/File:Lt18aug.JPGFile:Lt18aug.JPG2010-10-27T13:46:12Z<p>Macbereska: </p>
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<div></div>Macbereskahttp://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_sensorTeam:Edinburgh/Notebook/Blue light sensor2010-10-27T13:45:51Z<p>Macbereska: /* Blue Light Sensor */</p>
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<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial" class="dir">bacterial BRIDGEs</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Core_repressilator">the repressilator</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_producer">red light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_sensor">red sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_producer">blue light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Green_light_producer">green light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Green_light_sensor">green sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/BioBricks#Bacterial">submitted parts</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Bacterial">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/References">references</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling" class="dir">modelling BRIDGEs</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Kappa">kappa</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Genomic">the genomic model</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Bacterial">the bacterial model</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Signalling">the signalling model</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Tools">tools</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Modelling">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/References">references</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human" class="dir">human BRIDGEs</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Aspects">human aspects</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Communication">communication of science</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Terminology">terminology research</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Wiki">wiki</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Conversations">conversations</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Identity">identity</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Collaboration">collaboration</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Branding">branding research</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/SciFi">science fiction writing</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/FutureApps">future applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/SelfReflection">self-reflection</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Epic">the epic</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Human">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/References">references</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook" class="dir">lab notes&nbsp;&nbsp;&nbsp;</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Collaboration">collaboration</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Attribution">attribution</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/BRIDGE">BRIDGE</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_producer">red light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_sensor">red sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_producer">blue light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_producer">green light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_sensor">green sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Modelling">modelling</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Safety">safety</a></li><br />
<li><a href="http://www.openwetware.org/wiki/French_Lab">protocols</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Acknowledgements" class="dir">acknowledgements</a><br />
</li><br />
<br />
</ul><br />
</div><br />
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<span style="color:ivory;">These <b>notes</b> correspond to the part of the <b>project</b> detailed <a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_sensor">here</a>.</span><br />
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<br />
==Blue Light Sensor==<br />
<br><br />
<br />
'''14/07/10'''<br />
<br />
Received parts from Lausanne:<br />
*BBa_191006 (113 ng/µL) – LovTAP alone<br />
*BBa_191004 (166 ng/µL) – Readout <br />
*BBa_191003 (230 ng/µL) – whole system<br />
*BBa_191009 (85 ng/µL) – mutant <br />
*Sosnick vector containing TrpR (393 ng/µL) – vector <br />
<br />
<br />
<br />
'''16/07/10'''<br />
<br />
Transformations using 100 µL of cells, 900 µL broth<br />
*K191004 - amp<br />
*K191003 - kanamycin<br />
*K191009 – kanamycin<br />
*Sosnick vector with TrpR – amp<br />
<br />
Note: Labeling on tubes is unclear – K191009 and K191004 got mixed up, so plating both tubes on both Kan and amp<br />
<br />
<br />
'''22/07/10'''<br />
<br />
*Minipreps: K0998010 – 5x (1 didn’t grow, but did anyway)<br />
*Blue sensors (3 biobricks and Sosnick vector) – cultures set to grow overnight<br />
<br />
'''25/07/10'''<br />
Digest of the blue light sensor minipreps was performed with EcoRI and BamHI; samples:<br />
*3- K19100'''4'''; 900ul, (3)<br />
*4- K19100'''4'''; 100ul, (2)<br />
*5-K19100'''9'''; 100ul, (1)<br />
*6- K19100'''9'''; 100ul, (6)<br />
*7- K19100'''9'''; 900ul, (4)<br />
*8- K19100'''9'''; 100ul, (2)<br />
*9- K19100'''3'''; 900ul, (2)<br />
*10- K19100'''3'''; 900ul, (5)<br />
*11- K19100'''3'''; 100ul, (4)<br />
*12- K19100'''3'''; 100ul, (1)<br />
*13- K19100'''4'''; 100ul, (3)<br />
*14- K19100'''4'''; 900ul, (5)<br />
<br />
Lanes- 1- marker, 2-9 sampels as in order, 10-empty, 11, 12- marker instead of the samples- need to run last two again<br />
<br />
[[Image:109.JPG]]<br />
<br />
[[Image:111.JPG]]<br />
LAST TWO LANES- NSAMPLES NO 13 AND 14 FROM THE ABOVE LIST<br />
<br />
<br />
'''28/07/10'''<br />
<br />
Double transformants made using a novel cowboy procedure- double transformation with 4ul of each piece od DNA. Chances of succes- very very low... But at least it will be proved that it won't work!<br />
Trans. 1: BBa_K191004+ K191003- read out + LovTap<br />
Trans. 2: BBa_K191004+ K191009- read out + mutated LovTap<br />
<br />
'''30/7/10'''<br />
set up experiments to see if the blue light sensor will activate the response system under the daylight. Both liquid cultures and plates were set up, with red and white colonies- first supposed to turn white in the daylight and stay red in the dark (under the foil), second ones- no change of the colour. The same applies to the liquid cultures. However, the experiment failed- no proper growth was observed, and if there was a growth - it was very weak. No pigment observed, apart from the small points in the centre. <br />
Hypothesis- not stable plasmids-- cells look like they are not really resistant. Recorded growth:<br />
{|border="1"<br />
|<br />
|K191004+9 control<br />
|K191004+9<br />
|K191004+3 control<br />
|K191004+3<br />
|-<br />
|DARK<br />
| ++ , white<br />
| +++ , white<br />
| ++ , white<br />
| +++ , white with red points<br />
|-<br />
| LIGHT<br />
| + , white<br />
| +++ , white with red point<br />
| + -almost no growth <br />
| ++ white<br />
|}<br />
<br />
<br />
<br />
'''2/8/10'''<br />
<br />
Run the digest of the minipreps from the white and red cultures of double transformants. No proper DNA band in the solution- repeat the procedure or not? Don't know what to do.... again. <br />
[[File:Gel 3.jpg]]<br />
Collect the thoughts and ideas and start again tomorrow!<br />
<br />
'''6/8/10'''<br />
<br />
double transformants were prepared again, using non-cowboy procedure. 04 cells were first MADE COMPETENT, then transformed with 03 and 09, also 1 neg control was plated. Grow white on the intitial plate, then turned red if subcultured. <br />
<br />
'''7-8/08'''<br />
<br />
'Subbed these to duplicate fresh plates and incubated overnight at room temperature <br />
either under a fluorescent lamp or wrapped in foil - all white in both cases (and growth <br />
not great, probably because of low temperature)'. <br />
The plates were moved to 37C with the lamp /one under the light, one in the dark. so far -no results, checking in an hour/. <br />
<br />
Another idea- check for stability of the insert- we don't know if the plasmids are compatibile. Streaking from original plates to check if single colonies will be a mixture of white and red or just red- origin? no plasmid maps from Lausanne. <br />
by Chairman-->' If we can't find this information, I think our best course is to <br />
PCR out both inserts and combine them in a single vector, preferably pSB1C3.'<br />
<br />
<br />
'''9/8/2010'''<br />
<br />
to do:<br />
<br />
*primers for the parts to put them in a vector<br />
*check the lamp every hour!!<br />
<br />
go thruough Lausanne website to find the plasmid maps<br />
get back to Mexico about the parts and etc.<br />
find out the conditions for the blue light sensor assay!<br />
<br />
'''10/08/10'''<br />
* assay with dark/light liquid cultures - fluorescence measurment and OD, after 5 h in the shaker under lamp- see video and pictures<br />
*email to Mexico<br />
<br />
'''11/08/10'''<br />
<br />
*primer designed for combining LOVTap and Readout1 into 1 cell<br />
*repeated assay with the smae samples, after overngiht in RT and %5h under lamp (for the light samples) and RT without shaking<br />
*primary cultures streaked to amp/kan/IPTG plates- part 4+9 and 4+3 -three clones each<br />
<br />
'''12/08/10'''<br />
*results analysis after LOVTap experiment:check file, add graphs later.<br />
*email from Mexico about Blue promoter<br />
<br />
'''16/08/10'''<br />
* streaked single clone for 9+4 and 3+4 (clones number 5 and 2, respectively)- for tomorrow's experiment<br />
* minipreped mysterious double transformants and Sacb smples<br />
* tried out the box on the shaker- unscrewed the things. <br />
* made the graphs from the second reading<br />
* 03 and 09 LovTap taken for sequencing. <br />
<br />
to do: decide on the illumination length and think about controls and how many samples do you want to put. check Lausanne website<br />
t-test to check to importancy of the differences between the means.<br />
<br />
'''17/07/10'''<br />
* desinged experimental set up for the LovTap blue light response experiment /see next day/<br />
* BAD NEWS: there is a suspicion of 1bp deletion in LovTap!!! Chairman noticed it when he was checking the parts base by base. Should have done it much much earlier probably to save the time... <br />
* BAD NEWS: lack of the proper thinking put in doubt all the results from previous two experiments (that is why you can't find any files of it attached)- there was no IPTG added to the samples, therefore LOVTap expression was not induced- no point to do T-test right now... :( Well, at least I will learn from it and add it next time!<br />
<br />
<br />
'''18/08/10'''<br />
<br />
* tested LovTap under the blue light<br />
* conditions:<br />
Liquid cultures (same as for minipreps) were grown overnight in 37C with antibiotics. <br />
The next day, bottles with 3.5 ml of broth were inoculated with the 0.5 ml of appropriate cultures (0.5. ml of broth added as a control).<br />
*sample designation: (# refers to the clone number form the original plate). Original plates are the ones made on '''INSERT THE DATE LAZY''' and have 2 clones on each plates (clones1-3 are K191003&4 double transformants, clones 4-6 are K191009+4 double transformants). <br />
<br />
# K191003+4 (#1) + Amp + Kan + IPTG + LIGHT <br />
# K191003+4 (#1) + Amp + Kan IPTG + DARK <br />
# K191003+4 (#2) + Amp + Kan IPTG + LIGHT <br />
# K191003+4 (#2) + Amp + Kan IPTG + DARK <br />
# K191009+4 (#4) + Amp + Kan IPTG +LIGHT <br />
# K191009+4 (#4) + Amp + Kan IPTG +DARK <br />
# K191009+4 (#5) + Amp + Kan IPTG +LIGHT <br />
# K191009+4 (#5) + Amp + Kan IPTG +DARK <br />
# K191004 ONLY #2+ Amp + LIGHT <br />
# K191004 ONLY #2+ Amp + DARK <br />
# neg. control- broth, Amp + Kan + IPTG + LIGHT<br />
<br />
* samples designated DARK were wrapped in the aluminium foil straight after the inoculation; the cap was not wrapped (not see through anyway)so that samples could be easily taken without unwrapping the bottles from the foil. <br />
*All the samples were then taken to the shaker in the hot room and placed in prepared spots: for the dark- anywhere on an easily accessible holder, for light- in 2 rows of holders. <br />
*Light samples were covered with the cardboard box with blue LEDs attached on the sides, in a way to optimize light accession to the samples (ask Sarah for the details). Box was securely taped to the shaker. <br />
* Measurments were taken every half an hour: 200ul of the samples were placed in the numbered cuvette and then 800ul of water was added. Readings of fluroscence (green mode) were taken in standard fluorescence units (SFU, two readings each). Then optical density was measured (upstairs in the incubator room) at 360 nm wavelength. Control was used to calibrate the machine (set ref button). Also 2 readings were taken. Samples were shaken by inversion prior to taking measurment. <br />
* Experiment was stopped after 3 hours (6 sets of reading taken). <br />
<br />
*'''Result analysis'''<br />
<br />
[[Image:Lt18aug.JPG]]<br />
<br />
To generate the data for the graph:<br />
# means were calculated from 2 readings of optical density and fluorescence<br />
# (mean SFU minus background SFU) was divided by (mean OD minus background OD, if different from 0).<br />
# table was made for the samples at different points of time- e.g. sample 1 after 30 min, 60 min, 90 min, 120 min etc. <br />
# graph was made with series named the same as the samples<br />
<br />
[[File:Untitled.JPG]]<br />
<br />
**BAD BAD VERY BAD NEWS: after 2 hours of measurments the results of the sequencing were know and revealed that THERE IS A 1BP DELETION IN LOVTAP 03 AND 09 WHICH RESULTS IN FRAMESHIFT AND LACK OF THE FUNCTIONAL PRODUCT!!!! :(:(:( despite this information, it was decided that the experiment will be finished and analysed as initially planned.<br />
<br />
'''19/08/2010'''<br />
<br />
* Pcr- mutagenensis of LovTap- Mabel, adding missing bp. <br />
* Puryfing PCR product from the solution. <br />
<br />
<br />
'''20/8/2010'''<br />
* PCR run on the gel -correct band of ~5.5. kb size, suggest that the PCR worked well. couple of other fainter bands <br />
* Self -ligation overnight (MABEL self ligation- 12ul of H2O, 4ul of DNA, 2 ul of Ligase buffer, 1 ul of T4 DNA ligase, 1ul of T4 polynucleotide kinase. <br />
* transformation and setting up the liquid cultures for the minipreps over the weekend. <br />
<br />
[[Image:123.JPG]]<br />
<br />
'''23/08/2010'''<br />
* mini-prep DNA from the colonies after PCR- marker worn off in the shaker,so 3.2. and 9.1. got mixed up- probably sequencing necessary to distinguish the mutated version from non-mutated <br />
* digest minipreps with EcoRI, run the gel[photo]<br />
* run 5ul of the ligated 03 and 09 on the gel /also Richards samples/.<br />
<br />
[[Image:124.JPG]]<br />
<br />
'''24/08/2010'''<br />
* repeated digest of mutated LovTap samples: 3.1, 3.?, 3.3., 3.4 9.?, 9.2 with EcoRi<br />
* minipreped 6 more clones of mutated LovTap 03 and 09 from the same plate, labelled- 3.6, 3.7, 3.8, 9.4, 9.5, 9.6. Run the digest with EcoRI. Note: 3.7 sample is higly diluted as 80ul of EB was used instead of 40ul. oops. <br />
* delivered mutated 3.? and 9.2 minipreped clones for sequencing /only 2 reactions: each sample- forward/.<br />
<br />
[[Image:LT100.jpg]]<br />
<br />
'''04/10/10'''<br />
We may have a working LovTap construct + red/white (RFP) reporter system thanks to Mexico.<br />
<br />
<br />
Minor characterisation experiment:<br><br />
-LovTap constructed with red-white RFP reporter<br><br />
-If activated, the blue light sensor transformants should produce red colonies<br><br />
-Blue light sensor containing-colonies from transformant plate streaked onto two plates<br><br />
-One plate left in the light, the other left in the dark<br><br />
-If the one in the light grows red and the one in the dark doesn't, it is a good indication that the construct is working<br><br><br />
<br />
'''8/10/2010'''<br />
* repetaed LOVTap charactierzation- with TrpR mutant and WT under light/dark conidtion. <br />
* no clear response was seen. <br />
<br />
[[Image:8octLovtap.jpg]]<br />
<br />
[[Image:8octTRPR.jpg]]<br />
<br />
'''22/10/2010'''<br />
* checking temperature response of LOVTap<br />
* setting up samples with CMM minum medium, enriched in thymine, trace elements and glucose.<br />
CELLS FAIL TO GROW!:(</div>Macbereskahttp://2010.igem.org/Team:Edinburgh/Notebook/Red_light_sensorTeam:Edinburgh/Notebook/Red light sensor2010-10-27T12:12:05Z<p>Macbereska: /* Red Light Sensor */</p>
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<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Core_repressilator">the repressilator</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_producer">red light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_sensor">red sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_producer">blue light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Green_light_producer">green light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Green_light_sensor">green sensor</a></li><br />
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<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/References">references</a></li><br />
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<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling" class="dir">modelling BRIDGEs</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Kappa">kappa</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Genomic">the genomic model</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Bacterial">the bacterial model</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Signalling">the signalling model</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Tools">tools</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Modelling">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/References">references</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human" class="dir">human BRIDGEs</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Aspects">human aspects</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Communication">communication of science</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Terminology">terminology research</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Wiki">wiki</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Conversations">conversations</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Identity">identity</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Collaboration">collaboration</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Branding">branding research</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/SciFi">science fiction writing</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/FutureApps">future applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/SelfReflection">self-reflection</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Epic">the epic</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Human">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/References">references</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook" class="dir">lab notes&nbsp;&nbsp;&nbsp;</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Collaboration">collaboration</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Attribution">attribution</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/BRIDGE">BRIDGE</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_producer">red light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_sensor">red sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_producer">blue light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_producer">green light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_sensor">green sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Modelling">modelling</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Safety">safety</a></li><br />
<li><a href="http://www.openwetware.org/wiki/French_Lab">protocols</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Acknowledgements" class="dir">acknowledgements</a><br />
</li><br />
<br />
</ul><br />
</div><br />
<br />
<br><br />
<br><br />
<br><br />
<br />
<div id="windowbox" style="border: .2em solid #660000; padding: 5px; position:fixed; top:50%; right:30px; width:8%;"><br />
<span style="color:ivory;">These <b>notes</b> correspond to the part of the <b>project</b> detailed <a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_sensor">here</a>.</span><br />
<br />
</div><br />
<br />
</html><br />
<br />
== Red Light Sensor ==<br />
<br><br />
<br />
'''22/6/10'''<br />
<br />
Transformation of BBa_M30109; well 14 H, distribution plate 3 (2010)--> red light sensor <br />
<br />
'''24/6/10'''<br />
<br />
Subcultured transformants from ampicillin plates to new plates<br />
<br />
<br />
'''25/6/10'''<br />
<br />
1) DNA mini-preps were prepared from the transformants<br />
2) DNA mini-preps were cut with EcoRI enzyme to check for the presence of the plasmid (and DNA); agarose gel was prepared for DNA electrophoresis after cutting.<br />
3) Results from cutting showed no presence of the sensor.<br />
Troubleshooting: was the plate number correct? Was procedure performed correct?<br />
by:Maria, Will, Marta <br />
<br />
[[Image:1001.JPG]]<br />
<br />
'''28/6/10'''<br />
<br />
Tried to determine what was wrong with the red sensor that failed to appear in the gel. We definitely had the correct plate and well. We checked the seqeunce of the part and found that the sequence given does not match any given in BLAST. We suspect either that part of the plasmid was added to the distribution/registry or that the plasmid contains an illegal EcoR1 site, but are re-transforming cells just in case there was an experimental error.<br />
Due to failure of the previous transformation competent cells were retransformed with the two plasmids, as before. Two Chloramphenicol spread plates for each plasmid were made one of each pair containing 100micro-l of cells the other containing the 900micro-l remainder spun down and resuspended in 100micro-l of sterile LB.<br />
by: Richard, Lu<br />
<br />
<br />
'''29/6/10'''<br />
<br />
Streaking the colonies from the chloramphenicol plates on the new agar + chloramphenicol plates.<br />
<br />
<br />
'''30/6/10'''<br />
<br />
No growth on plates.<br />
<br />
<br />
'''5/7/10'''<br />
<br />
Made kanomycin 32mg/ml, IPTG 90mg/ml plates. Transformed cells with individual red light sensor parts (BBa_I15008 - 2010 distribution Plate 2 Well 13J; BBa-I15009 - distribution plate 1 Well 20F; BBa_I15010 - distribution Plate 3 Well 20D; & K098010 - distribution Plate 3 11N). Plated cells onto kanomycin plates and left to incubate overnight. <br />
by: Hannah and Richard<br />
<br />
<br />
'''6/7/10'''<br />
<br />
Richard/Hannah - made subcultures of red light sensor part transformants.<br />
<br />
*Agarose Gel Eleckrophoresis of composite red sensor M30109<br />
*The bands are not the right size<br />
*Sequencing done by the registry starts in the middle of the sequence (for forward sequence)<br />
*So it seems the plasmid doesn’t contain the right sequence<br />
*We will try to assemble it from individual parts<br />
<br />
[[Image:149.JPG]]<br />
<br />
'''8/7/10'''<br />
<br />
Plasmid minipreps of I15009 (the only red sensor part transformant that grew in liquid culture)<br />
by: Richard and Hannah<br />
<br />
<br />
'''9/7/10'''<br />
<br />
Gel run with I15009. <br />
<br />
{| border="1"<br />
| Lane 1 <br />
| Lane 2 <br />
| Lane 3 <br />
| Lane 4 <br />
| Lane 5 <br />
| Lane 6 <br />
| Lane 7 <br />
| Lane 8<br />
|-<br />
| Ladder <br />
| Pure SacB <br />
| Pure SacB & EcoRI <br />
| sacB & catR PCR <br />
| I15009 digest <br />
| S248T PCR Product <br />
| Purified 356K <br />
| Purified 356R<br />
|}<br />
<br />
*Insert Picture of Gel*<br />
<br />
'''13/7/2010'''<br />
NOTHING WORKS. No idea why...<br />
<br />
'''16/7/10'''<br />
<br />
Because the red light sensing parts were transformed with 50 µL or water instead of 15 µL during the first transformation, another attempted was tried.<br />
Transforming:<br />
*I15008 (all red light sensor parts)<br />
*I15009<br />
*I15010<br />
used 200 µL of cells, 800 µL of broth. Kan resistance.<br />
<br />
<br />
'''23/07/10'''<br />
<br />
Double digest of K098010. Ran gel. Lane was empty:<br />
<br />
[[Image:153.JPG]]<br />
*Not going to bother saying what is in each lane. <br />
<br />
'''30/7/10'''<br />
<br />
Primers were ordered in order to PCR out the Red Light Sensor Parts from the [http://partsregistry.org/wiki/index.php?title=Part:BBa_M30109 Bio Brick BBa_M30109]<br />
<br />
Forward Primer for PBad promoter [http://partsregistry.org/wiki/index.php?title=Part:BBa_I13453 Bio Brick BBa_I13453]<br><br />
Reverse Primer for Heme Oxygenase (ho1) [http://partsregistry.org/wiki/index.php?title=Part:BBa_I15008 Bio Brick BBa_I15008]<br><br />
Reverse Primer for Phycocyanobilin:Ferredoxin Oxidoreductase (PcyA) [http://partsregistry.org/wiki/index.php?title=Part:BBa_I15009 Bio Brick BBa_I15009]<br><br />
Forward Primer for TetR repressible promoter [http://partsregistry.org/wiki/index.php?title=Part:BBa_R0040 Bio Brick BBa_R0040]<br><br />
Reverse Primer for cph8 (Cph1/EnvZ fusion)Chimeric Cph1 light receptor/EnvZ protein [http://partsregistry.org/wiki/index.php?title=Part:BBa_I15010 Bio Brick BBa_I15010]<br><br />
<br />
<br />
'''3/8/10'''<br />
<br />
A PCR was run with the above primers in 3 distinct mixtures in an attempt to amplify out the Red Light Sensor parts using Bio Brick BBa_M30109 as a template.<br />
PCR 1 Contained The PBad promoter Forward Primer & the PcyA Reverse Primer<br><br />
PCR 2 Contained The TetR repressible promoter Forward Primer & the cph8 Reverse Primer <br><br />
PCR 3 Contained The PBad promoter Forward Primer & the cph8 Reverse Primer<br />
<br />
And a gel was run of the PCR products: Marker(Lane 1&6), only visible product- suggested PCR I15010 with RBS & promoter (Lane 3). <br />
[[Image:116.JPG]]<br />
<br />
Due to the successful amplification of the I15010 cph8 Red light sensor part, the DNA was ligated into plasmid psb1C3.<br />
<br />
<br />
'''4/8/10'''<br />
<br />
Competent cells were transformed with the ligated psb1C3 plasmid containing I15010 and plated out on Cml 40 + IPTG 90 plates and incubated O/N at 37 degrees Celsius.<br />
<br />
A PCR was run using the following primers:<br />
PCR 1 Contained The BBa_B0034 RBS Forward Primer & the ho1 Reverse Primer<br><br />
PCR 2 Contained The BBa_B0034 RBS Forward Primer & the PcyA Reverse Primer<br><br />
PCR 3 Contained The PBad promoter Forward Primer & the ho1 Reverse Primer<br><br />
PCR 4 Contained The PBad promoter Forward Primer & the PcyA Reverse Primer<br><br />
<br />
And a gel was run of the PCR products.<br />
<br />
*Insert picture of Gel*<br />
<br />
No successful amplification was seen.<br />
<br />
'''11/8/10'''<br />
<br />
Update from Maria. <br />
The transformations and PCR amplification of the HO1-pcyA fusion construct have nearly all failed. <br />
This weekend the kanamycin resistant version of the construct (K089010 in pSB3K3) was transformed and cultures grew.<br />
We already have a PCR product of the fused cph8-envZ sensing part. We now need to put the newly transformed K089010 in front of the sensor in order to rebuild the original sensor, which is well and truly fucked.<br />
<br />
[[Image:161.jpg]]<br />
<br />
'''16/8/10'''<br />
<br />
Successful PCR of K098010 using primers for EcoRI & I15009 reverse primer.<br />
[[Image:119.JPG]]<br />
<br />
<br />
'''17/8/10'''<br />
<br />
* The PCR product of the Harvard K098010 part was purified and then digested with both XbaI & SpeI as was the PCR product of cph8.<br />
* These digestions were then ligated together ON using in two orientations.<br />
<br />
'''18 &19/8/2010'''<br />
<br />
* Fusion PCR C + H with R004081 & I15009-1 <br />
* also running H + C same primers as negative control<br />
* run gel of PCR prod & 5 ul of ligation<br />
* luxAB -SpeI; lumP -XbaI, ;uxCDE -XbaI<br />
* lumP = P1024<br />
* luxAB + lumP and luxAB + lyxCDE ligation<br />
[[Image:120.JPG]]<br />
<br />
'''20/8/2010'''<br />
<br />
* Fusion PCR with ?????<br />
* get: <br />
<br />
'''24/8/2010'''<br />
* Digest cph8 (XbaI- yello) & Harvard (SpeI -brown)<br />
* Ligate Harvard and cph8 = harvard + cph*8<br />
* mutagenic PCR of lux CDE to mutate out site in LuxD<br />
* subculture luxAB lumP transformants<br />
* miniprep ON'S '''(?)''' set up<br />
<br />
<br />
'''25/8/2010'''<br />
<br />
* Minipreps of subcultures luxAB lumP,<br />
* digest & run gel<br />
* Purify luxCDE PCR product- from gel and froem the product; run the gel of purifications --> do MABEL self ligation- set 16C ON<br />
* Fusion PCR harv + cph8- run gel of product<br />
<br />
'''26/8/2010'''<br />
<br />
* Gel1 - minipreps 1-5, gluxCDE, PluxCDE<br />
* Gel2 - Minipreps 6-10, H + C ligation<br />
<br />
*Lane 7: Harvard + Cph8 ligation<br />
[[Image:129.JPG]]<br />
<br />
'''27/8/2010'''<br />
<br />
Transform cells with self ligated MABEL mutated luxCDE from gel and PCR product<br />
<br />
<br />
'''30/8/2010'''<br />
<br />
* Lu wants a broad sward (?).<br />
* Re-transform MABEL mutated<br />
* luxCDE from gel- PCR products<br />
* luxAB and luxABlumP double digest with EcoRi/PstI<br />
* Fusion PCR if H + cph8 using H promoter forward and cph8 reverse primer<br />
<br />
'''31/8/2010'''<br />
* Lane 1- marker, Lane 2- H+ Cph8 fPCR<br />
[[Image:133.JPG]]<br />
'''6/9/2010'''<br />
<br />
* ligated H+ C ON in psB1C3<br />
* Double digest luxCDE with EcoRI and PstI - run gel<br />
<br />
'''7/9/2010'''<br />
* Run digest luxCDE with XbaI --> gel<br />
* Gel of H + C ligation<br />
* transform cells with<br />
<br />
'''8/9/2010'''<br />
<br />
* PCR with biobrick pre/suff - primers luxCDE 1 and 4<br />
<br />
'''9/9/2010'''<br />
<br />
* Purify luxCDE PCR<br />
* Subculture transformants H + C<br />
<br />
'''10/9/2010'''<br />
<br />
* Add promoter to luxAB and luxABlumP<br />
* Liquid ON'S of H + C subculture transformants <br />
<br />
* Lab meeting: small--> <br />
**sacB - some preeliminary sucrose sensitivity characterisation data.<br />
** promoter: luxAB, luxAb-lumP- eission spectra, testing thing<br />
** red light luciferase: S184T, 356 K<br />
[[Image:143.JPG]]<br />
{| border="1"<br />
| Lane 1 <br />
| Lane 2 <br />
| Lane 3 <br />
| Lane 4<br />
|-<br />
| Ladder <br />
| Tna up<br />
| Cph8- fixing prefix <br />
| Cph8 - fixing PstI site <br />
|}<br />
<br />
'''13/9/2010'''<br />
* H + C + pSB1C3 minipreps<br />
[[Image:144.JPG]]<br />
<br />
'''14/9/2010'''<br />
* Digest H+ C minipreps with EcoRi. 100: 1,4,5. 900: 3,4,6<br />
[[Image:145.JPG]] <br />
<br />
'''???'''<br />
'''20/9/2010'''<br />
<br />
* Test luxCDE<br />
* luxCDE digest with XBaI and PstI (buffer H)--> purify and ligate to luxAB digested with SPEI PstI in buffer B after purification.<br />
* conclusion: purify digestions, ligate luxCDE into luxAB + promoter<br />
<br />
<br />
'''21/9/2010'''<br />
<br />
* Transform cells with luxAB + luxCDE ligation, plated on CML 40, IPTG 90<br />
* Made up 50 ml liquid cultures of luxAB lumP and luxAB with Cml40, IPTG 90<br />
<br />
<br><br><br />
<b>04/10/10</b><br />
<br><br />
Chris has managed to construct what we hope is a working red light sensor. It is being sent in for sequencing today. <br><br />
Minor characterisation experiment:<br><br />
-Red light sensor constructed with blue-white lacZ reporter<br><br />
-If activated, the red light sensor transformants should produce blue colonies<br><br />
-Red light sensor containing-colonies from transformant plate streaked onto two plates<br><br />
-One plate left in the light, the other left in the dark<br><br />
-If the one in the light grows blue and the one in the dark doesn't, it is a good indication that the construct is working<br><br><br />
<br />
'''12/10/10'''<br />
<br />
*Transform ''envZ'' mutant strain with YFP<br />
*Plate out onto 40mg/ml Cml<br />
<br />
'''13/10/10'''<br />
<br />
*Subcultured ''envZ'' transformants onto Cml 40 plates<br />
<br />
'''15/10/10'''<br />
<br />
*Liquid Cultures made of transformants with Cml 40 & grown ON<br />
<br />
'''16/10/10'''<br />
<br />
*Liquid ONs viewed under blue light, yellow fluorescence seen showing successful transformation.<br />
<br />
'''20/10/10'''<br />
<br />
*RLS characterisation begins.<br />
*Liquid ON's of<br />
<ul><br />
<li>JM109 – RLS.lacZ.YFP</li><br />
<li>envZ – RLS.lacZ.YFP</li><br />
<li>JM109 – YFP Control</li><br />
<li>envZ – YFP Control</li><br />
</ul><br />
were made in 5ml LB + Cml 40 and grown at 37 degrees with shaking in the dark.<br />
<br />
<p>100μl of ONs were used to inoculate two sterile LB + 40mg/ml Cml making up a total volume of 4ml in 5ml flasks which were then incubated in both light and dark, at 37C with shaking, as follows:</p><br />
<br />
<ul><br />
<li>JM109 – RLS.lacZ.YFP (Light / Dark)</li><br />
<li>envZ – RLS.lacZ.YFP (Light / Dark)</li><br />
<li>JM109 – YFP Control (Light / Dark)</li><br />
<li>envZ – YFP Control (Light / Dark)</li><br />
</ul><br />
<br />
<p>At 50 minute time intervals 200μl of each sample was taken and mixed with 800μl of sterile water in plastic cuvettes. The optical density of each sample was taken after the spectrophotometer was set with a sample of 200μl of sterile LB and 800μl water as a control, as was the luminescence, with readings for the background luminescence being taken for the sample of LB and water.</p><br />
<br />
<p>Two readings for each sample at each time interval were taken and then an average was calculated for each time interval for both optical density and luminescence.</p><br />
<br />
<p>For each sample at each time interval the average luminescence (normalised by the background luminescence) was divided by the optical density and plotted on a graph, shown below as Figure 2.</p><br />
<br />
<br />
[[Image:Ed10-RedLightSensorCharData.png]]<br />
<br />
* PCR of PhoA promoter (from genomic DNA).<br />
<br />
[[Image:171.jpg]]<br />
<br />
'''25/10/10'''<br />
<br />
*Liquid ON's made up of <br />
<li>JM109 – RLS.lacZ.YFP</li><br />
<li>envZ – RLS.lacZ.YFP</li><br />
<li>JM109 – RLS.lacZ</li><br />
<li>JM109 - lacZ Control</li><br />
in LB + 40 Cml</div>Macbereskahttp://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_sensorTeam:Edinburgh/Notebook/Blue light sensor2010-10-27T12:04:18Z<p>Macbereska: /* Blue Light Sensor */</p>
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<li><a href="https://2010.igem.org/Team:Edinburgh/BioBricks#Bacterial">submitted parts</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Bacterial">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/References">references</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling" class="dir">modelling BRIDGEs</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Kappa">kappa</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Genomic">the genomic model</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Bacterial">the bacterial model</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Signalling">the signalling model</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Tools">tools</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Modelling">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/References">references</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human" class="dir">human BRIDGEs</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Aspects">human aspects</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Communication">communication of science</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Terminology">terminology research</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Wiki">wiki</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Conversations">conversations</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Identity">identity</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Collaboration">collaboration</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Branding">branding research</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/SciFi">science fiction writing</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/FutureApps">future applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/SelfReflection">self-reflection</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Epic">the epic</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Human">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/References">references</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook" class="dir">lab notes&nbsp;&nbsp;&nbsp;</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Collaboration">collaboration</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Attribution">attribution</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/BRIDGE">BRIDGE</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_producer">red light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_sensor">red sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_producer">blue light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_producer">green light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_sensor">green sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Modelling">modelling</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Safety">safety</a></li><br />
<li><a href="http://www.openwetware.org/wiki/French_Lab">protocols</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Acknowledgements" class="dir">acknowledgements</a><br />
</li><br />
<br />
</ul><br />
</div><br />
<br />
<br><br />
<br><br />
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<span style="color:ivory;">These <b>notes</b> correspond to the part of the <b>project</b> detailed <a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_sensor">here</a>.</span><br />
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<br />
==Blue Light Sensor==<br />
<br><br />
<br />
'''14/07/10'''<br />
<br />
Received parts from Lausanne:<br />
*BBa_191006 (113 ng/µL) – LovTAP alone<br />
*BBa_191004 (166 ng/µL) – Readout <br />
*BBa_191003 (230 ng/µL) – whole system<br />
*BBa_191009 (85 ng/µL) – mutant <br />
*Sosnick vector containing TrpR (393 ng/µL) – vector <br />
<br />
<br />
<br />
'''16/07/10'''<br />
<br />
Transformations using 100 µL of cells, 900 µL broth<br />
*K191004 - amp<br />
*K191003 - kanamycin<br />
*K191009 – kanamycin<br />
*Sosnick vector with TrpR – amp<br />
<br />
Note: Labeling on tubes is unclear – K191009 and K191004 got mixed up, so plating both tubes on both Kan and amp<br />
<br />
<br />
'''22/07/10'''<br />
<br />
*Minipreps: K0998010 – 5x (1 didn’t grow, but did anyway)<br />
*Blue sensors (3 biobricks and Sosnick vector) – cultures set to grow overnight<br />
<br />
'''25/07/10'''<br />
Digest of the blue light sensor minipreps was performed with EcoRI and BamHI; samples:<br />
*3- K19100'''4'''; 900ul, (3)<br />
*4- K19100'''4'''; 100ul, (2)<br />
*5-K19100'''9'''; 100ul, (1)<br />
*6- K19100'''9'''; 100ul, (6)<br />
*7- K19100'''9'''; 900ul, (4)<br />
*8- K19100'''9'''; 100ul, (2)<br />
*9- K19100'''3'''; 900ul, (2)<br />
*10- K19100'''3'''; 900ul, (5)<br />
*11- K19100'''3'''; 100ul, (4)<br />
*12- K19100'''3'''; 100ul, (1)<br />
*13- K19100'''4'''; 100ul, (3)<br />
*14- K19100'''4'''; 900ul, (5)<br />
<br />
Lanes- 1- marker, 2-9 sampels as in order, 10-empty, 11, 12- marker instead of the samples- need to run last two again<br />
<br />
[[Image:109.JPG]]<br />
<br />
[[Image:111.JPG]]<br />
LAST TWO LANES- NSAMPLES NO 13 AND 14 FROM THE ABOVE LIST<br />
<br />
<br />
'''28/07/10'''<br />
<br />
Double transformants made using a novel cowboy procedure- double transformation with 4ul of each piece od DNA. Chances of succes- very very low... But at least it will be proved that it won't work!<br />
Trans. 1: BBa_K191004+ K191003- read out + LovTap<br />
Trans. 2: BBa_K191004+ K191009- read out + mutated LovTap<br />
<br />
'''30/7/10'''<br />
set up experiments to see if the blue light sensor will activate the response system under the daylight. Both liquid cultures and plates were set up, with red and white colonies- first supposed to turn white in the daylight and stay red in the dark (under the foil), second ones- no change of the colour. The same applies to the liquid cultures. However, the experiment failed- no proper growth was observed, and if there was a growth - it was very weak. No pigment observed, apart from the small points in the centre. <br />
Hypothesis- not stable plasmids-- cells look like they are not really resistant. Recorded growth:<br />
{|border="1"<br />
|<br />
|K191004+9 control<br />
|K191004+9<br />
|K191004+3 control<br />
|K191004+3<br />
|-<br />
|DARK<br />
| ++ , white<br />
| +++ , white<br />
| ++ , white<br />
| +++ , white with red points<br />
|-<br />
| LIGHT<br />
| + , white<br />
| +++ , white with red point<br />
| + -almost no growth <br />
| ++ white<br />
|}<br />
<br />
<br />
<br />
'''2/8/10'''<br />
<br />
Run the digest of the minipreps from the white and red cultures of double transformants. No proper DNA band in the solution- repeat the procedure or not? Don't know what to do.... again. <br />
[[File:Gel 3.jpg]]<br />
Collect the thoughts and ideas and start again tomorrow!<br />
<br />
'''6/8/10'''<br />
<br />
double transformants were prepared again, using non-cowboy procedure. 04 cells were first MADE COMPETENT, then transformed with 03 and 09, also 1 neg control was plated. Grow white on the intitial plate, then turned red if subcultured. <br />
<br />
'''7-8/08'''<br />
<br />
'Subbed these to duplicate fresh plates and incubated overnight at room temperature <br />
either under a fluorescent lamp or wrapped in foil - all white in both cases (and growth <br />
not great, probably because of low temperature)'. <br />
The plates were moved to 37C with the lamp /one under the light, one in the dark. so far -no results, checking in an hour/. <br />
<br />
Another idea- check for stability of the insert- we don't know if the plasmids are compatibile. Streaking from original plates to check if single colonies will be a mixture of white and red or just red- origin? no plasmid maps from Lausanne. <br />
by Chairman-->' If we can't find this information, I think our best course is to <br />
PCR out both inserts and combine them in a single vector, preferably pSB1C3.'<br />
<br />
<br />
'''9/8/2010'''<br />
<br />
to do:<br />
<br />
*primers for the parts to put them in a vector<br />
*check the lamp every hour!!<br />
<br />
go thruough Lausanne website to find the plasmid maps<br />
get back to Mexico about the parts and etc.<br />
find out the conditions for the blue light sensor assay!<br />
<br />
'''10/08/10'''<br />
* assay with dark/light liquid cultures - fluorescence measurment and OD, after 5 h in the shaker under lamp- see video and pictures<br />
*email to Mexico<br />
<br />
'''11/08/10'''<br />
<br />
*primer designed for combining LOVTap and Readout1 into 1 cell<br />
*repeated assay with the smae samples, after overngiht in RT and %5h under lamp (for the light samples) and RT without shaking<br />
*primary cultures streaked to amp/kan/IPTG plates- part 4+9 and 4+3 -three clones each<br />
<br />
'''12/08/10'''<br />
*results analysis after LOVTap experiment:check file, add graphs later.<br />
*email from Mexico about Blue promoter<br />
<br />
'''16/08/10'''<br />
* streaked single clone for 9+4 and 3+4 (clones number 5 and 2, respectively)- for tomorrow's experiment<br />
* minipreped mysterious double transformants and Sacb smples<br />
* tried out the box on the shaker- unscrewed the things. <br />
* made the graphs from the second reading<br />
* 03 and 09 LovTap taken for sequencing. <br />
<br />
to do: decide on the illumination length and think about controls and how many samples do you want to put. check Lausanne website<br />
t-test to check to importancy of the differences between the means.<br />
<br />
'''17/07/10'''<br />
* desinged experimental set up for the LovTap blue light response experiment /see next day/<br />
* BAD NEWS: there is a suspicion of 1bp deletion in LovTap!!! Chairman noticed it when he was checking the parts base by base. Should have done it much much earlier probably to save the time... <br />
* BAD NEWS: lack of the proper thinking put in doubt all the results from previous two experiments (that is why you can't find any files of it attached)- there was no IPTG added to the samples, therefore LOVTap expression was not induced- no point to do T-test right now... :( Well, at least I will learn from it and add it next time!<br />
<br />
<br />
'''18/08/10'''<br />
<br />
* tested LovTap under the blue light<br />
* conditions:<br />
Liquid cultures (same as for minipreps) were grown overnight in 37C with antibiotics. <br />
The next day, bottles with 3.5 ml of broth were inoculated with the 0.5 ml of appropriate cultures (0.5. ml of broth added as a control).<br />
*sample designation: (# refers to the clone number form the original plate). Original plates are the ones made on '''INSERT THE DATE LAZY''' and have 2 clones on each plates (clones1-3 are K191003&4 double transformants, clones 4-6 are K191009+4 double transformants). <br />
<br />
# K191003+4 (#1) + Amp + Kan + IPTG + LIGHT <br />
# K191003+4 (#1) + Amp + Kan IPTG + DARK <br />
# K191003+4 (#2) + Amp + Kan IPTG + LIGHT <br />
# K191003+4 (#2) + Amp + Kan IPTG + DARK <br />
# K191009+4 (#4) + Amp + Kan IPTG +LIGHT <br />
# K191009+4 (#4) + Amp + Kan IPTG +DARK <br />
# K191009+4 (#5) + Amp + Kan IPTG +LIGHT <br />
# K191009+4 (#5) + Amp + Kan IPTG +DARK <br />
# K191004 ONLY #2+ Amp + LIGHT <br />
# K191004 ONLY #2+ Amp + DARK <br />
# neg. control- broth, Amp + Kan + IPTG + LIGHT<br />
<br />
* samples designated DARK were wrapped in the aluminium foil straight after the inoculation; the cap was not wrapped (not see through anyway)so that samples could be easily taken without unwrapping the bottles from the foil. <br />
*All the samples were then taken to the shaker in the hot room and placed in prepared spots: for the dark- anywhere on an easily accessible holder, for light- in 2 rows of holders. <br />
*Light samples were covered with the cardboard box with blue LEDs attached on the sides, in a way to optimize light accession to the samples (ask Sarah for the details). Box was securely taped to the shaker. <br />
* Measurments were taken every half an hour: 200ul of the samples were placed in the numbered cuvette and then 800ul of water was added. Readings of fluroscence (green mode) were taken in standard fluorescence units (SFU, two readings each). Then optical density was measured (upstairs in the incubator room) at 360 nm wavelength. Control was used to calibrate the machine (set ref button). Also 2 readings were taken. Samples were shaken by inversion prior to taking measurment. <br />
* Experiment was stopped after 3 hours (6 sets of reading taken). <br />
<br />
*'''Result analysis'''<br />
see the Excel file attached: <br />
<br />
[[File:LovTap FAIL exp 18aug.xls]]<br />
<br />
To generate the data for the graph:<br />
# means were calculated from 2 readings of optical density and fluorescence<br />
# (mean SFU minus background SFU) was divided by (mean OD minus background OD, if different from 0).<br />
# table was made for the samples at different points of time- e.g. sample 1 after 30 min, 60 min, 90 min, 120 min etc. <br />
# graph was made with series named the same as the samples<br />
<br />
[[File:Untitled.JPG]]<br />
<br />
**BAD BAD VERY BAD NEWS: after 2 hours of measurments the results of the sequencing were know and revealed that THERE IS A 1BP DELETION IN LOVTAP 03 AND 09 WHICH RESULTS IN FRAMESHIFT AND LACK OF THE FUNCTIONAL PRODUCT!!!! :(:(:( despite this information, it was decided that the experiment will be finished and analysed as initially planned.<br />
<br />
'''19/08/2010'''<br />
<br />
* Pcr- mutagenensis of LovTap- Mabel, adding missing bp. <br />
* Puryfing PCR product from the solution. <br />
<br />
<br />
'''20/8/2010'''<br />
* PCR run on the gel -correct band of ~5.5. kb size, suggest that the PCR worked well. couple of other fainter bands <br />
* Self -ligation overnight (MABEL self ligation- 12ul of H2O, 4ul of DNA, 2 ul of Ligase buffer, 1 ul of T4 DNA ligase, 1ul of T4 polynucleotide kinase. <br />
* transformation and setting up the liquid cultures for the minipreps over the weekend. <br />
<br />
[[Image:123.JPG]]<br />
<br />
'''23/08/2010'''<br />
* mini-prep DNA from the colonies after PCR- marker worn off in the shaker,so 3.2. and 9.1. got mixed up- probably sequencing necessary to distinguish the mutated version from non-mutated <br />
* digest minipreps with EcoRI, run the gel[photo]<br />
* run 5ul of the ligated 03 and 09 on the gel /also Richards samples/.<br />
<br />
[[Image:124.JPG]]<br />
<br />
'''24/08/2010'''<br />
* repeated digest of mutated LovTap samples: 3.1, 3.?, 3.3., 3.4 9.?, 9.2 with EcoRi<br />
* minipreped 6 more clones of mutated LovTap 03 and 09 from the same plate, labelled- 3.6, 3.7, 3.8, 9.4, 9.5, 9.6. Run the digest with EcoRI. Note: 3.7 sample is higly diluted as 80ul of EB was used instead of 40ul. oops. <br />
* delivered mutated 3.? and 9.2 minipreped clones for sequencing /only 2 reactions: each sample- forward/.<br />
<br />
[[Image:LT100.jpg]]<br />
<br />
'''04/10/10'''<br />
We may have a working LovTap construct + red/white (RFP) reporter system thanks to Mexico.<br />
<br />
<br />
Minor characterisation experiment:<br><br />
-LovTap constructed with red-white RFP reporter<br><br />
-If activated, the blue light sensor transformants should produce red colonies<br><br />
-Blue light sensor containing-colonies from transformant plate streaked onto two plates<br><br />
-One plate left in the light, the other left in the dark<br><br />
-If the one in the light grows red and the one in the dark doesn't, it is a good indication that the construct is working<br><br><br />
<br />
'''8/10/2010'''<br />
* repetaed LOVTap charactierzation- with TrpR mutant and WT under light/dark conidtion. <br />
* no clear response was seen. <br />
<br />
[[Image:8octLovtap.jpg]]<br />
<br />
[[Image:8octTRPR.jpg]]<br />
<br />
'''22/10/2010'''<br />
* checking temperature response of LOVTap<br />
* setting up samples with CMM minum medium, enriched in thymine, trace elements and glucose.<br />
CELLS FAIL TO GROW!:(</div>Macbereskahttp://2010.igem.org/Team:Edinburgh/Notebook/BRIDGETeam:Edinburgh/Notebook/BRIDGE2010-10-27T12:03:24Z<p>Macbereska: /* BRIDGE */</p>
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<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Aspects">human aspects</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Communication">communication of science</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Terminology">terminology research</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Wiki">wiki</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Conversations">conversations</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Identity">identity</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Collaboration">collaboration</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Branding">branding research</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/SciFi">science fiction writing</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/FutureApps">future applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/SelfReflection">self-reflection</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Epic">the epic</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Human">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/References">references</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook" class="dir">lab notes&nbsp;&nbsp;&nbsp;</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Collaboration">collaboration</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Attribution">attribution</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/BRIDGE">BRIDGE</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_producer">red light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_sensor">red sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_producer">blue light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_producer">green light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_sensor">green sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Modelling">modelling</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Safety">safety</a></li><br />
<li><a href="http://www.openwetware.org/wiki/French_Lab">protocols</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Acknowledgements" class="dir">acknowledgements</a><br />
</li><br />
<br />
</ul><br />
</div><br />
<br />
<br><br />
<br><br />
<br><br />
<br />
<div id="windowbox" style="border: .2em solid #660000; padding: 5px; position:fixed; top:50%; right:30px; width:8%;"><br />
<span style="color:ivory;">These <b>notes</b> correspond to the part of the <b>project</b> detailed <a href="https://2010.igem.org/Team:Edinburgh/Project">here</a>.</span><br />
<br />
</div><br />
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</html><br />
<br />
== BRIDGE ==<br />
<br><br />
<br />
'''5/7/2010'''<br />
<br />
* sacB digest with EcoRI -band around kb '''(gel 1)'''<br />
[[Image:103.JPG]]<br />
<br />
<br />
'''6/7/2010'''<br />
* sacB digest with EcoRi/PstI. Band also around 5 kb. <br />
[[Image:1005.JPG]]<br />
<br />
'''7/7/2010''' <br />
<br />
* PCR of sacB to check insert. Used sacB tube 1 and 3.<br />
* Restriction digest of PCR with EcoRI and NotI. <br />
[[Image:106.JPG]]<br />
* lane 1- marker<br />
* lane 2, 3, 4 - sacB 1, 2 & 3 cut with NotI; band size: slightly bigger than 4 kb<br />
* Lane 5- PCR of LuxAB; band size around 1.5. kb<br />
* lane 6- Vector of sacB (pSB1C3, 2072bp, aroun 3kb with RFP) cut with NotI; band size around 1.5. kb<br />
* Lane 7- PCR of sacB with gene sepcific primers; band size around 1.5. kb<br />
<br />
* Trouble shooting: sacB cut with NotI give about 4kb, whereas cutting with EcoRI gives 5.5. kb... hmhmhm.<br />
there should be 2 NotI sites on either side of the insert but we only get 1 band--> is there only 1 NotI site? Furthermore, PstI/EcoRI digests give only 1 band (same as cut only with EcoRI), so maybe there is no PstI/NotI site?<br />
<br />
* Purifying sacB from PCR product to start making BRIDGE- row F in the BOX 1.<br />
<br />
'''9/7/2010'''<br />
* Making working solutions of primers from stock: 4ul of H20; 1 ul of stock solution<br />
* Primers: forward 72.3 C, revers- 77.6 C; length of the product: 2.6-2.7 kb<br />
{|border="1"<br />
|Lane 1<br />
|Lane 2<br />
|Lane 3<br />
|Lane 4<br />
|Lane 5<br />
|Lane 6<br />
|Lane 7<br />
|Lane8<br />
|-<br />
| ladder<br />
| sacB <br />
| sacB digest wtih EcoRI-HF<br />
| sacB +<br />
| digest `5009<br />
| S248T PCR product<br />
| Pure 356K<br />
| Pure 356R<br />
|}<br />
<br />
[[Image:152.JPG]]<br />
'''to do on Monday:'''<br />
* redo sacB- catR ligation<br />
* Run 5 ul of ligation on gel to check for large bands<br />
* redo RLS part transformations<br />
<br />
<br />
'''12/7/2010''' <br />
<br />
* Transformants of luxAB-edi1 failed- probably smth wrong at the purification stage...<br />
<br />
[[Image:107.JPG]]<br />
<br />
{|border="1"<br />
|Lane 1<br />
|Lane 2<br />
|Lane 3<br />
|Lane 4<br />
|Lane 5<br />
|Lane 6<br />
|-<br />
| ladder<br />
| luxAB-ediI digest<br />
| luxAB-ediI ligation<br />
| sacB digest<br />
| catR digest<br />
| sacB- catR lgation<br />
|-<br />
| expec. no of bands<br />
| 2<br />
| 1 or 3 <br />
| 1<br />
| 1<br />
| 1 band= to 4+5<br />
|}<br />
<br />
* If all failed- purification procedure failed<br />
* If 3 and 6 failed- ligation failed<br />
* If all fine then PCR/transformation failed<br />
conclusion:...<br />
<br />
* 3 and 6 failed- ligation problem (not enough DNA, ligase).<br />
* Band 1- luxAB but no ediI (?)<br />
<br />
* Repeated digest (sacB with XbaI, catR with SpeI, LuxAB & EdiI with SpeI & SacI) and purification.<br />
* Set up ligations with 2ul ligase instead of 1 ul (1ul of H2O less). If this fails- perhaps only 2-3 h ligation ina RT (SpeI is set up in both digest- could be common cause)<br />
<br />
'''13/7/2010'''<br />
*First, run PCR of catR-sacB ligation (use DNA'''?????'''' MK1- with sacBr1 (19.6.10); MK2- sacBr1(10)<br />
* Gel electrophoresis of: digested catR, digested sacB, ligated sacb-catR, digested LuxAB-ediI and ligated luxAB-ediI along with sacB-catR PCR<br />
<br />
[[Image:108.JPG]]<br />
<br />
{|border="1"<br />
|Lane 1<br />
|Lane 2<br />
|Lane 3<br />
|Lane 4<br />
|Lane 5<br />
|Lane 6<br />
|Lane 7<br />
|-<br />
| ladder<br />
| ediI<br />
| digested LuxAB-ediI<br />
| ligated luxAB-ediI<br />
| ligated catR & sacB<br />
| PCR 1 (MK1)<br />
| PCR 2 (MK2)<br />
|}<br />
<br />
''''AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAARGH!!!!!!!!!!!!!!!1''''<br />
<br />
* Almost completely convinced it's the ligase... or me (MK). Officially taking myself off the project. xx<br />
* (Spotted 2 purified sacBs in the freezer... maybe try the original if this one fails...?)<br />
'''3/8/2010'''<br />
*minipreps: marker (lane 1& 8) & sacb 8.1-8.6( Lanes 2-7)<br />
[[Image:115.JPG]]<br />
''''9/8/2010''''<br />
<br />
Gel with :<br />
* cat + sacB (PCR product) (6)<br />
* vector + cat + sacB (7) (both done by CF)<br />
<br />
'''20/8/2010'''<br />
<br />
* Made plates variety of sucrose combinations--> 40 cml<br />
* 0%<br />
* 0.1%<br />
* 0.25%<br />
* 0.5%<br />
* 1.0%<br />
<br />
*Plated E6 miniprep, which should show some sucrose sensitivity (1plate at each concentration)<br />
*Control- E5, 1 plate at each concentration<br />
<br />
'''30/8/2010'''<br />
* Primers have arrived for up and downstream sequences of TnaA region.<br />
'''8/9/2010'''<br />
* Lane 1 &8- marker, Lane 6- cat +sacB PCR; Lane 7- vector+cat+sacB<br />
[[Image:141.JPG]]<br />
<br />
'''13/9/2010'''<br />
Plane for week begininng 13th:<br />
* Amplify RFP with primers --> RBS-F2; YFP - R2 ---> to amplify RFP<br />
* Digest UP with SpeI and B-D with XbaI<br />
* Ligate UP to BRIDGE-DOWN<br />
* Digest RFP with XbaI and SpeI<br />
* Ligate UP and RFP<br />
* Ligate DOWN and RFP<br />
<br />
* Also- registry editing for RLS (definately dodgy sequence), LovTap<br />
* Justification of reseubmission<br />
* Look up absorption/emission rates and transmission through medium for Donal<br />
<br />
'''20/9/2010'''<br />
<br />
* Achieved: cat/sacB- traA down ligation<br />
* Attempted: traA up- cat/sacB/down ligation; traA up- RFP ligation; RFP- traA down ligation/ ALL FAILED<br />
* Running: purified digest of:<br />
- cat/sacB + SpeI <br><br />
- tnaA down- XbaI<br><br />
- tnaA up + SpeI<br><br />
- cat/sacB/down + XbaI <br><br />
- RFP + XbaI<br><br />
and also running: UP- BRIDGE- DOWN ligation; UP - RFP ligation<br />
<br />
Have: PCR of cat/sacB, RFP, BRIDGE-DOWN, traA up, traA down; ligation of BRIDGE-DOWN<br />
<br />
<br />
[[Image:146.JPG]]<br />
<br />
Run gel: <br />
{| border="1"<br />
| Lane 1 <br />
| Lane 2 <br />
| Lane 3 <br />
| Lane 4 <br />
| Lane 5 <br />
| Lane 6 <br />
| Lane 7 <br />
| Lane 8<br />
|-<br />
| Ladder <br />
| Pure SacB <br />
| Pure SacB & EcoRI <br />
| sacB & catR PCR <br />
| I15009 digest <br />
| S284T PCR Product <br />
| Purified 356K <br />
| Purified 356R<br />
|}<br />
<br />
<b>25/10/10</b><br />
<br><br />
Repetitive failure of purifications with both traditional method and kits, unsure what is going wrong. Have decided to tell Chris. Off to Firbush on Wednesday.<br><br><br />
<b>01/10/10</b><br />
<br><br />
In my absence (at Firbush with the rest of the Biotechnologists) Chris has managed to construct the tnaA UP-cat-sacB-tnaA DOWN segment that we need for the first part of the BRIDGE protocol. I will continue to build the tnaA UP-RFP-tnaA DOWN segment. <br />
<br><br />
Set up ligations of RFP-DOWN and UP-RFP, left in 16C incubator overnight. <br />
<br><br><br />
<b>02/10/10</b><br />
<br><br />
Lab on a Saturday again, fun fun fun...<br />
<br><br />
Retrieved and amplified ligations and ran on gel. UP-RFP appears to be significantly shorter than RFP-DOWN and indeed shorter than it ought to be (should be about 2kb, is only around 1.5kb). <br><br />
Purified both products, then digested RFP-DOWN with XbaI. (Used 2hour incubation time to write presentation for Wednesday).<br><br />
Set up ligation of UP with RFP-DOWN and left in incubator overnight.<br><br />
<br><br />
<b>03/10/10</b><br />
<br><br />
Bus to Darwin for 11:20am; enter Darwin; lift to 8th floor; retrieve ligations from waterbath in cold room; walk down to 7th floor; put ligation in freezer; lift to ground floor; leave Darwin; catch 11:40am bus back to central.<br />
<br><br><br />
<b>04/10/10</b><br />
<br><br />
PCR of ligation of UP-RFP-DOWN. Made and ran gel along with purified PCRs from Saturday, just in case they're still not working.<br><br />
They worked! The purifications were perfect. There is a band about the size of UP-RFP-DOWN in it's lane, but it's quite faint. I will probably repeat the PCR with a lower annealing temperature (around 50) this afternoon. I suspect the UP primers do not anneal properly above 54C.<br />
<br><br><br />
<b>07/10/10</b><br />
<br><br />
Chris set up overnight cultures of DH5-alpha cells and JM109 cells, both containing the lambda-red plasmid, in 2.5ml LB with tetracycline.<br><br />
Repeated PCR of up-RFP-down ligation as well as PCR of 3 subcultures suspected to contain the cat-sacB construct, performed using the tnaA UP forward primer and the tnaA DOWN reverse primer. <br />
<br><br><br />
<b>08/10/10</b><br><br />
Took 2 5ml LB bottles:<br><br />
- Added 1.7 microlitres of tet15 and 0.3ml DH5-alpha overnight culture to one<br><br />
- Added 5 microlitres of tet15 and 0.3ml of JM109 overnight culture to the other<br><br><br />
Grew second cultures for 2 hours at 30C with shaking <br><br />
Measured optical density after 2 hours:<br><br />
- Water = 0.00<br><br />
- DH5-alpha = 0.251<br><br />
- JM109 = 0.320<br><br><br />
Added 100microlitres arabinose to both cultures and grew at 37C for one and a half hours with shaking.<br><br><br />
Prepared cells for electroporation:<br><br />
1- Added 1.5ml of liquid culture to an eppendorf<br><br />
2- Spun down cells at 11,000rpm for 30s<br><br />
3- Removed supernatant, added 1ml of cold sterile water and resuspended cells<br><br />
4- Repeated steps 2 and 3 twice more<br><br />
5- Added 40microlitres of suspended cells and 1microlitre of DNA construct (<i>up-cat-sacB-down</i>) to chilled electroporation cuvette (also prepared one cuvette of each cell culture without DNA for controls)<br><br><br />
Performed electroporation<br><br />
Suspended cells in cuvette by adding 0.5ml LB and added to eppendorf containing 1.0ml LB<br><br />
Left in 37C incubator for one hour, no sign of growth - spun cells down and no visible pellet, resuspended in 0.9ml and decided to leave overnight<br><br><br />
Will plate up cells on chloramphenicol tomorrow and they should have grown by Monday.<br><br><br />
Annoyingly, the Red/ET recombination plasmid is lost at 37C, but it was worth following the procedure to see if it actually works first time. If we need to repeat it, the plates can be grown at 30C so that there is no need to retransform with the recombination plasmid.<br><br><br />
<b>09/10/10</b><br />
<br>Cells left to grow for ~20 hours<br><br />
Plated from 100microlitres from 900microlitre suspension onto 4 chloramphenicol plates marked JM109/lambda + cat/sacB and -cat/sacB (control) and DH5-alpha/lambda + cat/sacB and - cat/sacB (control)<br><br />
Spun cells down at 7000rpm for 2 minutes - gave decent sized pellet. Removed 700microlitres of supernatant and resuspended cells<br><br />
Plated last 100microlitres onto a further 4 chloramphenicol plates <br><br />
Plates will be left in 37C incubator - cells were concentrated enough on second set of plates so that if they are cml resistant they will grow by tomorrow. <br><br><br />
<b>10/10/10</b><br><br />
Significant growth of red colonies on DH5-alpha+ (concentrated) but none on DH5-alpha- (not-concentrated). This is highly confusing as there should be no RFP in the cells. We now believe this to be a contamination.<br />
<br><br />
There is growth on both of the negative plates of the JM109 cells, but none on the positive plates. This could be a labelling error.<br><br />
There is no growth on any of the other plates.<br><br><br />
<b>11/10/10</b><br><br />
No growth on any of the plates that had not grown yesterday. <br><br />
Colonies on JM109- plates do not smell of indole. If they are E.coli then this is promising. Subcultured 4 colonies from each plate onto cml plates.<br><br><br />
Ran gels of PCRs performed on 07/10. No bands in the three lanes from subcultures, but a strong band from the PCR of the ligation.<br><br><br />
<b>12/10/10</b><br><br />
The subcultures from yesterday have not grown particularly well. The cells are white, unlike normal E.coli, and the colonies are uncharacteristically runny. Ran PCR of subcultures to determine whether they contain the insert. (with insert ~2.5kb, without ~1.5kb)<br><br />
Ran this PCR on a gel. Gave no bands. First attempt at BRIDGE did not work. <br><br><br />
Alterations to be made on second attempt:<br />
<br>1- Resuspend cells in 20-50microlitres after cleaning to increase transformation efficiency<br><br />
2- Grow at 30C instead of 37C after electroporation so as not to lose recombination plasmid<br><br />
3- Use normal chloramphenicol plates rather than cml+iptg+xgal.<br />
<br />
<br><br><br />
<b>14/10/10</b><br><br />
Prepared overnight cultures:<br />
<br><br />
- DH5-alpha/lambda in 5ml LB with 1.5microlitres tet15<br><br />
- JM109/lambda in 5ml LB with 5microlitres tet15<br><br><br />
Grew cells at 30C, 200rpm<br><br><br />
<br />
[[Image:171.jpg]]<br />
<br />
* tnaA PCR, JM109, MK2+4, CF 6.<br />
<b>15/10/10</b><br><br />
Prepared secondary liquid cultures:<br />
<br><br />
- 5ml LB + 1.5microlitres tet15 inoculated with 0.3ml overnight DH5-alpha/lambda culture<br><br />
- 5ml LB + 5microlitres tet15 inoculated with 0.3ml overnight culture JM109/lambda<br><br><br />
Grew cells at 30C, 200rpm for 2 hours<br><br><br />
Transferred 1.5ml of both cultures to individual eppendorfs and spun down cells for 3 minutes at 8000rpm. Removed supernatant and added another 1.5ml. Spun down cells again and removed supernatant.<br><br />
Suspended cells in 1ml cold sterile water and spun them down for 1 minute at 10000rpm before removing supernatant.<br />
Repeated this twice more.<br />
<br><br />
Resuspended cells in 100microlitires cold sterile water.<br><br><br />
Added 40microlitres of the relevant cell culture to cold sterile electroporation cuvettes labelled J+, J-, D+ and D-.<br><br />
Added 2microlitres DNA to the J+ cuvette.<br><br />
Added 1microlitre DNA to the D+ cuvetter.<br><br />
Prepared 8 eppendorfs, 4 containing 1.5ml LB.<br><br><br />
Electroporated all 4 samples.<br><br />
The voltage decay readings for the J-, D+ and D- cells were all above 4.0, but the reading for the J+ was 3.30. This probably indicates that there is too much DNA in the sample as the charge dissapated too quickly. From now on only 1microlitre of DNA will be used.<br><br><br />
Immediately after electroporation, 500microlitres of LB was added to the cuvette from an eppendorf to resuspend the cells and transferred back to the eppendorf. The LB was mixed and 750microlitres were transferred to a clean eppendorf.<br><br><br />
For each transformation, half the cells were grown at 30C overnight and half were grown at 37C overnight.<br />
<br><br><br />
<b>16/10/10</b><br><br />
Spun down cells from each of the 8 eppendorfs at 8000rpm for 3 minutes and removed 600microlitres of supernatant. <br><br />
Spread transformants onto chloramphenicol (one plate each) and grew at relevant temperatures for 48 hours. <br><br><br />
<b>18/10/10</b><br><br />
Only growth on plates appears to be non-E.coli. Major contamination has only occurred on 30C plates. Whatever it is, it seems to grow better at 30C. <br><br />
Since this is the second time we have seen this type of contamination the next attempt at the protocol will be combined with taking samples of cells at each step.<br><br><br />
<b>19/10/10</b><br><br />
Made more tet15 plates, as will need about 40 this week.<br><br />
Prepared overnight cultures of DH5-alpha/lambda and JM109/lamda, both grown at 30C.<br><br><br />
<b>20/10/10</b><br><br />
Took a sample of each overnight culture and spread it onto two tetracycline plates (1), one grown at 30C the other grown at 37C. The reasoning here is that any contaminants at this stage must be tetracycline resistant.<br><br><br />
Added 0.3ml overnight culture to fresh LB with tetracyline. Grew for 2 hours at 30C with shaking.<br />
<br><br><br />
Sampled each culture again, this time plating to both chloramphenicol and tetracyline plates. (2)<br><br />
NB: JM109 is not growing as well as DH5alpha in liquid culture. We may need to change the concentration of tet in one or both slightly.<br><br><br />
Something of a mini catastrophe...I knocked over my DH5alpha liquid culture and lost half the contents. Have added LB and a proportionate amount of tet15 and left both in the 30C shaker for another hour. This should recover enough cells to continue, however it also means the LB may contaminate the DH5alpha...if it is the LB, then only DH5alpha will be contaminated.<br><br><br />
In the mean time, am making 20-26 plates of cml.<br><br><br />
Added 100microlitres of 20% L-arabinose to each culture and left for one hour at 37C with shaking. Prepared cold water, cuvettes and eppendorfs with LB.<br><br><br />
Sampled cultures again, plating to chloramphenicol only. (3)<br><br />
Washed cells with cold water.<br><br />
Sampled cells again and plated to chloramphenicol. (4)<br><br><br />
<br><br><br />
Electroporated two sets with the cat/sacB construct and and two without. Transferred to LB in eppendorfs for recovery and grew each set of four at both 30C and 37C for an hour.<br><br><br />
Sampled cells again and plated to chloramphenicol. (5)<br><br />
Innoculated 5ml liquid cultures with 5microlitres of cml and 600microlitres of the transformed sample and grew overnight at 30C and 37C with shaking.<br><br><br />
<b>21/10/10</b><br />
<br><br />
No growth on any of the chloramphenicol plates from yesterdays samples. This suggests the usual contaminant has not yet reached the cultures. There is growth on each tetracycline plate, which is expected as the cells still contain the Red/ET plasmid at this point.<br><br><br />
Unfortunately there is no growth in the positive chloramphenicol liquid cultures. There is growth in the D- at 37C culture which presumably is one of our contaminants. I will keep an eye on the D- 37C plate and possibly plate out the liquid culture to examine the colonies. This contamination must have occurred during the final stage of the protocol from yesterday and must be human error as it is an isolated incident. <br><br><br />
<b>22/10/10</b><br><br />
Set up secondary liquid cultures in tet15 (3.5microlitres for JM109 as it seems to be struggling to grow) and left to grow in 30C shaker for 3 hours.<br />
<br><br />
Plated out D- 37C cell culture that survived chloramphenicol to determine strain.<br><br />
<br><br />
Checked chloramphenicol sample plates again:<br><br />
Sample 2- White colony growth on both JM109 plates but none on DH5alpha<br><br />
Sample 3- No growth (contaminants have disappeared, killed by arabinose?)<br><br />
Sample 4- White colony growth 30C plates only, two micrococcal colonies on J 37C<br><br />
Sample 5- A few very small white colonies on 30C plates D-, D+ and J-, no growth on D- at 37C<br><br><br />
The bulk of the contamination appears to occur at the washing step, but whatever they are, they don't seem to be able to survive cml40 in a liquid culture. Confusingly, whatever has contaminated the D- culture at 37C doesn't seem to be on the plates. This probably means it is an isolated event. I don't know if the same goes for the contaminants in sample 2. These are more confusing as they disappear after the arabinose is added. It is possible that they do not survive either in arabinose or at 37C in liquid.<br><br><br />
Next step to remove contaminants:<br><br />
Previously the washing step had been performed in the lab away from the clean hood. From now on, any stage where the cells are exposed will take place under the clean hood.<br><br />
A control will be set up at this step to confirm that contamination is occurring here and not previously. A second set of eppendorfs will be washed repeatedly with water and then 100microlitres of the water will be plated along with the actual samples. <br><br />
Growth on the negative control will indicate that either the water or the eppendorfs is contaminated. <br><br />
No growth on either will indicate that the use of the clean hood makes a difference.<br><br><br />
Next step in improving the efficacy of BRIDGE:<br><br />
The cat gene we're using is not very strongly induced. Couple this with the fact that DH5alpha and JM109 both struggle to grow at regular concentrations of antibiotics and we can assume that any recombinants we do get won't be able to grow in cml40. This time I will grow them overnight in a lower concentration of chloramphenicol. 3microlitres for JM109, 2microlitres for DH5alpha. <br><br><br />
This might be the last time I will be able to attempt BRIDGE before the wiki freeze. Fingers crossed.<br><br><br />
Maximised biomass this time by growing JM109 in 3.5microlitres tet15 and DH5alpha in 1.75microlitres as usual, for 2 and a half hours after adding 0.5ml overnight culture rather than 0.3ml. <br><br><br />
After the induction step spun down whole cell culture and kept cells under clean hood whenever they were exposed. <br>Also took negative controls at washing step of sterile water and LB, both kept in eppendorfs for 10minutes.<br>If there is growth on:<br />
<br><br />
- Sterile water only - sterile water is contaminated<br><br />
- LB only - LB is contaminated<br><br />
- Both - eppendorfs are contaminated<br>Also took samples of actual cell cultures at this step.<br><br><br />
Electroporated cells as normal. Grew at recovery step for 2 hours.<br><br><br />
Grew all 8 samples in cml40: JM109 in 3.5microlitres, DH5alpha in 1.75microlitres.<br><br />
Grew positive transformants in tet15 (normal concentrations) to determine if the cells were still alive. <br><br />
If the positive cells grow in:<br><br />
- cml and tet - recombinants<br><br />
- cml but not tet - contaminant<br><br />
- tet but not cml - non-recombinant<br><br />
- neither - cells have died<br><br><br />
<br />
Transformed JM109 cells with pSB1A2+cat to characterise cat and Edinbrick as a negative pSB1A2 control. Plated cells to ampicillin. <br><br><br />
<b>23/10/10</b><br><br />
The negative controls from the washing step have not grown, but then neither have the samples, so no contamination on anything. <br><br><br />
There is growth in 3 of the 4 tetracyline cultures from yesterday (JM109 has not grown at 37C, possibly because of inability of plasmid to replicate). This means the cells are not, in 3 cases, being killed by the electrporation. <br>There is no growth in any of the negative controls, so no indication of contamination. <br><br />
No growth in any of the cml positive cultures which indicates that the recombinase genes are not being induced properly. <br><br />
<br>The D- 37C contamination from Wednesday turned out to have RFP in it. This is quite confusing but, as stated earlier, all contaminations have been removed from the procedure.<br><br><br />
Good growth of cat and Edinbrick transformants on ampicillin. Have subcultured 4 of each, again to ampicillin.<br><br><br />
<b>24/10/10</b><br />
<br><br />
Subcultured the cat and Edinbrick transformants from ampicillin to cml40. <br><br><br />
<b>25/10/10</b><br><br />
No significant growth on subcultures.<br><br><br />
Set up titre of cml resistance using positive 30C transformants and negative controls. All grown in tetracycline and in increasing concentrations of chloramphenicol. Grown overnight at 30C and 200rpm.<br><br><br />
<b>26/10/10</b><br><br />
Growth on Edinbrick on cml40 but not on cat. Could be mislabelling. Have re-attempted subculture from transformant plates but if initial plates are labelled wrong then the results will be the same.<br><br><br />
Results from titre. Conclusion: recombination isn't working as cat has been known to show resistance to cml10-40.<br><br><br />
<br />
<br />
{|border="1" style="text-align: center"<br />
|-<br />
!<br />
! colspan="2" | Ju109 (tet15 / 5ul)<br />
! colspan="2" | DH5a (tet3.75 / 1.75ul)<br />
|-<br />
! Cml<br />
! + cat/sacB<br />
! control<br />
! + cat/sacB<br />
! control<br />
|-<br />
| 0 / 0 ul || + || + || + || +<br />
|-<br />
| 4 / 0.5 ul || - || - || - || -<br />
|-<br />
| 8 / 1 ul || - || - || - || -<br />
|-<br />
| 12 / 1.5 ul || - || - || - || -<br />
|-<br />
| 16 / 2 ul || - || - || - || -<br />
|-<br />
|| 20 / 2.5 ul || - || - || - || -<br />
|-<br />
| 24 / 3 ul || - || - || - || -<br />
|}</div>Macbereskahttp://2010.igem.org/File:LT100.jpgFile:LT100.jpg2010-10-27T12:02:18Z<p>Macbereska: </p>
<hr />
<div></div>Macbereskahttp://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_sensorTeam:Edinburgh/Notebook/Blue light sensor2010-10-27T11:53:33Z<p>Macbereska: /* Blue Light Sensor */</p>
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<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_producer">blue light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_producer">green light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_sensor">green sensor</a></li><br />
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<span style="color:ivory;">These <b>notes</b> correspond to the part of the <b>project</b> detailed <a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_sensor">here</a>.</span><br />
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<br />
==Blue Light Sensor==<br />
<br><br />
<br />
'''14/07/10'''<br />
<br />
Received parts from Lausanne:<br />
*BBa_191006 (113 ng/µL) – LovTAP alone<br />
*BBa_191004 (166 ng/µL) – Readout <br />
*BBa_191003 (230 ng/µL) – whole system<br />
*BBa_191009 (85 ng/µL) – mutant <br />
*Sosnick vector containing TrpR (393 ng/µL) – vector <br />
<br />
<br />
<br />
'''16/07/10'''<br />
<br />
Transformations using 100 µL of cells, 900 µL broth<br />
*K191004 - amp<br />
*K191003 - kanamycin<br />
*K191009 – kanamycin<br />
*Sosnick vector with TrpR – amp<br />
<br />
Note: Labeling on tubes is unclear – K191009 and K191004 got mixed up, so plating both tubes on both Kan and amp<br />
<br />
<br />
'''22/07/10'''<br />
<br />
*Minipreps: K0998010 – 5x (1 didn’t grow, but did anyway)<br />
*Blue sensors (3 biobricks and Sosnick vector) – cultures set to grow overnight<br />
<br />
'''25/07/10'''<br />
Digest of the blue light sensor minipreps was performed with EcoRI and BamHI; samples:<br />
*3- K19100'''4'''; 900ul, (3)<br />
*4- K19100'''4'''; 100ul, (2)<br />
*5-K19100'''9'''; 100ul, (1)<br />
*6- K19100'''9'''; 100ul, (6)<br />
*7- K19100'''9'''; 900ul, (4)<br />
*8- K19100'''9'''; 100ul, (2)<br />
*9- K19100'''3'''; 900ul, (2)<br />
*10- K19100'''3'''; 900ul, (5)<br />
*11- K19100'''3'''; 100ul, (4)<br />
*12- K19100'''3'''; 100ul, (1)<br />
*13- K19100'''4'''; 100ul, (3)<br />
*14- K19100'''4'''; 900ul, (5)<br />
<br />
Lanes- 1- marker, 2-9 sampels as in order, 10-empty, 11, 12- marker instead of the samples- need to run last two again<br />
<br />
[[Image:109.JPG]]<br />
<br />
[[Image:111.JPG]]<br />
LAST TWO LANES- NSAMPLES NO 13 AND 14 FROM THE ABOVE LIST<br />
<br />
<br />
'''28/07/10'''<br />
<br />
Double transformants made using a novel cowboy procedure- double transformation with 4ul of each piece od DNA. Chances of succes- very very low... But at least it will be proved that it won't work!<br />
Trans. 1: BBa_K191004+ K191003- read out + LovTap<br />
Trans. 2: BBa_K191004+ K191009- read out + mutated LovTap<br />
<br />
'''30/7/10'''<br />
set up experiments to see if the blue light sensor will activate the response system under the daylight. Both liquid cultures and plates were set up, with red and white colonies- first supposed to turn white in the daylight and stay red in the dark (under the foil), second ones- no change of the colour. The same applies to the liquid cultures. However, the experiment failed- no proper growth was observed, and if there was a growth - it was very weak. No pigment observed, apart from the small points in the centre. <br />
Hypothesis- not stable plasmids-- cells look like they are not really resistant. Recorded growth:<br />
{|border="1"<br />
|<br />
|K191004+9 control<br />
|K191004+9<br />
|K191004+3 control<br />
|K191004+3<br />
|-<br />
|DARK<br />
| ++ , white<br />
| +++ , white<br />
| ++ , white<br />
| +++ , white with red points<br />
|-<br />
| LIGHT<br />
| + , white<br />
| +++ , white with red point<br />
| + -almost no growth <br />
| ++ white<br />
|}<br />
<br />
<br />
<br />
'''2/8/10'''<br />
<br />
Run the digest of the minipreps from the white and red cultures of double transformants. No proper DNA band in the solution- repeat the procedure or not? Don't know what to do.... again. <br />
[[File:Gel 3.jpg]]<br />
Collect the thoughts and ideas and start again tomorrow!<br />
<br />
'''6/8/10'''<br />
<br />
double transformants were prepared again, using non-cowboy procedure. 04 cells were first MADE COMPETENT, then transformed with 03 and 09, also 1 neg control was plated. Grow white on the intitial plate, then turned red if subcultured. <br />
<br />
'''7-8/08'''<br />
<br />
'Subbed these to duplicate fresh plates and incubated overnight at room temperature <br />
either under a fluorescent lamp or wrapped in foil - all white in both cases (and growth <br />
not great, probably because of low temperature)'. <br />
The plates were moved to 37C with the lamp /one under the light, one in the dark. so far -no results, checking in an hour/. <br />
<br />
Another idea- check for stability of the insert- we don't know if the plasmids are compatibile. Streaking from original plates to check if single colonies will be a mixture of white and red or just red- origin? no plasmid maps from Lausanne. <br />
by Chairman-->' If we can't find this information, I think our best course is to <br />
PCR out both inserts and combine them in a single vector, preferably pSB1C3.'<br />
<br />
<br />
'''9/8/2010'''<br />
<br />
to do:<br />
<br />
*primers for the parts to put them in a vector<br />
*check the lamp every hour!!<br />
<br />
go thruough Lausanne website to find the plasmid maps<br />
get back to Mexico about the parts and etc.<br />
find out the conditions for the blue light sensor assay!<br />
<br />
'''10/08/10'''<br />
* assay with dark/light liquid cultures - fluorescence measurment and OD, after 5 h in the shaker under lamp- see video and pictures<br />
*email to Mexico<br />
<br />
'''11/08/10'''<br />
<br />
*primer designed for combining LOVTap and Readout1 into 1 cell<br />
*repeated assay with the smae samples, after overngiht in RT and %5h under lamp (for the light samples) and RT without shaking<br />
*primary cultures streaked to amp/kan/IPTG plates- part 4+9 and 4+3 -three clones each<br />
<br />
'''12/08/10'''<br />
*results analysis after LOVTap experiment:check file, add graphs later.<br />
*email from Mexico about Blue promoter<br />
<br />
'''16/08/10'''<br />
* streaked single clone for 9+4 and 3+4 (clones number 5 and 2, respectively)- for tomorrow's experiment<br />
* minipreped mysterious double transformants and Sacb smples<br />
* tried out the box on the shaker- unscrewed the things. <br />
* made the graphs from the second reading<br />
* 03 and 09 LovTap taken for sequencing. <br />
<br />
to do: decide on the illumination length and think about controls and how many samples do you want to put. check Lausanne website<br />
t-test to check to importancy of the differences between the means.<br />
<br />
'''17/07/10'''<br />
* desinged experimental set up for the LovTap blue light response experiment /see next day/<br />
* BAD NEWS: there is a suspicion of 1bp deletion in LovTap!!! Chairman noticed it when he was checking the parts base by base. Should have done it much much earlier probably to save the time... <br />
* BAD NEWS: lack of the proper thinking put in doubt all the results from previous two experiments (that is why you can't find any files of it attached)- there was no IPTG added to the samples, therefore LOVTap expression was not induced- no point to do T-test right now... :( Well, at least I will learn from it and add it next time!<br />
<br />
<br />
'''18/08/10'''<br />
<br />
* tested LovTap under the blue light<br />
* conditions:<br />
Liquid cultures (same as for minipreps) were grown overnight in 37C with antibiotics. <br />
The next day, bottles with 3.5 ml of broth were inoculated with the 0.5 ml of appropriate cultures (0.5. ml of broth added as a control).<br />
*sample designation: (# refers to the clone number form the original plate). Original plates are the ones made on '''INSERT THE DATE LAZY''' and have 2 clones on each plates (clones1-3 are K191003&4 double transformants, clones 4-6 are K191009+4 double transformants). <br />
<br />
# K191003+4 (#1) + Amp + Kan + IPTG + LIGHT <br />
# K191003+4 (#1) + Amp + Kan IPTG + DARK <br />
# K191003+4 (#2) + Amp + Kan IPTG + LIGHT <br />
# K191003+4 (#2) + Amp + Kan IPTG + DARK <br />
# K191009+4 (#4) + Amp + Kan IPTG +LIGHT <br />
# K191009+4 (#4) + Amp + Kan IPTG +DARK <br />
# K191009+4 (#5) + Amp + Kan IPTG +LIGHT <br />
# K191009+4 (#5) + Amp + Kan IPTG +DARK <br />
# K191004 ONLY #2+ Amp + LIGHT <br />
# K191004 ONLY #2+ Amp + DARK <br />
# neg. control- broth, Amp + Kan + IPTG + LIGHT<br />
<br />
* samples designated DARK were wrapped in the aluminium foil straight after the inoculation; the cap was not wrapped (not see through anyway)so that samples could be easily taken without unwrapping the bottles from the foil. <br />
*All the samples were then taken to the shaker in the hot room and placed in prepared spots: for the dark- anywhere on an easily accessible holder, for light- in 2 rows of holders. <br />
*Light samples were covered with the cardboard box with blue LEDs attached on the sides, in a way to optimize light accession to the samples (ask Sarah for the details). Box was securely taped to the shaker. <br />
* Measurments were taken every half an hour: 200ul of the samples were placed in the numbered cuvette and then 800ul of water was added. Readings of fluroscence (green mode) were taken in standard fluorescence units (SFU, two readings each). Then optical density was measured (upstairs in the incubator room) at 360 nm wavelength. Control was used to calibrate the machine (set ref button). Also 2 readings were taken. Samples were shaken by inversion prior to taking measurment. <br />
* Experiment was stopped after 3 hours (6 sets of reading taken). <br />
<br />
*'''Result analysis'''<br />
see the Excel file attached: <br />
<br />
[[File:LovTap FAIL exp 18aug.xls]]<br />
<br />
To generate the data for the graph:<br />
# means were calculated from 2 readings of optical density and fluorescence<br />
# (mean SFU minus background SFU) was divided by (mean OD minus background OD, if different from 0).<br />
# table was made for the samples at different points of time- e.g. sample 1 after 30 min, 60 min, 90 min, 120 min etc. <br />
# graph was made with series named the same as the samples<br />
<br />
[[File:Untitled.JPG]]<br />
<br />
**BAD BAD VERY BAD NEWS: after 2 hours of measurments the results of the sequencing were know and revealed that THERE IS A 1BP DELETION IN LOVTAP 03 AND 09 WHICH RESULTS IN FRAMESHIFT AND LACK OF THE FUNCTIONAL PRODUCT!!!! :(:(:( despite this information, it was decided that the experiment will be finished and analysed as initially planned.<br />
<br />
'''19/08/2010'''<br />
<br />
* Pcr- mutagenensis of LovTap- Mabel, adding missing bp. <br />
* Puryfing PCR product from the solution. <br />
<br />
<br />
'''20/8/2010'''<br />
* PCR run on the gel -correct band of ~5.5. kb size, suggest that the PCR worked well. couple of other fainter bands <br />
* Self -ligation overnight (MABEL self ligation- 12ul of H2O, 4ul of DNA, 2 ul of Ligase buffer, 1 ul of T4 DNA ligase, 1ul of T4 polynucleotide kinase. <br />
* transformation and setting up the liquid cultures for the minipreps over the weekend. <br />
<br />
[[Image:123.JPG]]<br />
<br />
'''23/08/2010'''<br />
* mini-prep DNA from the colonies after PCR- marker worn off in the shaker,so 3.2. and 9.1. got mixed up- probably sequencing necessary to distinguish the mutated version from non-mutated <br />
* digest minipreps with EcoRI, run the gel[photo]<br />
* run 5ul of the ligated 03 and 09 on the gel /also Richards samples/.<br />
<br />
[[Image:124.JPG]]<br />
<br />
'''24/08/2010'''<br />
* repeated digest of mutated LovTap samples: 3.1, 3.?, 3.3., 3.4 9.?, 9.2 with EcoRi<br />
* minipreped 6 more clones of mutated LovTap 03 and 09 from the same plate, labelled- 3.6, 3.7, 3.8, 9.4, 9.5, 9.6. Run the digest with EcoRI. Note: 3.7 sample is higly diluted as 80ul of EB was used instead of 40ul. oops. <br />
* delivered mutated 3.? and 9.2 minipreped clones for sequencing /only 2 reactions: each sample- forward/.<br />
<br />
'''04/10/10'''<br />
We may have a working LovTap construct + red/white (RFP) reporter system thanks to Mexico.<br />
<br />
<br />
Minor characterisation experiment:<br><br />
-LovTap constructed with red-white RFP reporter<br><br />
-If activated, the blue light sensor transformants should produce red colonies<br><br />
-Blue light sensor containing-colonies from transformant plate streaked onto two plates<br><br />
-One plate left in the light, the other left in the dark<br><br />
-If the one in the light grows red and the one in the dark doesn't, it is a good indication that the construct is working<br><br><br />
<br />
'''8/10/2010'''<br />
* repetaed LOVTap charactierzation- with TrpR mutant and WT under light/dark conidtion. <br />
* no clear response was seen. <br />
<br />
[[Image:8octLovtap.jpg]]<br />
<br />
[[Image:8octTRPR.jpg]]<br />
<br />
'''22/10/2010'''<br />
* checking temperature response of LOVTap<br />
* setting up samples with CMM minum medium, enriched in thymine, trace elements and glucose.<br />
CELLS FAIL TO GROW!:(</div>Macbereskahttp://2010.igem.org/File:8octTRPR.jpgFile:8octTRPR.jpg2010-10-27T11:52:35Z<p>Macbereska: </p>
<hr />
<div></div>Macbereskahttp://2010.igem.org/File:8octLovtap.jpgFile:8octLovtap.jpg2010-10-27T11:51:15Z<p>Macbereska: </p>
<hr />
<div></div>Macbereskahttp://2010.igem.org/File:123.JPGFile:123.JPG2010-10-27T11:44:07Z<p>Macbereska: </p>
<hr />
<div></div>Macbereskahttp://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_sensorTeam:Edinburgh/Notebook/Blue light sensor2010-10-27T11:42:52Z<p>Macbereska: /* Blue Light Sensor */</p>
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<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_sensor">blue sensor</a></li><br />
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<span style="color:ivory;">These <b>notes</b> correspond to the part of the <b>project</b> detailed <a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_sensor">here</a>.</span><br />
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<br />
==Blue Light Sensor==<br />
<br><br />
<br />
'''14/07/10'''<br />
<br />
Received parts from Lausanne:<br />
*BBa_191006 (113 ng/µL) – LovTAP alone<br />
*BBa_191004 (166 ng/µL) – Readout <br />
*BBa_191003 (230 ng/µL) – whole system<br />
*BBa_191009 (85 ng/µL) – mutant <br />
*Sosnick vector containing TrpR (393 ng/µL) – vector <br />
<br />
<br />
<br />
'''16/07/10'''<br />
<br />
Transformations using 100 µL of cells, 900 µL broth<br />
*K191004 - amp<br />
*K191003 - kanamycin<br />
*K191009 – kanamycin<br />
*Sosnick vector with TrpR – amp<br />
<br />
Note: Labeling on tubes is unclear – K191009 and K191004 got mixed up, so plating both tubes on both Kan and amp<br />
<br />
<br />
'''22/07/10'''<br />
<br />
*Minipreps: K0998010 – 5x (1 didn’t grow, but did anyway)<br />
*Blue sensors (3 biobricks and Sosnick vector) – cultures set to grow overnight<br />
<br />
'''25/07/10'''<br />
Digest of the blue light sensor minipreps was performed with EcoRI and BamHI; samples:<br />
*3- K19100'''4'''; 900ul, (3)<br />
*4- K19100'''4'''; 100ul, (2)<br />
*5-K19100'''9'''; 100ul, (1)<br />
*6- K19100'''9'''; 100ul, (6)<br />
*7- K19100'''9'''; 900ul, (4)<br />
*8- K19100'''9'''; 100ul, (2)<br />
*9- K19100'''3'''; 900ul, (2)<br />
*10- K19100'''3'''; 900ul, (5)<br />
*11- K19100'''3'''; 100ul, (4)<br />
*12- K19100'''3'''; 100ul, (1)<br />
*13- K19100'''4'''; 100ul, (3)<br />
*14- K19100'''4'''; 900ul, (5)<br />
<br />
Lanes- 1- marker, 2-9 sampels as in order, 10-empty, 11, 12- marker instead of the samples- need to run last two again<br />
<br />
[[Image:109.JPG]]<br />
<br />
[[Image:111.JPG]]<br />
LAST TWO LANES- NSAMPLES NO 13 AND 14 FROM THE ABOVE LIST<br />
<br />
<br />
'''28/07/10'''<br />
<br />
Double transformants made using a novel cowboy procedure- double transformation with 4ul of each piece od DNA. Chances of succes- very very low... But at least it will be proved that it won't work!<br />
Trans. 1: BBa_K191004+ K191003- read out + LovTap<br />
Trans. 2: BBa_K191004+ K191009- read out + mutated LovTap<br />
<br />
'''30/7/10'''<br />
set up experiments to see if the blue light sensor will activate the response system under the daylight. Both liquid cultures and plates were set up, with red and white colonies- first supposed to turn white in the daylight and stay red in the dark (under the foil), second ones- no change of the colour. The same applies to the liquid cultures. However, the experiment failed- no proper growth was observed, and if there was a growth - it was very weak. No pigment observed, apart from the small points in the centre. <br />
Hypothesis- not stable plasmids-- cells look like they are not really resistant. Recorded growth:<br />
{|border="1"<br />
|<br />
|K191004+9 control<br />
|K191004+9<br />
|K191004+3 control<br />
|K191004+3<br />
|-<br />
|DARK<br />
| ++ , white<br />
| +++ , white<br />
| ++ , white<br />
| +++ , white with red points<br />
|-<br />
| LIGHT<br />
| + , white<br />
| +++ , white with red point<br />
| + -almost no growth <br />
| ++ white<br />
|}<br />
<br />
<br />
<br />
'''2/8/10'''<br />
<br />
Run the digest of the minipreps from the white and red cultures of double transformants. No proper DNA band in the solution- repeat the procedure or not? Don't know what to do.... again. <br />
[[File:Gel 3.jpg]]<br />
Collect the thoughts and ideas and start again tomorrow!<br />
<br />
'''6/8/10'''<br />
<br />
double transformants were prepared again, using non-cowboy procedure. 04 cells were first MADE COMPETENT, then transformed with 03 and 09, also 1 neg control was plated. Grow white on the intitial plate, then turned red if subcultured. <br />
<br />
'''7-8/08'''<br />
<br />
'Subbed these to duplicate fresh plates and incubated overnight at room temperature <br />
either under a fluorescent lamp or wrapped in foil - all white in both cases (and growth <br />
not great, probably because of low temperature)'. <br />
The plates were moved to 37C with the lamp /one under the light, one in the dark. so far -no results, checking in an hour/. <br />
<br />
Another idea- check for stability of the insert- we don't know if the plasmids are compatibile. Streaking from original plates to check if single colonies will be a mixture of white and red or just red- origin? no plasmid maps from Lausanne. <br />
by Chairman-->' If we can't find this information, I think our best course is to <br />
PCR out both inserts and combine them in a single vector, preferably pSB1C3.'<br />
<br />
<br />
'''9/8/2010'''<br />
<br />
to do:<br />
<br />
*primers for the parts to put them in a vector<br />
*check the lamp every hour!!<br />
<br />
go thruough Lausanne website to find the plasmid maps<br />
get back to Mexico about the parts and etc.<br />
find out the conditions for the blue light sensor assay!<br />
<br />
'''10/08/10'''<br />
* assay with dark/light liquid cultures - fluorescence measurment and OD, after 5 h in the shaker under lamp- see video and pictures<br />
*email to Mexico<br />
<br />
'''11/08/10'''<br />
<br />
*primer designed for combining LOVTap and Readout1 into 1 cell<br />
*repeated assay with the smae samples, after overngiht in RT and %5h under lamp (for the light samples) and RT without shaking<br />
*primary cultures streaked to amp/kan/IPTG plates- part 4+9 and 4+3 -three clones each<br />
<br />
'''12/08/10'''<br />
*results analysis after LOVTap experiment:check file, add graphs later.<br />
*email from Mexico about Blue promoter<br />
<br />
'''16/08/10'''<br />
* streaked single clone for 9+4 and 3+4 (clones number 5 and 2, respectively)- for tomorrow's experiment<br />
* minipreped mysterious double transformants and Sacb smples<br />
* tried out the box on the shaker- unscrewed the things. <br />
* made the graphs from the second reading<br />
* 03 and 09 LovTap taken for sequencing. <br />
<br />
to do: decide on the illumination length and think about controls and how many samples do you want to put. check Lausanne website<br />
t-test to check to importancy of the differences between the means.<br />
<br />
'''17/07/10'''<br />
* desinged experimental set up for the LovTap blue light response experiment /see next day/<br />
* BAD NEWS: there is a suspicion of 1bp deletion in LovTap!!! Chairman noticed it when he was checking the parts base by base. Should have done it much much earlier probably to save the time... <br />
* BAD NEWS: lack of the proper thinking put in doubt all the results from previous two experiments (that is why you can't find any files of it attached)- there was no IPTG added to the samples, therefore LOVTap expression was not induced- no point to do T-test right now... :( Well, at least I will learn from it and add it next time!<br />
<br />
<br />
'''18/08/10'''<br />
<br />
* tested LovTap under the blue light<br />
* conditions:<br />
Liquid cultures (same as for minipreps) were grown overnight in 37C with antibiotics. <br />
The next day, bottles with 3.5 ml of broth were inoculated with the 0.5 ml of appropriate cultures (0.5. ml of broth added as a control).<br />
*sample designation: (# refers to the clone number form the original plate). Original plates are the ones made on '''INSERT THE DATE LAZY''' and have 2 clones on each plates (clones1-3 are K191003&4 double transformants, clones 4-6 are K191009+4 double transformants). <br />
<br />
# K191003+4 (#1) + Amp + Kan + IPTG + LIGHT <br />
# K191003+4 (#1) + Amp + Kan IPTG + DARK <br />
# K191003+4 (#2) + Amp + Kan IPTG + LIGHT <br />
# K191003+4 (#2) + Amp + Kan IPTG + DARK <br />
# K191009+4 (#4) + Amp + Kan IPTG +LIGHT <br />
# K191009+4 (#4) + Amp + Kan IPTG +DARK <br />
# K191009+4 (#5) + Amp + Kan IPTG +LIGHT <br />
# K191009+4 (#5) + Amp + Kan IPTG +DARK <br />
# K191004 ONLY #2+ Amp + LIGHT <br />
# K191004 ONLY #2+ Amp + DARK <br />
# neg. control- broth, Amp + Kan + IPTG + LIGHT<br />
<br />
* samples designated DARK were wrapped in the aluminium foil straight after the inoculation; the cap was not wrapped (not see through anyway)so that samples could be easily taken without unwrapping the bottles from the foil. <br />
*All the samples were then taken to the shaker in the hot room and placed in prepared spots: for the dark- anywhere on an easily accessible holder, for light- in 2 rows of holders. <br />
*Light samples were covered with the cardboard box with blue LEDs attached on the sides, in a way to optimize light accession to the samples (ask Sarah for the details). Box was securely taped to the shaker. <br />
* Measurments were taken every half an hour: 200ul of the samples were placed in the numbered cuvette and then 800ul of water was added. Readings of fluroscence (green mode) were taken in standard fluorescence units (SFU, two readings each). Then optical density was measured (upstairs in the incubator room) at 360 nm wavelength. Control was used to calibrate the machine (set ref button). Also 2 readings were taken. Samples were shaken by inversion prior to taking measurment. <br />
* Experiment was stopped after 3 hours (6 sets of reading taken). <br />
<br />
*'''Result analysis'''<br />
see the Excel file attached: <br />
<br />
[[File:LovTap FAIL exp 18aug.xls]]<br />
<br />
To generate the data for the graph:<br />
# means were calculated from 2 readings of optical density and fluorescence<br />
# (mean SFU minus background SFU) was divided by (mean OD minus background OD, if different from 0).<br />
# table was made for the samples at different points of time- e.g. sample 1 after 30 min, 60 min, 90 min, 120 min etc. <br />
# graph was made with series named the same as the samples<br />
<br />
[[File:Untitled.JPG]]<br />
<br />
**BAD BAD VERY BAD NEWS: after 2 hours of measurments the results of the sequencing were know and revealed that THERE IS A 1BP DELETION IN LOVTAP 03 AND 09 WHICH RESULTS IN FRAMESHIFT AND LACK OF THE FUNCTIONAL PRODUCT!!!! :(:(:( despite this information, it was decided that the experiment will be finished and analysed as initially planned.<br />
<br />
'''19/08/2010'''<br />
<br />
* Pcr- mutagenensis of LovTap- Mabel, adding missing bp. <br />
* Puryfing PCR product from the solution. <br />
<br />
<br />
'''20/8/2010'''<br />
* PCR run on the gel -correct band of ~5.5. kb size, suggest that the PCR worked well. couple of other fainter bands <br />
* Self -ligation overnight (MABEL self ligation- 12ul of H2O, 4ul of DNA, 2 ul of Ligase buffer, 1 ul of T4 DNA ligase, 1ul of T4 polynucleotide kinase. <br />
* transformation and setting up the liquid cultures for the minipreps over the weekend. <br />
<br />
[[Image:123.JPG]]<br />
<br />
'''23/08/2010'''<br />
* mini-prep DNA from the colonies after PCR- marker worn off in the shaker,so 3.2. and 9.1. got mixed up- probably sequencing necessary to distinguish the mutated version from non-mutated <br />
* digest minipreps with EcoRI, run the gel[photo]<br />
* run 5ul of the ligated 03 and 09 on the gel /also Richards samples/.<br />
<br />
[[Image:124.JPG]]<br />
<br />
'''24/08/2010'''<br />
* repeated digest of mutated LovTap samples: 3.1, 3.?, 3.3., 3.4 9.?, 9.2 with EcoRi<br />
* minipreped 6 more clones of mutated LovTap 03 and 09 from the same plate, labelled- 3.6, 3.7, 3.8, 9.4, 9.5, 9.6. Run the digest with EcoRI. Note: 3.7 sample is higly diluted as 80ul of EB was used instead of 40ul. oops. <br />
* delivered mutated 3.? and 9.2 minipreped clones for sequencing /only 2 reactions: each sample- forward/.<br />
<br />
'''04/10/10'''<br />
We may have a working LovTap construct + red/white (RFP) reporter system thanks to Mexico.<br />
<br />
<br />
Minor characterisation experiment:<br><br />
-LovTap constructed with red-white RFP reporter<br><br />
-If activated, the blue light sensor transformants should produce red colonies<br><br />
-Blue light sensor containing-colonies from transformant plate streaked onto two plates<br><br />
-One plate left in the light, the other left in the dark<br><br />
-If the one in the light grows red and the one in the dark doesn't, it is a good indication that the construct is working<br><br><br />
<br />
'''8/10/2010'''<br />
* repetaed LOVTap charactierzation- with TrpR mutant and WT under light/dark conidtion. <br />
* no clear response was seen. <br />
<br />
<br />
'''22/10/2010'''<br />
* checking temperature response of LOVTap<br />
* setting up samples with CMM minum medium, enriched in thymine, trace elements and glucose.<br />
CELLS FAIL TO GROW!:(</div>Macbereskahttp://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_sensorTeam:Edinburgh/Notebook/Blue light sensor2010-10-27T11:40:01Z<p>Macbereska: /* Blue Light Sensor */</p>
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<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_producer">blue light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_producer">green light</a></li><br />
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<span style="color:ivory;">These <b>notes</b> correspond to the part of the <b>project</b> detailed <a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_sensor">here</a>.</span><br />
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==Blue Light Sensor==<br />
<br><br />
<br />
'''14/07/10'''<br />
<br />
Received parts from Lausanne:<br />
*BBa_191006 (113 ng/µL) – LovTAP alone<br />
*BBa_191004 (166 ng/µL) – Readout <br />
*BBa_191003 (230 ng/µL) – whole system<br />
*BBa_191009 (85 ng/µL) – mutant <br />
*Sosnick vector containing TrpR (393 ng/µL) – vector <br />
<br />
<br />
<br />
'''16/07/10'''<br />
<br />
Transformations using 100 µL of cells, 900 µL broth<br />
*K191004 - amp<br />
*K191003 - kanamycin<br />
*K191009 – kanamycin<br />
*Sosnick vector with TrpR – amp<br />
<br />
Note: Labeling on tubes is unclear – K191009 and K191004 got mixed up, so plating both tubes on both Kan and amp<br />
<br />
<br />
'''22/07/10'''<br />
<br />
*Minipreps: K0998010 – 5x (1 didn’t grow, but did anyway)<br />
*Blue sensors (3 biobricks and Sosnick vector) – cultures set to grow overnight<br />
<br />
'''25/07/10'''<br />
Digest of the blue light sensor minipreps was performed with EcoRI and BamHI; samples:<br />
*3- K19100'''4'''; 900ul, (3)<br />
*4- K19100'''4'''; 100ul, (2)<br />
*5-K19100'''9'''; 100ul, (1)<br />
*6- K19100'''9'''; 100ul, (6)<br />
*7- K19100'''9'''; 900ul, (4)<br />
*8- K19100'''9'''; 100ul, (2)<br />
*9- K19100'''3'''; 900ul, (2)<br />
*10- K19100'''3'''; 900ul, (5)<br />
*11- K19100'''3'''; 100ul, (4)<br />
*12- K19100'''3'''; 100ul, (1)<br />
*13- K19100'''4'''; 100ul, (3)<br />
*14- K19100'''4'''; 900ul, (5)<br />
<br />
Lanes- 1- marker, 2-9 sampels as in order, 10-empty, 11, 12- marker instead of the samples- need to run last two again<br />
<br />
[[Image:109.JPG]]<br />
<br />
[[Image:111.JPG]]<br />
LAST TWO LANES- NSAMPLES NO 13 AND 14 FROM THE ABOVE LIST<br />
<br />
<br />
'''28/07/10'''<br />
<br />
Double transformants made using a novel cowboy procedure- double transformation with 4ul of each piece od DNA. Chances of succes- very very low... But at least it will be proved that it won't work!<br />
Trans. 1: BBa_K191004+ K191003- read out + LovTap<br />
Trans. 2: BBa_K191004+ K191009- read out + mutated LovTap<br />
<br />
'''30/7/10'''<br />
set up experiments to see if the blue light sensor will activate the response system under the daylight. Both liquid cultures and plates were set up, with red and white colonies- first supposed to turn white in the daylight and stay red in the dark (under the foil), second ones- no change of the colour. The same applies to the liquid cultures. However, the experiment failed- no proper growth was observed, and if there was a growth - it was very weak. No pigment observed, apart from the small points in the centre. <br />
Hypothesis- not stable plasmids-- cells look like they are not really resistant. Recorded growth:<br />
{|border="1"<br />
|<br />
|K191004+9 control<br />
|K191004+9<br />
|K191004+3 control<br />
|K191004+3<br />
|-<br />
|DARK<br />
| ++ , white<br />
| +++ , white<br />
| ++ , white<br />
| +++ , white with red points<br />
|-<br />
| LIGHT<br />
| + , white<br />
| +++ , white with red point<br />
| + -almost no growth <br />
| ++ white<br />
|}<br />
<br />
<br />
<br />
'''2/8/10'''<br />
<br />
Run the digest of the minipreps from the white and red cultures of double transformants. No proper DNA band in the solution- repeat the procedure or not? Don't know what to do.... again. <br />
[[File:Gel 3.jpg]]<br />
Collect the thoughts and ideas and start again tomorrow!<br />
<br />
'''6/8/10'''<br />
<br />
double transformants were prepared again, using non-cowboy procedure. 04 cells were first MADE COMPETENT, then transformed with 03 and 09, also 1 neg control was plated. Grow white on the intitial plate, then turned red if subcultured. <br />
<br />
'''7-8/08'''<br />
<br />
'Subbed these to duplicate fresh plates and incubated overnight at room temperature <br />
either under a fluorescent lamp or wrapped in foil - all white in both cases (and growth <br />
not great, probably because of low temperature)'. <br />
The plates were moved to 37C with the lamp /one under the light, one in the dark. so far -no results, checking in an hour/. <br />
<br />
Another idea- check for stability of the insert- we don't know if the plasmids are compatibile. Streaking from original plates to check if single colonies will be a mixture of white and red or just red- origin? no plasmid maps from Lausanne. <br />
by Chairman-->' If we can't find this information, I think our best course is to <br />
PCR out both inserts and combine them in a single vector, preferably pSB1C3.'<br />
<br />
<br />
'''9/8/2010'''<br />
<br />
to do:<br />
<br />
*primers for the parts to put them in a vector<br />
*check the lamp every hour!!<br />
<br />
go thruough Lausanne website to find the plasmid maps<br />
get back to Mexico about the parts and etc.<br />
find out the conditions for the blue light sensor assay!<br />
<br />
'''10/08/10'''<br />
* assay with dark/light liquid cultures - fluorescence measurment and OD, after 5 h in the shaker under lamp- see video and pictures<br />
*email to Mexico<br />
<br />
'''11/08/10'''<br />
<br />
*primer designed for combining LOVTap and Readout1 into 1 cell<br />
*repeated assay with the smae samples, after overngiht in RT and %5h under lamp (for the light samples) and RT without shaking<br />
*primary cultures streaked to amp/kan/IPTG plates- part 4+9 and 4+3 -three clones each<br />
<br />
'''12/08/10'''<br />
*results analysis after LOVTap experiment:check file, add graphs later.<br />
*email from Mexico about Blue promoter<br />
<br />
'''16/08/10'''<br />
* streaked single clone for 9+4 and 3+4 (clones number 5 and 2, respectively)- for tomorrow's experiment<br />
* minipreped mysterious double transformants and Sacb smples<br />
* tried out the box on the shaker- unscrewed the things. <br />
* made the graphs from the second reading<br />
* 03 and 09 LovTap taken for sequencing. <br />
<br />
to do: decide on the illumination length and think about controls and how many samples do you want to put. check Lausanne website<br />
t-test to check to importancy of the differences between the means.<br />
<br />
'''17/07/10'''<br />
* desinged experimental set up for the LovTap blue light response experiment /see next day/<br />
* BAD NEWS: there is a suspicion of 1bp deletion in LovTap!!! Chairman noticed it when he was checking the parts base by base. Should have done it much much earlier probably to save the time... <br />
* BAD NEWS: lack of the proper thinking put in doubt all the results from previous two experiments (that is why you can't find any files of it attached)- there was no IPTG added to the samples, therefore LOVTap expression was not induced- no point to do T-test right now... :( Well, at least I will learn from it and add it next time!<br />
<br />
<br />
'''18/08/10'''<br />
<br />
* tested LovTap under the blue light<br />
* conditions:<br />
Liquid cultures (same as for minipreps) were grown overnight in 37C with antibiotics. <br />
The next day, bottles with 3.5 ml of broth were inoculated with the 0.5 ml of appropriate cultures (0.5. ml of broth added as a control).<br />
*sample designation: (# refers to the clone number form the original plate). Original plates are the ones made on '''INSERT THE DATE LAZY''' and have 2 clones on each plates (clones1-3 are K191003&4 double transformants, clones 4-6 are K191009+4 double transformants). <br />
<br />
# K191003+4 (#1) + Amp + Kan + IPTG + LIGHT <br />
# K191003+4 (#1) + Amp + Kan IPTG + DARK <br />
# K191003+4 (#2) + Amp + Kan IPTG + LIGHT <br />
# K191003+4 (#2) + Amp + Kan IPTG + DARK <br />
# K191009+4 (#4) + Amp + Kan IPTG +LIGHT <br />
# K191009+4 (#4) + Amp + Kan IPTG +DARK <br />
# K191009+4 (#5) + Amp + Kan IPTG +LIGHT <br />
# K191009+4 (#5) + Amp + Kan IPTG +DARK <br />
# K191004 ONLY #2+ Amp + LIGHT <br />
# K191004 ONLY #2+ Amp + DARK <br />
# neg. control- broth, Amp + Kan + IPTG + LIGHT<br />
<br />
* samples designated DARK were wrapped in the aluminium foil straight after the inoculation; the cap was not wrapped (not see through anyway)so that samples could be easily taken without unwrapping the bottles from the foil. <br />
*All the samples were then taken to the shaker in the hot room and placed in prepared spots: for the dark- anywhere on an easily accessible holder, for light- in 2 rows of holders. <br />
*Light samples were covered with the cardboard box with blue LEDs attached on the sides, in a way to optimize light accession to the samples (ask Sarah for the details). Box was securely taped to the shaker. <br />
* Measurments were taken every half an hour: 200ul of the samples were placed in the numbered cuvette and then 800ul of water was added. Readings of fluroscence (green mode) were taken in standard fluorescence units (SFU, two readings each). Then optical density was measured (upstairs in the incubator room) at 360 nm wavelength. Control was used to calibrate the machine (set ref button). Also 2 readings were taken. Samples were shaken by inversion prior to taking measurment. <br />
* Experiment was stopped after 3 hours (6 sets of reading taken). <br />
<br />
*'''Result analysis'''<br />
see the Excel file attached: <br />
<br />
[[File:LovTap FAIL exp 18aug.xls]]<br />
<br />
To generate the data for the graph:<br />
# means were calculated from 2 readings of optical density and fluorescence<br />
# (mean SFU minus background SFU) was divided by (mean OD minus background OD, if different from 0).<br />
# table was made for the samples at different points of time- e.g. sample 1 after 30 min, 60 min, 90 min, 120 min etc. <br />
# graph was made with series named the same as the samples<br />
<br />
[[File:Untitled.JPG]]<br />
<br />
**BAD BAD VERY BAD NEWS: after 2 hours of measurments the results of the sequencing were know and revealed that THERE IS A 1BP DELETION IN LOVTAP 03 AND 09 WHICH RESULTS IN FRAMESHIFT AND LACK OF THE FUNCTIONAL PRODUCT!!!! :(:(:( despite this information, it was decided that the experiment will be finished and analysed as initially planned.<br />
<br />
'''19/08/2010'''<br />
<br />
* Pcr- mutagenensis of LovTap- Mabel, adding missing bp. <br />
* Puryfing PCR product from the solution. <br />
<br />
<br />
'''20/8/2010'''<br />
* PCR run on the gel -correct band of ~5.5. kb size, suggest that the PCR worked well. couple of other fainter bands <br />
* Self -ligation overnight (MABEL self ligation- 12ul of H2O, 4ul of DNA, 2 ul of Ligase buffer, 1 ul of T4 DNA ligase, 1ul of T4 polynucleotide kinase. <br />
* transformation and setting up the liquid cultures for the minipreps over the weekend. <br />
<br />
'''23/08/2010'''<br />
* mini-prep DNA from the colonies after PCR- marker worn off in the shaker,so 3.2. and 9.1. got mixed up- probably sequencing necessary to distinguish the mutated version from non-mutated <br />
* digest minipreps with EcoRI, run the gel<br />
* run 5ul of the ligated 03 and 09 on the gel /also Richards samples/.<br />
<br />
'''24/08/2010'''<br />
* repeated digest of mutated LovTap samples: 3.1, 3.?, 3.3., 3.4 9.?, 9.2 with EcoRi<br />
* minipreped 6 more clones of mutated LovTap 03 and 09 from the same plate, labelled- 3.6, 3.7, 3.8, 9.4, 9.5, 9.6. Run the digest with EcoRI. Note: 3.7 sample is higly diluted as 80ul of EB was used instead of 40ul. oops. <br />
* delivered mutated 3.? and 9.2 minipreped clones for sequencing /only 2 reactions: each sample- forward/.<br />
<br />
'''04/10/10'''<br />
We may have a working LovTap construct + red/white (RFP) reporter system thanks to Mexico.<br />
<br />
<br />
Minor characterisation experiment:<br><br />
-LovTap constructed with red-white RFP reporter<br><br />
-If activated, the blue light sensor transformants should produce red colonies<br><br />
-Blue light sensor containing-colonies from transformant plate streaked onto two plates<br><br />
-One plate left in the light, the other left in the dark<br><br />
-If the one in the light grows red and the one in the dark doesn't, it is a good indication that the construct is working<br><br><br />
<br />
'''8/10/2010'''<br />
* repetaed LOVTap charactierzation- with TrpR mutant and WT under light/dark conidtion. <br />
* no clear response was seen. <br />
<br />
<br />
'''22/10/2010'''<br />
* checking temperature response of LOVTap<br />
* setting up samples with CMM minum medium, enriched in thymine, trace elements and glucose.<br />
CELLS FAIL TO GROW!:(</div>Macbereskahttp://2010.igem.org/File:152.JPGFile:152.JPG2010-10-27T10:57:38Z<p>Macbereska: </p>
<hr />
<div></div>Macbereskahttp://2010.igem.org/File:146.JPGFile:146.JPG2010-10-27T10:56:49Z<p>Macbereska: </p>
<hr />
<div></div>Macbereskahttp://2010.igem.org/Team:Edinburgh/Notebook/Red_light_sensorTeam:Edinburgh/Notebook/Red light sensor2010-10-26T23:42:56Z<p>Macbereska: /* Red Light Sensor */</p>
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<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Human">results</a></li><br />
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<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook" class="dir">lab notes&nbsp;&nbsp;&nbsp;</a><br />
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<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Collaboration">collaboration</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Attribution">attribution</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/BRIDGE">BRIDGE</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_producer">red light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_sensor">red sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_producer">blue light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_producer">green light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_sensor">green sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Modelling">modelling</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Safety">safety</a></li><br />
<li><a href="http://www.openwetware.org/wiki/French_Lab">protocols</a></li><br />
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<li><a href="https://2010.igem.org/Team:Edinburgh/Acknowledgements" class="dir">acknowledgements</a><br />
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<div id="windowbox" style="border: .2em solid #660000; padding: 5px; position:fixed; top:50%; right:30px; width:8%;"><br />
<span style="color:ivory;">Throughout this wiki there are words in <b>bold</b> that indicate a relevance to <b>human aspects</b>. It will become obvious that <b>human aspects</b> are a part of almost everything in <b>iGEM</b>.</span><br />
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<br />
== Red Light Sensor ==<br />
<br><br />
<br />
'''22/6/10'''<br />
<br />
Transformation of BBa_M30109; well 14 H, distribution plate 3 (2010)--> red light sensor <br />
<br />
'''24/6/10'''<br />
<br />
Subcultured transformants from ampicillin plates to new plates<br />
<br />
<br />
'''25/6/10'''<br />
<br />
1) DNA mini-preps were prepared from the transformants<br />
2) DNA mini-preps were cut with EcoRI enzyme to check for the presence of the plasmid (and DNA); agarose gel was prepared for DNA electrophoresis after cutting.<br />
3) Results from cutting showed no presence of the sensor.<br />
Troubleshooting: was the plate number correct? Was procedure performed correct?<br />
by:Maria, Will, Marta <br />
<br />
<br />
'''28/6/10'''<br />
<br />
Tried to determine what was wrong with the red sensor that failed to appear in the gel. We definitely had the correct plate and well. We checked the seqeunce of the part and found that the sequence given does not match any given in BLAST. We suspect either that part of the plasmid was added to the distribution/registry or that the plasmid contains an illegal EcoR1 site, but are re-transforming cells just in case there was an experimental error.<br />
Due to failure of the previous transformation competent cells were retransformed with the two plasmids, as before. Two Chloramphenicol spread plates for each plasmid were made one of each pair containing 100micro-l of cells the other containing the 900micro-l remainder spun down and resuspended in 100micro-l of sterile LB.<br />
by: Richard, Lu<br />
<br />
<br />
'''29/6/10'''<br />
<br />
Streaking the colonies from the chloramphenicol plates on the new agar + chloramphenicol plates.<br />
<br />
<br />
'''30/6/10'''<br />
<br />
No growth on plates.<br />
<br />
<br />
'''5/7/10'''<br />
<br />
Made kanomycin 32mg/ml, IPTG 90mg/ml plates. Transformed cells with individual red light sensor parts (BBa_I15008 - 2010 distribution Plate 2 Well 13J; BBa-I15009 - distribution plate 1 Well 20F; BBa_I15010 - distribution Plate 3 Well 20D; & K098010 - distribution Plate 3 11N). Plated cells onto kanomycin plates and left to incubate overnight. <br />
by: Hannah and Richard<br />
<br />
<br />
'''6/7/10'''<br />
<br />
Richard/Hannah - made subcultures of red light sensor part transformants.<br />
<br />
*Agarose Gel Eleckrophoresis of composite red sensor M30109<br />
*The bands are not the right size<br />
*Sequencing done by the registry starts in the middle of the sequence (for forward sequence)<br />
*So it seems the plasmid doesn’t contain the right sequence<br />
*We will try to assemble it from individual parts<br />
<br />
[[Image:149.JPG]]<br />
<br />
'''8/7/10'''<br />
<br />
Plasmid minipreps of I15009 (the only red sensor part transformant that grew in liquid culture)<br />
by: Richard and Hannah<br />
<br />
<br />
'''9/7/10'''<br />
<br />
Gel run with I15009. <br />
<br />
{| border="1"<br />
| Lane 1 <br />
| Lane 2 <br />
| Lane 3 <br />
| Lane 4 <br />
| Lane 5 <br />
| Lane 6 <br />
| Lane 7 <br />
| Lane 8<br />
|-<br />
| Ladder <br />
| Pure SacB <br />
| Pure SacB & EcoRI <br />
| sacB & catR PCR <br />
| I15009 digest <br />
| S248T PCR Product <br />
| Purified 356K <br />
| Purified 356R<br />
|}<br />
<br />
*Insert Picture of Gel*<br />
<br />
'''13/7/2010'''<br />
NOTHING WORKS. No idea why...<br />
<br />
'''16/7/10'''<br />
<br />
Because the red light sensing parts were transformed with 50 µL or water instead of 15 µL during the first transformation, another attempted was tried.<br />
Transforming:<br />
*I15008 (all red light sensor parts)<br />
*I15009<br />
*I15010<br />
used 200 µL of cells, 800 µL of broth. Kan resistance.<br />
<br />
<br />
'''23/07/10'''<br />
<br />
Double digest of K098010. Ran gel. Lane was empty:<br />
<br />
[[Image:153.JPG]]<br />
*Not going to bother saying what is in each lane. <br />
<br />
'''30/7/10'''<br />
<br />
Primers were ordered in order to PCR out the Red Light Sensor Parts from the [http://partsregistry.org/wiki/index.php?title=Part:BBa_M30109 Bio Brick BBa_M30109]<br />
<br />
Forward Primer for PBad promoter [http://partsregistry.org/wiki/index.php?title=Part:BBa_I13453 Bio Brick BBa_I13453]<br><br />
Reverse Primer for Heme Oxygenase (ho1) [http://partsregistry.org/wiki/index.php?title=Part:BBa_I15008 Bio Brick BBa_I15008]<br><br />
Reverse Primer for Phycocyanobilin:Ferredoxin Oxidoreductase (PcyA) [http://partsregistry.org/wiki/index.php?title=Part:BBa_I15009 Bio Brick BBa_I15009]<br><br />
Forward Primer for TetR repressible promoter [http://partsregistry.org/wiki/index.php?title=Part:BBa_R0040 Bio Brick BBa_R0040]<br><br />
Reverse Primer for cph8 (Cph1/EnvZ fusion)Chimeric Cph1 light receptor/EnvZ protein [http://partsregistry.org/wiki/index.php?title=Part:BBa_I15010 Bio Brick BBa_I15010]<br><br />
<br />
<br />
'''3/8/10'''<br />
<br />
A PCR was run with the above primers in 3 distinct mixtures in an attempt to amplify out the Red Light Sensor parts using Bio Brick BBa_M30109 as a template.<br />
PCR 1 Contained The PBad promoter Forward Primer & the PcyA Reverse Primer<br><br />
PCR 2 Contained The TetR repressible promoter Forward Primer & the cph8 Reverse Primer <br><br />
PCR 3 Contained The PBad promoter Forward Primer & the cph8 Reverse Primer<br />
<br />
And a gel was run of the PCR products: Marker(Lane 1&6), only visible product- suggested PCR I15010 with RBS & promoter (Lane 3). <br />
[[Image:116.JPG]]<br />
<br />
Due to the successful amplification of the I15010 cph8 Red light sensor part, the DNA was ligated into plasmid psb1C3.<br />
<br />
<br />
'''4/8/10'''<br />
<br />
Competent cells were transformed with the ligated psb1C3 plasmid containing I15010 and plated out on Cml 40 + IPTG 90 plates and incubated O/N at 37 degrees Celsius.<br />
<br />
A PCR was run using the following primers:<br />
PCR 1 Contained The BBa_B0034 RBS Forward Primer & the ho1 Reverse Primer<br><br />
PCR 2 Contained The BBa_B0034 RBS Forward Primer & the PcyA Reverse Primer<br><br />
PCR 3 Contained The PBad promoter Forward Primer & the ho1 Reverse Primer<br><br />
PCR 4 Contained The PBad promoter Forward Primer & the PcyA Reverse Primer<br><br />
<br />
And a gel was run of the PCR products.<br />
<br />
*Insert picture of Gel*<br />
<br />
No successful amplification was seen.<br />
<br />
'''11/8/10'''<br />
<br />
Update from Maria. <br />
The transformations and PCR amplification of the HO1-pcyA fusion construct have nearly all failed. <br />
This weekend the kanamycin resistant version of the construct (K089010 in pSB3K3) was transformed and cultures grew.<br />
We already have a PCR product of the fused cph8-envZ sensing part. We now need to put the newly transformed K089010 in front of the sensor in order to rebuild the original sensor, which is well and truly fucked.<br />
<br />
[[Image:161.jpg]]<br />
<br />
'''16/8/10'''<br />
<br />
Successful PCR of K098010 using primers for EcoRI & I15009 reverse primer.<br />
[[Image:119.JPG]]<br />
<br />
<br />
'''17/8/10'''<br />
<br />
* The PCR product of the Harvard K098010 part was purified and then digested with both XbaI & SpeI as was the PCR product of cph8.<br />
* These digestions were then ligated together ON using in two orientations.<br />
<br />
'''18 &19/8/2010'''<br />
<br />
* Fusion PCR C + H with R004081 & I15009-1 <br />
* also running H + C same primers as negative control<br />
* run gel of PCR prod & 5 ul of ligation<br />
* luxAB -SpeI; lumP -XbaI, ;uxCDE -XbaI<br />
* lumP = P1024<br />
* luxAB + lumP and luxAB + lyxCDE ligation<br />
[[Image:120.JPG]]<br />
<br />
'''20/8/2010'''<br />
<br />
* Fusion PCR with ?????<br />
* get: <br />
<br />
'''24/8/2010'''<br />
* Digest cph8 (XbaI- yello) & Harvard (SpeI -brown)<br />
* Ligate Harvard and cph8 = harvard + cph*8<br />
* mutagenic PCR of lux CDE to mutate out site in LuxD<br />
* subculture luxAB lumP transformants<br />
* miniprep ON'S '''(?)''' set up<br />
<br />
<br />
'''25/8/2010'''<br />
<br />
* Minipreps of subcultures luxAB lumP,<br />
* digest & run gel<br />
* Purify luxCDE PCR product- from gel and froem the product; run the gel of purifications --> do MABEL self ligation- set 16C ON<br />
* Fusion PCR harv + cph8- run gel of product<br />
<br />
'''26/8/2010'''<br />
<br />
* Gel1 - minipreps 1-5, gluxCDE, PluxCDE<br />
* Gel2 - Minipreps 6-10, H + C ligation<br />
<br />
*Lane 7: Harvard + Cph8 ligation<br />
[[Image:129.JPG]]<br />
<br />
'''27/8/2010'''<br />
<br />
Transform cells with self ligated MABEL mutated luxCDE from gel and PCR product<br />
<br />
<br />
'''30/8/2010'''<br />
<br />
* Lu wants a broad sward (?).<br />
* Re-transform MABEL mutated<br />
* luxCDE from gel- PCR products<br />
* luxAB and luxABlumP double digest with EcoRi/PstI<br />
* Fusion PCR if H + cph8 using H promoter forward and cph8 reverse primer<br />
<br />
'''31/8/2010'''<br />
* Lane 1- marker, Lane 2- H+ Cph8 fPCR<br />
[[Image:133.JPG]]<br />
'''6/9/2010'''<br />
<br />
* ligated H+ C ON in psB1C3<br />
* Double digest luxCDE with EcoRI and PstI - run gel<br />
<br />
'''7/9/2010'''<br />
* Run digest luxCDE with XbaI --> gel<br />
* Gel of H + C ligation<br />
* transform cells with<br />
<br />
'''8/9/2010'''<br />
<br />
* PCR with biobrick pre/suff - primers luxCDE 1 and 4<br />
<br />
'''9/9/2010'''<br />
<br />
* Purify luxCDE PCR<br />
* Subculture transformants H + C<br />
<br />
'''10/9/2010'''<br />
<br />
* Add promoter to luxAB and luxABlumP<br />
* Liquid ON'S of H + C subculture transformants <br />
<br />
* Lab meeting: small--> <br />
**sacB - some preeliminary sucrose sensitivity characterisation data.<br />
** promoter: luxAB, luxAb-lumP- eission spectra, testing thing<br />
** red light luciferase: S184T, 356 K<br />
[[Image:143.JPG]]<br />
{| border="1"<br />
| Lane 1 <br />
| Lane 2 <br />
| Lane 3 <br />
| Lane 4<br />
|-<br />
| Ladder <br />
| Tna up<br />
| Cph8- fixing prefix <br />
| Cph8 - fixing PstI site <br />
|}<br />
<br />
'''13/9/2010'''<br />
* H + C + pSB1C3 minipreps<br />
[[Image:144.JPG]]<br />
<br />
'''14/9/2010'''<br />
* Digest H+ C minipreps with EcoRi. 100: 1,4,5. 900: 3,4,6<br />
[[Image:145.JPG]] <br />
<br />
'''???'''<br />
'''20/9/2010'''<br />
<br />
* Test luxCDE<br />
* luxCDE digest with XBaI and PstI (buffer H)--> purify and ligate to luxAB digested with SPEI PstI in buffer B after purification.<br />
* conclusion: purify digestions, ligate luxCDE into luxAB + promoter<br />
<br />
<br />
'''21/9/2010'''<br />
<br />
* Transform cells with luxAB + luxCDE ligation, plated on CML 40, IPTG 90<br />
* Made up 50 ml liquid cultures of luxAB lumP and luxAB with Cml40, IPTG 90<br />
<br />
<br><br><br />
<b>04/10/10</b><br />
<br><br />
Chris has managed to construct what we hope is a working red light sensor. It is being sent in for sequencing today. <br><br />
Minor characterisation experiment:<br><br />
-Red light sensor constructed with blue-white lacZ reporter<br><br />
-If activated, the red light sensor transformants should produce blue colonies<br><br />
-Red light sensor containing-colonies from transformant plate streaked onto two plates<br><br />
-One plate left in the light, the other left in the dark<br><br />
-If the one in the light grows blue and the one in the dark doesn't, it is a good indication that the construct is working<br><br><br />
<br />
'''12/10/10'''<br />
<br />
*Transform ''envZ'' mutant strain with YFP<br />
*Plate out onto 40mg/ml Cml<br />
<br />
'''13/10/10'''<br />
<br />
*Subcultured ''envZ'' transformants onto Cml 40 plates<br />
<br />
'''15/10/10'''<br />
<br />
*Liquid Cultures made of transformants with Cml 40 & grown ON<br />
<br />
'''16/10/10'''<br />
<br />
*Liquid ONs viewed under blue light, yellow fluorescence seen showing successful transformation.<br />
<br />
'''20/10/10'''<br />
<br />
*RLS characterisation begins.<br />
*Liquid ON's of<br />
<ul><br />
<li>JM109 – RLS.lacZ.YFP</li><br />
<li>envZ – RLS.lacZ.YFP</li><br />
<li>JM109 – YFP Control</li><br />
<li>envZ – YFP Control</li><br />
</ul><br />
were made in 5ml LB + Cml 40 and grown at 37 degrees with shaking in the dark.<br />
<br />
<p>100μl of ONs were used to inoculate two sterile LB + 40mg/ml Cml making up a total volume of 4ml in 5ml flasks which were then incubated in both light and dark, at 37C with shaking, as follows:</p><br />
<br />
<ul><br />
<li>JM109 – RLS.lacZ.YFP (Light / Dark)</li><br />
<li>envZ – RLS.lacZ.YFP (Light / Dark)</li><br />
<li>JM109 – YFP Control (Light / Dark)</li><br />
<li>envZ – YFP Control (Light / Dark)</li><br />
</ul><br />
<br />
<p>At 50 minute time intervals 200μl of each sample was taken and mixed with 800μl of sterile water in plastic cuvettes. The optical density of each sample was taken after the spectrophotometer was set with a sample of 200μl of sterile LB and 800μl water as a control, as was the luminescence, with readings for the background luminescence being taken for the sample of LB and water.</p><br />
<br />
<p>Two readings for each sample at each time interval were taken and then an average was calculated for each time interval for both optical density and luminescence.</p><br />
<br />
<p>For each sample at each time interval the average luminescence (normalised by the background luminescence) was divided by the optical density and plotted on a graph, shown below as Figure 2.</p><br />
<br />
<br />
[[Image:Ed10-RedLightSensorCharData.png]]<br />
<br />
* PCR of PhoA promoter (from genomic DNA).<br />
<br />
[[Image:171.jpg]]<br />
<br />
'''25/10/10'''<br />
<br />
*Liquid ON's made up of <br />
<li>JM109 – RLS.lacZ.YFP</li><br />
<li>envZ – RLS.lacZ.YFP</li><br />
<li>JM109 – RLS.lacZ</li><br />
<li>JM109 - lacZ Control</li><br />
in LB + 40 Cml</div>Macbereskahttp://2010.igem.org/Team:Edinburgh/Notebook/BRIDGETeam:Edinburgh/Notebook/BRIDGE2010-10-26T23:41:11Z<p>Macbereska: /* BRIDGE */</p>
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<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Future">future work</a></li><br />
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<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Aspects">human aspects</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Communication">communication of science</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Terminology">terminology research</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Wiki">wiki</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Conversations">conversations</a></li><br />
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<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Collaboration">collaboration</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Branding">branding research</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/SciFi">science fiction writing</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/FutureApps">future applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/SelfReflection">self-reflection</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Epic">the epic</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Human">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/References">references</a></li><br />
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<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook" class="dir">lab notes&nbsp;&nbsp;&nbsp;</a><br />
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<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Collaboration">collaboration</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Attribution">attribution</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/BRIDGE">BRIDGE</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_producer">red light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_sensor">red sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_producer">blue light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_producer">green light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_sensor">green sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Modelling">modelling</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Safety">safety</a></li><br />
<li><a href="http://www.openwetware.org/wiki/French_Lab">protocols</a></li><br />
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<li><a href="https://2010.igem.org/Team:Edinburgh/Acknowledgements" class="dir">acknowledgements</a><br />
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<span style="color:ivory;">Throughout this wiki there are words in <b>bold</b> that indicate a relevance to <b>human aspects</b>. It will become obvious that <b>human aspects</b> are a part of almost everything in <b>iGEM</b>.</span><br />
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<br />
== BRIDGE ==<br />
<br><br />
<br />
'''5/7/2010'''<br />
<br />
* sacB digest with EcoRI -band around kb '''(gel 1)'''<br />
[[Image:103.JPG]]<br />
<br />
<br />
'''6/7/2010'''<br />
* sacB digest with EcoRi/PstI. Band also around 5 kb. <br />
[[Image:1005.JPG]]<br />
<br />
'''7/7/2010''' <br />
<br />
* PCR of sacB to check insert. Used sacB tube 1 and 3.<br />
* Restriction digest of PCR with EcoRI and NotI. <br />
[[Image:106.JPG]]<br />
* lane 1- marker<br />
* lane 2, 3, 4 - sacB 1, 2 & 3 cut with NotI; band size: slightly bigger than 4 kb<br />
* Lane 5- PCR of LuxAB; band size around 1.5. kb<br />
* lane 6- Vector of sacB (pSB1C3, 2072bp, aroun 3kb with RFP) cut with NotI; band size around 1.5. kb<br />
* Lane 7- PCR of sacB with gene sepcific primers; band size around 1.5. kb<br />
<br />
* Trouble shooting: sacB cut with NotI give about 4kb, whereas cutting with EcoRI gives 5.5. kb... hmhmhm.<br />
there should be 2 NotI sites on either side of the insert but we only get 1 band--> is there only 1 NotI site? Furthermore, PstI/EcoRI digests give only 1 band (same as cut only with EcoRI), so maybe there is no PstI/NotI site?<br />
<br />
* Purifying sacB from PCR product to start making BRIDGE- row F in the BOX 1.<br />
<br />
'''9/7/2010'''<br />
* Making working solutions of primers from stock: 4ul of H20; 1 ul of stock solution<br />
* Primers: forward 72.3 C, revers- 77.6 C; length of the product: 2.6-2.7 kb<br />
{|border="1"<br />
|Lane 1<br />
|Lane 2<br />
|Lane 3<br />
|Lane 4<br />
|Lane 5<br />
|Lane 6<br />
|Lane 7<br />
|Lane8<br />
|-<br />
| ladder<br />
| sacB <br />
| sacB digest wtih EcoRI-HF<br />
| sacB +<br />
| digest `5009<br />
| S248T PCR product<br />
| Pure 356K<br />
| Pure 356R<br />
|}<br />
<br />
[[Image:152.JPG]]<br />
'''to do on Monday:'''<br />
* redo sacB- catR ligation<br />
* Run 5 ul of ligation on gel to check for large bands<br />
* redo RLS part transformations<br />
<br />
<br />
'''12/7/2010''' <br />
<br />
* Transformants of luxAB-edi1 failed- probably smth wrong at the purification stage...<br />
* [[Image:107.JPG]]<br />
<br />
{|border="1"<br />
|Lane 1<br />
|Lane 2<br />
|Lane 3<br />
|Lane 4<br />
|Lane 5<br />
|Lane 6<br />
|-<br />
| ladder<br />
| luxAB-ediI digest<br />
| luxAB-ediI ligation<br />
| sacB digest<br />
| catR digest<br />
| sacB- catR lgation<br />
|-<br />
| expec. no of bands<br />
| 2<br />
| 1 or 3 <br />
| 1<br />
| 1<br />
| 1 band= to 4+5<br />
|}<br />
<br />
* If all failed- purification procedure failed<br />
* If 3 and 6 failed- ligation failed<br />
* If all fine then PCR/transformation failed<br />
conclusion:...<br />
<br />
* 3 and 6 failed- ligation problem (not enough DNA, ligase).<br />
* Band 1- luxAB but no ediI (?)<br />
<br />
* Repeated digest (sacB with XbaI, catR with SpeI, LuxAB & EdiI with SpeI & SacI) and purification.<br />
* Set up ligations with 2ul ligase instead of 1 ul (1ul of H2O less). If this fails- perhaps only 2-3 h ligation ina RT (SpeI is set up in both digest- could be common cause)<br />
<br />
'''13/7/2010'''<br />
*First, run PCR of catR-sacB ligation (use DNA'''?????'''' MK1- with sacBr1 (19.6.10); MK2- sacBr1(10)<br />
* Gel electrophoresis of: digested catR, digested sacB, ligated sacb-catR, digested LuxAB-ediI and ligated luxAB-ediI along with sacB-catR PCR<br />
<br />
[[Image:108.JPG]]<br />
{|border="1"<br />
|Lane 1<br />
|Lane 2<br />
|Lane 3<br />
|Lane 4<br />
|Lane 5<br />
|Lane 6<br />
|Lane 7<br />
|-<br />
| ladder<br />
| ediI<br />
| digested LuxAB-ediI<br />
| ligated luxAB-ediI<br />
| ligated catR & sacB<br />
| PCR 1 (MK1)<br />
| PCR 2 (MK2)<br />
|}<br />
<br />
''''AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAARGH!!!!!!!!!!!!!!!1''''<br />
<br />
* Almost completely convinced it's the ligase... or me (MK). Officially taking myself off the project. xx<br />
* (Spotted 2 purified sacBs in the freezer... maybe try the original if this one fails...?)<br />
'''3/8/2010'''<br />
*minipreps: marker (lane 1& 8) & sacb 8.1-8.6( Lanes 2-7)<br />
[[Image:115.JPG]]<br />
''''9/8/2010''''<br />
<br />
Gel with :<br />
* cat + sacB (PCR product) (6)<br />
* vector + cat + sacB (7) (both done by CF)<br />
<br />
'''20/8/2010'''<br />
<br />
* Made plates variety of sucrose combinations--> 40 cml<br />
* 0%<br />
* 0.1%<br />
* 0.25%<br />
* 0.5%<br />
* 1.0%<br />
<br />
*Plated E6 miniprep, which should show some sucrose sensitivity (1plate at each concentration)<br />
*Control- E5, 1 plate at each concentration<br />
<br />
'''30/8/2010'''<br />
* Primers have arrived for up and downstream sequences of TnaA region.<br />
'''8/9/2010'''<br />
* Lane 1 &8- marker, Lane 6- cat +sacB PCR; Lane 7- vector+cat+sacB<br />
[[Image:141.JPG]]<br />
<br />
'''13/9/2010'''<br />
Plane for week begininng 13th:<br />
* Amplify RFP with primers --> RBS-F2; YFP - R2 ---> to amplify RFP<br />
* Digest UP with SpeI and B-D with XbaI<br />
* Ligate UP to BRIDGE-DOWN<br />
* Digest RFP with XbaI and SpeI<br />
* Ligate UP and RFP<br />
* Ligate DOWN and RFP<br />
<br />
* Also- registry editing for RLS (definately dodgy sequence), LovTap<br />
* Justification of reseubmission<br />
* Look up absorption/emission rates and transmission through medium for Donal<br />
<br />
'''20/9/2010'''<br />
<br />
* Achieved: cat/sacB- traA down ligation<br />
* Attempted: traA up- cat/sacB/down ligation; traA up- RFP ligation; RFP- traA down ligation/ ALL FAILED<br />
* Running: purified digest of:<br />
- cat/sacB + SpeI <br><br />
- tnaA down- XbaI<br><br />
- tnaA up + SpeI<br><br />
- cat/sacB/down + XbaI <br><br />
- RFP + XbaI<br><br />
and also running: UP- BRIDGE- DOWN ligation; UP - RFP ligation<br />
<br />
Have: PCR of cat/sacB, RFP, BRIDGE-DOWN, traA up, traA down; ligation of BRIDGE-DOWN<br />
<br />
<br />
[[Image:146.JPG]]<br />
<br />
Run gel: <br />
{| border="1"<br />
| Lane 1 <br />
| Lane 2 <br />
| Lane 3 <br />
| Lane 4 <br />
| Lane 5 <br />
| Lane 6 <br />
| Lane 7 <br />
| Lane 8<br />
|-<br />
| Ladder <br />
| Pure SacB <br />
| Pure SacB & EcoRI <br />
| sacB & catR PCR <br />
| I15009 digest <br />
| S284T PCR Product <br />
| Purified 356K <br />
| Purified 356R<br />
|}<br />
<br />
<b>25/10/10</b><br />
<br><br />
Repetitive failure of purifications with both traditional method and kits, unsure what is going wrong. Have decided to tell Chris. Off to Firbush on Wednesday.<br><br><br />
<b>01/10/10</b><br />
<br><br />
In my absence (at Firbush with the rest of the Biotechnologists) Chris has managed to construct the tnaA UP-cat-sacB-tnaA DOWN segment that we need for the first part of the BRIDGE protocol. I will continue to build the tnaA UP-RFP-tnaA DOWN segment. <br />
<br><br />
Set up ligations of RFP-DOWN and UP-RFP, left in 16C incubator overnight. <br />
<br><br><br />
<b>02/10/10</b><br />
<br><br />
Lab on a Saturday again, fun fun fun...<br />
<br><br />
Retrieved and amplified ligations and ran on gel. UP-RFP appears to be significantly shorter than RFP-DOWN and indeed shorter than it ought to be (should be about 2kb, is only around 1.5kb). <br><br />
Purified both products, then digested RFP-DOWN with XbaI. (Used 2hour incubation time to write presentation for Wednesday).<br><br />
Set up ligation of UP with RFP-DOWN and left in incubator overnight.<br><br />
<br><br />
<b>03/10/10</b><br />
<br><br />
Bus to Darwin for 11:20am; enter Darwin; lift to 8th floor; retrieve ligations from waterbath in cold room; walk down to 7th floor; put ligation in freezer; lift to ground floor; leave Darwin; catch 11:40am bus back to central.<br />
<br><br><br />
<b>04/10/10</b><br />
<br><br />
PCR of ligation of UP-RFP-DOWN. Made and ran gel along with purified PCRs from Saturday, just in case they're still not working.<br><br />
They worked! The purifications were perfect. There is a band about the size of UP-RFP-DOWN in it's lane, but it's quite faint. I will probably repeat the PCR with a lower annealing temperature (around 50) this afternoon. I suspect the UP primers do not anneal properly above 54C.<br />
<br><br><br />
<b>07/10/10</b><br />
<br><br />
Chris set up overnight cultures of DH5-alpha cells and JM109 cells, both containing the lambda-red plasmid, in 2.5ml LB with tetracycline.<br><br />
Repeated PCR of up-RFP-down ligation as well as PCR of 3 subcultures suspected to contain the cat-sacB construct, performed using the tnaA UP forward primer and the tnaA DOWN reverse primer. <br />
<br><br><br />
<b>08/10/10</b><br><br />
Took 2 5ml LB bottles:<br><br />
- Added 1.7 microlitres of tet15 and 0.3ml DH5-alpha overnight culture to one<br><br />
- Added 5 microlitres of tet15 and 0.3ml of JM109 overnight culture to the other<br><br><br />
Grew second cultures for 2 hours at 30C with shaking <br><br />
Measured optical density after 2 hours:<br><br />
- Water = 0.00<br><br />
- DH5-alpha = 0.251<br><br />
- JM109 = 0.320<br><br><br />
Added 100microlitres arabinose to both cultures and grew at 37C for one and a half hours with shaking.<br><br><br />
Prepared cells for electroporation:<br><br />
1- Added 1.5ml of liquid culture to an eppendorf<br><br />
2- Spun down cells at 11,000rpm for 30s<br><br />
3- Removed supernatant, added 1ml of cold sterile water and resuspended cells<br><br />
4- Repeated steps 2 and 3 twice more<br><br />
5- Added 40microlitres of suspended cells and 1microlitre of DNA construct (<i>up-cat-sacB-down</i>) to chilled electroporation cuvette (also prepared one cuvette of each cell culture without DNA for controls)<br><br><br />
Performed electroporation<br><br />
Suspended cells in cuvette by adding 0.5ml LB and added to eppendorf containing 1.0ml LB<br><br />
Left in 37C incubator for one hour, no sign of growth - spun cells down and no visible pellet, resuspended in 0.9ml and decided to leave overnight<br><br><br />
Will plate up cells on chloramphenicol tomorrow and they should have grown by Monday.<br><br><br />
Annoyingly, the Red/ET recombination plasmid is lost at 37C, but it was worth following the procedure to see if it actually works first time. If we need to repeat it, the plates can be grown at 30C so that there is no need to retransform with the recombination plasmid.<br><br><br />
<b>09/10/10</b><br />
<br>Cells left to grow for ~20 hours<br><br />
Plated from 100microlitres from 900microlitre suspension onto 4 chloramphenicol plates marked JM109/lambda + cat/sacB and -cat/sacB (control) and DH5-alpha/lambda + cat/sacB and - cat/sacB (control)<br><br />
Spun cells down at 7000rpm for 2 minutes - gave decent sized pellet. Removed 700microlitres of supernatant and resuspended cells<br><br />
Plated last 100microlitres onto a further 4 chloramphenicol plates <br><br />
Plates will be left in 37C incubator - cells were concentrated enough on second set of plates so that if they are cml resistant they will grow by tomorrow. <br><br><br />
<b>10/10/10</b><br><br />
Significant growth of red colonies on DH5-alpha+ (concentrated) but none on DH5-alpha- (not-concentrated). This is highly confusing as there should be no RFP in the cells. We now believe this to be a contamination.<br />
<br><br />
There is growth on both of the negative plates of the JM109 cells, but none on the positive plates. This could be a labelling error.<br><br />
There is no growth on any of the other plates.<br><br><br />
<b>11/10/10</b><br><br />
No growth on any of the plates that had not grown yesterday. <br><br />
Colonies on JM109- plates do not smell of indole. If they are E.coli then this is promising. Subcultured 4 colonies from each plate onto cml plates.<br><br><br />
Ran gels of PCRs performed on 07/10. No bands in the three lanes from subcultures, but a strong band from the PCR of the ligation.<br><br><br />
<b>12/10/10</b><br><br />
The subcultures from yesterday have not grown particularly well. The cells are white, unlike normal E.coli, and the colonies are uncharacteristically runny. Ran PCR of subcultures to determine whether they contain the insert. (with insert ~2.5kb, without ~1.5kb)<br><br />
Ran this PCR on a gel. Gave no bands. First attempt at BRIDGE did not work. <br><br><br />
Alterations to be made on second attempt:<br />
<br>1- Resuspend cells in 20-50microlitres after cleaning to increase transformation efficiency<br><br />
2- Grow at 30C instead of 37C after electroporation so as not to lose recombination plasmid<br><br />
3- Use normal chloramphenicol plates rather than cml+iptg+xgal.<br />
<br />
<br><br><br />
<b>14/10/10</b><br><br />
Prepared overnight cultures:<br />
<br><br />
- DH5-alpha/lambda in 5ml LB with 1.5microlitres tet15<br><br />
- JM109/lambda in 5ml LB with 5microlitres tet15<br><br><br />
Grew cells at 30C, 200rpm<br><br><br />
<br />
[[Image:171.jpg]]<br />
<br />
* tnaA PCR, JM109, MK2+4, CF 6.<br />
<b>15/10/10</b><br><br />
Prepared secondary liquid cultures:<br />
<br><br />
- 5ml LB + 1.5microlitres tet15 inoculated with 0.3ml overnight DH5-alpha/lambda culture<br><br />
- 5ml LB + 5microlitres tet15 inoculated with 0.3ml overnight culture JM109/lambda<br><br><br />
Grew cells at 30C, 200rpm for 2 hours<br><br><br />
Transferred 1.5ml of both cultures to individual eppendorfs and spun down cells for 3 minutes at 8000rpm. Removed supernatant and added another 1.5ml. Spun down cells again and removed supernatant.<br><br />
Suspended cells in 1ml cold sterile water and spun them down for 1 minute at 10000rpm before removing supernatant.<br />
Repeated this twice more.<br />
<br><br />
Resuspended cells in 100microlitires cold sterile water.<br><br><br />
Added 40microlitres of the relevant cell culture to cold sterile electroporation cuvettes labelled J+, J-, D+ and D-.<br><br />
Added 2microlitres DNA to the J+ cuvette.<br><br />
Added 1microlitre DNA to the D+ cuvetter.<br><br />
Prepared 8 eppendorfs, 4 containing 1.5ml LB.<br><br><br />
Electroporated all 4 samples.<br><br />
The voltage decay readings for the J-, D+ and D- cells were all above 4.0, but the reading for the J+ was 3.30. This probably indicates that there is too much DNA in the sample as the charge dissapated too quickly. From now on only 1microlitre of DNA will be used.<br><br><br />
Immediately after electroporation, 500microlitres of LB was added to the cuvette from an eppendorf to resuspend the cells and transferred back to the eppendorf. The LB was mixed and 750microlitres were transferred to a clean eppendorf.<br><br><br />
For each transformation, half the cells were grown at 30C overnight and half were grown at 37C overnight.<br />
<br><br><br />
<b>16/10/10</b><br><br />
Spun down cells from each of the 8 eppendorfs at 8000rpm for 3 minutes and removed 600microlitres of supernatant. <br><br />
Spread transformants onto chloramphenicol (one plate each) and grew at relevant temperatures for 48 hours. <br><br><br />
<b>18/10/10</b><br><br />
Only growth on plates appears to be non-E.coli. Major contamination has only occurred on 30C plates. Whatever it is, it seems to grow better at 30C. <br><br />
Since this is the second time we have seen this type of contamination the next attempt at the protocol will be combined with taking samples of cells at each step.<br><br><br />
<b>19/10/10</b><br><br />
Made more tet15 plates, as will need about 40 this week.<br><br />
Prepared overnight cultures of DH5-alpha/lambda and JM109/lamda, both grown at 30C.<br><br><br />
<b>20/10/10</b><br><br />
Took a sample of each overnight culture and spread it onto two tetracycline plates (1), one grown at 30C the other grown at 37C. The reasoning here is that any contaminants at this stage must be tetracycline resistant.<br><br><br />
Added 0.3ml overnight culture to fresh LB with tetracyline. Grew for 2 hours at 30C with shaking.<br />
<br><br><br />
Sampled each culture again, this time plating to both chloramphenicol and tetracyline plates. (2)<br><br />
NB: JM109 is not growing as well as DH5alpha in liquid culture. We may need to change the concentration of tet in one or both slightly.<br><br><br />
Something of a mini catastrophe...I knocked over my DH5alpha liquid culture and lost half the contents. Have added LB and a proportionate amount of tet15 and left both in the 30C shaker for another hour. This should recover enough cells to continue, however it also means the LB may contaminate the DH5alpha...if it is the LB, then only DH5alpha will be contaminated.<br><br><br />
In the mean time, am making 20-26 plates of cml.<br><br><br />
Added 100microlitres of 20% L-arabinose to each culture and left for one hour at 37C with shaking. Prepared cold water, cuvettes and eppendorfs with LB.<br><br><br />
Sampled cultures again, plating to chloramphenicol only. (3)<br><br />
Washed cells with cold water.<br><br />
Sampled cells again and plated to chloramphenicol. (4)<br><br><br />
<br><br><br />
Electroporated two sets with the cat/sacB construct and and two without. Transferred to LB in eppendorfs for recovery and grew each set of four at both 30C and 37C for an hour.<br><br><br />
Sampled cells again and plated to chloramphenicol. (5)<br><br />
Innoculated 5ml liquid cultures with 5microlitres of cml and 600microlitres of the transformed sample and grew overnight at 30C and 37C with shaking.<br><br><br />
<b>21/10/10</b><br />
<br><br />
No growth on any of the chloramphenicol plates from yesterdays samples. This suggests the usual contaminant has not yet reached the cultures. There is growth on each tetracycline plate, which is expected as the cells still contain the Red/ET plasmid at this point.<br><br><br />
Unfortunately there is no growth in the positive chloramphenicol liquid cultures. There is growth in the D- at 37C culture which presumably is one of our contaminants. I will keep an eye on the D- 37C plate and possibly plate out the liquid culture to examine the colonies. This contamination must have occurred during the final stage of the protocol from yesterday and must be human error as it is an isolated incident. <br><br><br />
<b>22/10/10</b><br><br />
Set up secondary liquid cultures in tet15 (3.5microlitres for JM109 as it seems to be struggling to grow) and left to grow in 30C shaker for 3 hours.<br />
<br><br />
Plated out D- 37C cell culture that survived chloramphenicol to determine strain.<br><br />
<br><br />
Checked chloramphenicol sample plates again:<br><br />
Sample 2- White colony growth on both JM109 plates but none on DH5alpha<br><br />
Sample 3- No growth (contaminants have disappeared, killed by arabinose?)<br><br />
Sample 4- White colony growth 30C plates only, two micrococcal colonies on J 37C<br><br />
Sample 5- A few very small white colonies on 30C plates D-, D+ and J-, no growth on D- at 37C<br><br><br />
The bulk of the contamination appears to occur at the washing step, but whatever they are, they don't seem to be able to survive cml40 in a liquid culture. Confusingly, whatever has contaminated the D- culture at 37C doesn't seem to be on the plates. This probably means it is an isolated event. I don't know if the same goes for the contaminants in sample 2. These are more confusing as they disappear after the arabinose is added. It is possible that they do not survive either in arabinose or at 37C in liquid.<br><br><br />
Next step to remove contaminants:<br><br />
Previously the washing step had been performed in the lab away from the clean hood. From now on, any stage where the cells are exposed will take place under the clean hood.<br><br />
A control will be set up at this step to confirm that contamination is occurring here and not previously. A second set of eppendorfs will be washed repeatedly with water and then 100microlitres of the water will be plated along with the actual samples. <br><br />
Growth on the negative control will indicate that either the water or the eppendorfs is contaminated. <br><br />
No growth on either will indicate that the use of the clean hood makes a difference.<br><br><br />
Next step in improving the efficacy of BRIDGE:<br><br />
The cat gene we're using is not very strongly induced. Couple this with the fact that DH5alpha and JM109 both struggle to grow at regular concentrations of antibiotics and we can assume that any recombinants we do get won't be able to grow in cml40. This time I will grow them overnight in a lower concentration of chloramphenicol. 3microlitres for JM109, 2microlitres for DH5alpha. <br><br><br />
This might be the last time I will be able to attempt BRIDGE before the wiki freeze. Fingers crossed.<br><br><br />
Maximised biomass this time by growing JM109 in 3.5microlitres tet15 and DH5alpha in 1.75microlitres as usual, for 2 and a half hours after adding 0.5ml overnight culture rather than 0.3ml. <br><br><br />
After the induction step spun down whole cell culture and kept cells under clean hood whenever they were exposed. <br>Also took negative controls at washing step of sterile water and LB, both kept in eppendorfs for 10minutes.<br>If there is growth on:<br />
<br><br />
- Sterile water only - sterile water is contaminated<br><br />
- LB only - LB is contaminated<br><br />
- Both - eppendorfs are contaminated<br>Also took samples of actual cell cultures at this step.<br><br><br />
Electroporated cells as normal. Grew at recovery step for 2 hours.<br><br><br />
Grew all 8 samples in cml40: JM109 in 3.5microlitres, DH5alpha in 1.75microlitres.<br><br />
Grew positive transformants in tet15 (normal concentrations) to determine if the cells were still alive. <br><br />
If the positive cells grow in:<br><br />
- cml and tet - recombinants<br><br />
- cml but not tet - contaminant<br><br />
- tet but not cml - non-recombinant<br><br />
- neither - cells have died<br><br><br />
<br />
Transformed JM109 cells with pSB1A2+cat to characterise cat and Edinbrick as a negative pSB1A2 control. Plated cells to ampicillin. <br><br><br />
<b>23/10/10</b><br><br />
The negative controls from the washing step have not grown, but then neither have the samples, so no contamination on anything. <br><br><br />
There is growth in 3 of the 4 tetracyline cultures from yesterday (JM109 has not grown at 37C, possibly because of inability of plasmid to replicate). This means the cells are not, in 3 cases, being killed by the electrporation. <br>There is no growth in any of the negative controls, so no indication of contamination. <br><br />
No growth in any of the cml positive cultures which indicates that the recombinase genes are not being induced properly. <br><br />
<br>The D- 37C contamination from Wednesday turned out to have RFP in it. This is quite confusing but, as stated earlier, all contaminations have been removed from the procedure.<br><br><br />
Good growth of cat and Edinbrick transformants on ampicillin. Have subcultured 4 of each, again to ampicillin.<br><br><br />
<b>24/10/10</b><br />
<br><br />
Subcultured the cat and Edinbrick transformants from ampicillin to cml40. <br><br><br />
<b>25/10/10</b><br><br />
No significant growth on subcultures.<br><br><br />
Set up titre of cml resistance using positive 30C transformants and negative controls. All grown in tetracycline and in increasing concentrations of chloramphenicol. Grown overnight at 30C and 200rpm.<br><br><br />
<b>26/10/10</b><br><br />
Growth on Edinbrick on cml40 but not on cat. Could be mislabelling. Have re-attempted subculture from transformant plates but if initial plates are labelled wrong then the results will be the same.<br><br><br />
Results from titre. Conclusion: recombination isn't working as cat has been known to show resistance to cml10-40.<br><br><br />
<br />
<br />
{|border="1" style="text-align: center"<br />
|-<br />
!<br />
! colspan="2" | Ju109 (tet15 / 5ul)<br />
! colspan="2" | DH5a (tet3.75 / 1.75ul)<br />
|-<br />
! Cml<br />
! + cat/sacB<br />
! control<br />
! + cat/sacB<br />
! control<br />
|-<br />
| 0 / 0 ul || + || + || + || +<br />
|-<br />
| 4 / 0.5 ul || - || - || - || -<br />
|-<br />
| 8 / 1 ul || - || - || - || -<br />
|-<br />
| 12 / 1.5 ul || - || - || - || -<br />
|-<br />
| 16 / 2 ul || - || - || - || -<br />
|-<br />
|| 20 / 2.5 ul || - || - || - || -<br />
|-<br />
| 24 / 3 ul || - || - || - || -<br />
|}</div>Macbereskahttp://2010.igem.org/Team:Edinburgh/Notebook/Red_light_producerTeam:Edinburgh/Notebook/Red light producer2010-10-26T23:39:00Z<p>Macbereska: /* Red Light Producer */</p>
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<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook" class="dir">lab notes&nbsp;&nbsp;&nbsp;</a><br />
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<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Collaboration">collaboration</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Attribution">attribution</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/BRIDGE">BRIDGE</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_producer">red light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_sensor">red sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_producer">blue light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_producer">green light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_sensor">green sensor</a></li><br />
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<li><a href="http://www.openwetware.org/wiki/French_Lab">protocols</a></li><br />
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<span style="color:ivory;">Throughout this wiki there are words in <b>bold</b> that indicate a relevance to <b>human aspects</b>. It will become obvious that <b>human aspects</b> are a part of almost everything in <b>iGEM</b>.</span><br />
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<br />
== Red Light Producer ==<br />
<br><br />
<br />
'''29/6/10'''<br />
<br />
Now preparing primers to make a set of different spectra bioluminescent proteins. Need to order primers for Site directed mutagenesis of the Firely luciferase to make it red (MABEL protocol), and to make click beetle luciferase Rluc8 (which emits light at about 420nm in presence of bisdeoxycoelenterazine - about the same price a coelenterazine though)<br />
<br />
'''8/7/2010'''<br />
* Red luciferase mutagenesis primers arrived- 3 sets:<br />
** S248T<br />
** 356 K<br />
** 356 R<br />
<br />
* Followed the protocol with 34ul water + 0.5 template DNA<br />
* Put the primers and template in green box '''(???)'''<br />
*Ran Gel: 5 µL PCR product, 5 µL water, 2.5 µL buffer<br />
[[Image:151.JPG]]<br />
<br />
{| border="1"<br />
| Marker<br />
| S284T <br />
| 356K <br />
| 356R<br />
|}<br />
<br />
<br />
[[File:07-08.JPG]]<br />
<br />
<br />
*356K and 356R look fine<br />
*going to run S284T at a higher ** temp tomorrow to see if it works better<br />
*all PCR tubes stored in freezer<br />
<br />
<br />
<br />
'''09/7/10'''<br />
<br />
Redo PCR of Red Luciferase<br />
*Everything except template DNA + Kod polymerase kept on ice<br />
*Ran PCR<br />
*Template DNA used PyeaR and P. pyralis luciferase BBa_K216015<br />
*Purification done for 356K and 356R; stored in iGEM box (-20C).<br />
<br />
[[File:07-09straight.JPG]]<br />
<br />
'''9/7/2010'''<br />
*Ligations set up overnight- might have put too much ligase in 356K... Incubated at 15C.<br />
*Oure 356R and 356K are in the freezer- row 10 (G &H)?<br />
<br />
'''13/07/10'''<br />
Goal of the day: <br />
<br />
Transformations:<br />
*356R (2): one from 16 °C and one from room temp(RT)<br />
*356K (2): one from 16 °C and one from room temp(RT)<br />
*S284T (2): one with 25/5 ligase, one with 1/7 ligase<br />
<br />
* I had a burger for the lunch. It was very nice.<br />
* Now we are transforming cells:<br />
** 356R (2)& 356K (2): one form 16C, one from RT<br />
** S248T (2): 1 with 25/5 ligase, other with 1.7.10 ligase/<br />
Empty edinbrick vector for control. <br />
* LuxAB + edinbrick ligation.<br />
<br />
* Using 100 ul transformed cells + 800 ul LB for all. Incubation started half 5. <br />
'''22/07/10'''<br />
<br />
Liquid Cultures (amp 80):<br />
*356R (RT) – 1 with NaNO3 and 1 without<br />
**(16°C) – 2 with NaNO3 and 2 without<br />
*356K (RT) – 1 with NaNO3 and 1 without<br />
**(16°C) – 2 with NaNO3 and 2 without <br />
*S284T (100 µL and 25/5 ligase) – 2 with NaNO3 and 2 without<br />
**(900 µL and 25/5 ligase) – 3 with NaNO3 and 3 without<br />
**(900 µL and 1/7 ligase) – 5 with NaNO3 and 5 without<br />
<br />
NaNO3 = 30mM Sodium Nitrate<br />
<br />
'''23/07/10'''<br />
<br />
Double digest of K098010<br />
<br />
'''28/07/10'''<br />
<br />
Primer design to produce a stable red light luciferase. The mutations for increasing bioluminescence come from [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W9V-4NHM57C-1&_user=809099&_coverDate=07%2F15%2F2007&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1414502247&_rerunOrigin=google&_acct=C000043939&_version=1&_urlVersion=0&_userid=809099&md5=00d9c92f6452bd276c6ea606ed7d3bbf Fujii et al, 2007] and the paper from [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W9V-4X6MSYB-7&_user=809099&_coverDate=01%2F15%2F2010&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1414503636&_rerunOrigin=google&_acct=C000043939&_version=1&_urlVersion=0&_userid=809099&md5=297be52832193b4d026953632a1edf3c Branchini et al, 2010]<br />
<br />
'''29/7/2010'''<br />
<br />
*Double digest with EcoRI HF&PstI in buffer 2 (neb).<br />
* 2 bands: with and without vector?<br />
* did not leave the digest for too long... <br />
[[Image:157.jpg]]<br />
<br />
{| border="1"<br />
| Lane 1<br />
| Lane 2<br />
| Lane 3<br />
| Lane 4<br />
| Lane 5<br />
| Lane 6<br />
|-<br />
| Ladder <br />
| S284T (4) <br />
| S284T (6)<br />
| 356R (6, 900)<br />
| 356R (6)<br />
| 356K (4)<br />
|}<br />
<br />
'''2/8/2010'''<br />
* Red luciferases EcoRI HF&PstI double digests- lanes 2-7 in the second miniprep set. <br />
*Ladder didn't work too well ,though...<br />
[[Image:113.JPG]]<br />
* Transform last years luciferase<br />
* Look at papers for expression in E. coli<br />
* Replace B0034 by stronger RBS?<br />
* Confirm that we order bright luc.<br />
* Single digest of constructs<br />
* Test glowing with positive control<br />
<br />
'''9/8/2010'''<br />
<br />
[[Image:159.jpg]]<br />
<br />
Digest: <br />
*WT1<br />
*356 R: (2)<br />
* 356 K: (4)<br />
* S284T: (6)<br />
* 3 eith EcoRI; 3 with EcoRi + PstI<br />
<br />
<br />
'''10/8/2010'''<br />
* Sequencing of red mutant luciferases. <br />
* Forward primers for S284T and reverse for 356R and K should do. <br />
* Concentration of primers for sequencing: 5uM.<br />
* sequencing tubE: 6 ul liquid: 3ul DNA + 2ul water, 1 ul primer mix.<br />
* when using miniprep DNA- remember to spin down the dirt- 1min, max speed, then take the DNA from the top to avoid disturbing any dirt that is settled. <br />
<br />
<br />
'''12/8/2010'''<br />
* Got the sequences for red luciferases<br />
*see: if we can detect Red Light with the luminometer?<br />
* Red Luciferases 356R and S284T were nNOT MUTANTS- just WT<br />
* set up ;iquid cultures with 30 ul nitrate for: <br />
** 356R - no 3,1 from RT plate, no 5 from 16C plate; no 1 from Master plate<br />
** S284T - 5 from masterplate; 2, 4, 6 from masterplate<br />
** 356K: 4 from masterplate<br />
<br />
'''13/8/2010'''<br />
Big day today... TESTING YESTERDAYS PLAN- LUMINESCENCE<br />
<br />
'''16/8/2010'''<br />
* Sequecning of: <br />
** WR1- 356R -use S284T forward primer<br />
** WR2 -S284T -use 356 R reverse primer<br />
** WR3- LovTap 03<br />
**WR4 -LovTap 09<br />
<br />
* RESULTS: ONLY 356K WAS A REAL MUTANT; S284T AND 356R TURNED OUT TO BE STILL A WT<br />
<br />
'''19/8/2010'''<br />
* star6: 56,869<br />
* star4: 13,471<br />
* 356K: 263,778<br />
* star 2: 3,717<br />
* WT: 1,617832<br />
<br />
*Gel of the ligations: marker (lane 1), 356K (lane 2,3), S284T (lane 3,4), 358Rs (lane 5,6).<br />
<br />
[[Image:163.jpg]]<br />
<br />
'''25/8/2010'''<br />
* Next step:- redo ligations for 356R; redo PCR for S284T<br />
* Ligations<br />
*Did MABEL PCR PPyInc + S284T primers<br />
*used 0.5 ul template DNA<br />
*Gel: marker (Lane1), ligation of PCR product for 356R (Lane2), Ligation of 07 PCR for S284T (lane3), Pure PCR from S284T. <br />
<br />
[[Image:164.jpg]]<br />
<br />
'''26/8/2010'''<br />
* check the Fuji et. al paper, see what sequence they used to start off with. <br />
* check IAV in other luciferase<br />
* Fix scar before RBS and in teh second stop codon. <br />
* compared to WT, luciferase sequence has: <br />
** N 50 P<br />
** N 99 G<br />
** SKL- IAV<br />
<br />
[[Image:165.jpg]]<br />
<br />
'''27/8/2010'''<br />
* Ligations from yesterday out of freezer.<br />
* Running on gel<br />
* Column 3 9with 356R) has less DNA in it- not enough from ligation and messed up loading of gel). <br />
[[Image:132.JPG]]<br />
{| border="1"<br />
| Lane 1<br />
| Lane 2<br />
| Lane 3<br />
| Lane 4<br />
<br />
|-<br />
| Ladder <br />
| New S284T PCR - new ligase and the buffer<br />
| 356 R ligation<br />
| S284T PCR + old ligase<br />
|}<br />
<br />
'''30/8/2010'''<br />
* Primers have arrived for fixing the luciferase- removing 6 bases<br />
* Primers have arrived for up and downstream sequences of TnaA region.<br />
* Send Chairman sequence for T7 promoter<br />
* transformed 356R and S284T ligations from Friday<br />
* Used PCR product as a control<br />
* Plated out the transformed cells. Used 900 rather than 100 if possible<br />
<br />
<br />
'''31/8/2010'''<br />
* We have S284T transformants but <br />
** 14 colonies on ligation plate<br />
** 4 colonies on control where we used PCR product instead of ligation<br />
<br />
* For sequencing- <br />
** if sequencing a S284T mutant, use 356R/K reverse primer<br />
** if sequencing 356R mutant, use S284T forward primer.<br />
<br />
'''2/9/2010'''<br />
* minipreped 14 colonies (1-14)<br />
* run the gel with samples digested with EcoRI<br />
* here: samples 1-7<br />
[[Image:135.JPG]]<br />
<br />
'''3/9/2010'''<br />
* S284T 8-14 (from yesterday)<br />
[[Image:136.JPG]]<br />
<br />
'''8/9/2010'''<br />
* wWT of luciferase (left)<br />
* mutation of S284T (right)<br />
<br />
[[Image:168.jpg]]<br />
<br />
'''9/9/2010'''<br />
<br />
[[Image:170.jpg]]<br />
<br />
{| border="1"<br />
| Lane 1<br />
| Lane 2<br />
| Lane 3<br />
| Lane 4<br />
| Lane 5<br />
|-<br />
| Ladder <br />
| WT <br />
| S284T 1<br />
| luxCDE 9:4<br />
| LuxCDE 9:1<br />
|}<br />
<br />
'''20/9/2010'''<br />
<br />
*send for sequencing: 356K, S284T, WT (samples from 16/9: WT- 900, 2; also 16/9 S284T- 900, 4.)<br />
* all in psB1C3 vector. <br />
* Transform 356K and S284T with RBS and promoter (lacZ, LacI, RBS). <br />
* Run gel of 365K in psb1C3 of the digest. --> control: RFP + psb1C3<br />
* then add promoter<br />
<br />
'''23/9/2010'''<br />
<br />
* Added in incubator room liquid culture '''?)''' LacP + RBS + Luciferase for S284T, 356K and WT. <br />
* miniprep- 6 bottles<br />
* GLOWING<br />
<br />
* S248T- <br />
**9.2; 5 <br />
** 9.4; 1 <br />
**5; 4<br />
* 356<br />
** 3.3<br />
* WT<br />
** 3.2; 4 <br />
** 1.4, 5</div>Macbereskahttp://2010.igem.org/Team:Edinburgh/Notebook/Red_light_producerTeam:Edinburgh/Notebook/Red light producer2010-10-26T23:38:09Z<p>Macbereska: /* Red Light Producer */</p>
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<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook" class="dir">lab notes&nbsp;&nbsp;&nbsp;</a><br />
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<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Attribution">attribution</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/BRIDGE">BRIDGE</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_producer">red light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_sensor">red sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_producer">blue light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_producer">green light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_sensor">green sensor</a></li><br />
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<span style="color:ivory;">Throughout this wiki there are words in <b>bold</b> that indicate a relevance to <b>human aspects</b>. It will become obvious that <b>human aspects</b> are a part of almost everything in <b>iGEM</b>.</span><br />
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<br />
== Red Light Producer ==<br />
<br><br />
<br />
'''29/6/10'''<br />
<br />
Now preparing primers to make a set of different spectra bioluminescent proteins. Need to order primers for Site directed mutagenesis of the Firely luciferase to make it red (MABEL protocol), and to make click beetle luciferase Rluc8 (which emits light at about 420nm in presence of bisdeoxycoelenterazine - about the same price a coelenterazine though)<br />
<br />
'''8/7/2010'''<br />
* Red luciferase mutagenesis primers arrived- 3 sets:<br />
** S248T<br />
** 356 K<br />
** 356 R<br />
<br />
* Followed the protocol with 34ul water + 0.5 template DNA<br />
* Put the primers and template in green box '''(???)'''<br />
*Ran Gel: 5 µL PCR product, 5 µL water, 2.5 µL buffer<br />
[[Image:151.JPG]]<br />
<br />
{| border="1"<br />
| Marker<br />
| S284T <br />
| 356K <br />
| 356R<br />
|}<br />
<br />
<br />
[[File:07-08.JPG]]<br />
<br />
<br />
*356K and 356R look fine<br />
*going to run S284T at a higher ** temp tomorrow to see if it works better<br />
*all PCR tubes stored in freezer<br />
<br />
<br />
<br />
'''09/7/10'''<br />
<br />
Redo PCR of Red Luciferase<br />
*Everything except template DNA + Kod polymerase kept on ice<br />
*Ran PCR<br />
*Template DNA used PyeaR and P. pyralis luciferase BBa_K216015<br />
*Purification done for 356K and 356R; stored in iGEM box (-20C).<br />
<br />
[[File:07-09straight.JPG]]<br />
<br />
'''9/7/2010'''<br />
*Ligations set up overnight- might have put too much ligase in 356K... Incubated at 15C.<br />
*Oure 356R and 356K are in the freezer- row 10 (G &H)?<br />
<br />
'''13/07/10'''<br />
Goal of the day: <br />
<br />
Transformations:<br />
*356R (2): one from 16 °C and one from room temp(RT)<br />
*356K (2): one from 16 °C and one from room temp(RT)<br />
*S284T (2): one with 25/5 ligase, one with 1/7 ligase<br />
<br />
* I had a burger for the lunch. It was very nice.<br />
* Now we are transforming cells:<br />
** 356R (2)& 356K (2): one form 16C, one from RT<br />
** S248T (2): 1 with 25/5 ligase, other with 1.7.10 ligase/<br />
Empty edinbrick vector for control. <br />
* LuxAB + edinbrick ligation.<br />
<br />
* Using 100 ul transformed cells + 800 ul LB for all. Incubation started half 5. <br />
'''22/07/10'''<br />
<br />
Liquid Cultures (amp 80):<br />
*356R (RT) – 1 with NaNO3 and 1 without<br />
**(16°C) – 2 with NaNO3 and 2 without<br />
*356K (RT) – 1 with NaNO3 and 1 without<br />
**(16°C) – 2 with NaNO3 and 2 without <br />
*S284T (100 µL and 25/5 ligase) – 2 with NaNO3 and 2 without<br />
**(900 µL and 25/5 ligase) – 3 with NaNO3 and 3 without<br />
**(900 µL and 1/7 ligase) – 5 with NaNO3 and 5 without<br />
<br />
NaNO3 = 30mM Sodium Nitrate<br />
<br />
'''23/07/10'''<br />
<br />
Double digest of K098010<br />
<br />
'''28/07/10'''<br />
<br />
Primer design to produce a stable red light luciferase. The mutations for increasing bioluminescence come from [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W9V-4NHM57C-1&_user=809099&_coverDate=07%2F15%2F2007&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1414502247&_rerunOrigin=google&_acct=C000043939&_version=1&_urlVersion=0&_userid=809099&md5=00d9c92f6452bd276c6ea606ed7d3bbf Fujii et al, 2007] and the paper from [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W9V-4X6MSYB-7&_user=809099&_coverDate=01%2F15%2F2010&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1414503636&_rerunOrigin=google&_acct=C000043939&_version=1&_urlVersion=0&_userid=809099&md5=297be52832193b4d026953632a1edf3c Branchini et al, 2010]<br />
<br />
'''29/7/2010'''<br />
<br />
*Double digest with EcoRI HF&PstI in buffer 2 (neb).<br />
* 2 bands: with and without vector?<br />
* did not leave the digest for too long... <br />
[[Image:157.jpg]]<br />
<br />
{| border="1"<br />
| Lane 1<br />
| Lane 2<br />
| Lane 3<br />
| Lane 4<br />
| Lane 5<br />
| Lane 6<br />
|-<br />
| Ladder <br />
| S284T (4) <br />
| S284T (6)<br />
| 356R (6, 900)<br />
| 356R (6)<br />
| 356K (4)<br />
|}<br />
<br />
'''2/8/2010'''<br />
* Red luciferases EcoRI HF&PstI double digests- lanes 2-7 in the second miniprep set. <br />
*Ladder didn't work too well ,though...<br />
[[Image:113.JPG]]<br />
* Transform last years luciferase<br />
* Look at papers for expression in E. coli<br />
* Replace B0034 by stronger RBS?<br />
* Confirm that we order bright luc.<br />
* Single digest of constructs<br />
* Test glowing with positive control<br />
<br />
'''9/8/2010'''<br />
<br />
[[Image:159.jpg]]<br />
<br />
Digest: <br />
*WT1<br />
*356 R: (2)<br />
* 356 K: (4)<br />
* S284T: (6)<br />
* 3 eith EcoRI; 3 with EcoRi + PstI<br />
<br />
<br />
'''10/8/2010'''<br />
* Sequencing of red mutant luciferases. <br />
* Forward primers for S284T and reverse for 356R and K should do. <br />
* Concentration of primers for sequencing: 5uM.<br />
* sequencing tubE: 6 ul liquid: 3ul DNA + 2ul water, 1 ul primer mix.<br />
* when using miniprep DNA- remember to spin down the dirt- 1min, max speed, then take the DNA from the top to avoid disturbing any dirt that is settled. <br />
<br />
<br />
'''12/8/2010'''<br />
* Got the sequences for red luciferases<br />
*see: if we can detect Red Light with the luminometer?<br />
* Red Luciferases 356R and S284T were nNOT MUTANTS- just WT<br />
* set up ;iquid cultures with 30 ul nitrate for: <br />
** 356R - no 3,1 from RT plate, no 5 from 16C plate; no 1 from Master plate<br />
** S284T - 5 from masterplate; 2, 4, 6 from masterplate<br />
** 356K: 4 from masterplate<br />
<br />
'''13/8/2010'''<br />
Big day today... TESTING YESTERDAYS PLAN- LUMINESCENCE<br />
<br />
'''16/8/2010'''<br />
* Sequecning of: <br />
** WR1- 356R -use S284T forward primer<br />
** WR2 -S284T -use 356 R reverse primer<br />
** WR3- LovTap 03<br />
**WR4 -LovTap 09<br />
<br />
* RESULTS: ONLY 356K WAS A REAL MUTANT; S284T AND 356R TURNED OUT TO BE STILL A WT<br />
<br />
'''19/8/2010'''<br />
* star6: 56,869<br />
* star4: 13,471<br />
* 356K: 263,778<br />
* star 2: 3,717<br />
* WT: 1,617832<br />
<br />
*Gel of the ligations: marker (lane 1), 356K (lane 2,3), S284T (lane 3,4), 358Rs (lane 5,6).<br />
<br />
[[Image:163.jpg]]<br />
<br />
'''25/8/2010'''<br />
* Next step:- redo ligations for 356R; redo PCR for S284T<br />
* Ligations<br />
*Did MABEL PCR PPyInc + S284T primers<br />
*used 0.5 ul template DNA<br />
*Gel: marker (Lane1), ligation of PCR product for 356R (Lane2), Ligation of 07 PCR for S284T (lane3), Pure PCR from S284T. <br />
<br />
[[Image:164.jpg]]<br />
<br />
'''26/8/2010'''<br />
* check the Fuji et. al paper, see what sequence they used to start off with. <br />
* check IAV in other luciferase<br />
* Fix scar before RBS and in teh second stop codon. <br />
* compared to WT, luciferase sequence has: <br />
** N 50 P<br />
** N 99 G<br />
** SKL- IAV<br />
<br />
[[Image:165.jpg]]<br />
<br />
'''27/8/2010'''<br />
* Ligations from yesterday out of freezer.<br />
* Running on gel<br />
* Column 3 9with 356R) has less DNA in it- not enough from ligation and messed up loading of gel). <br />
[[Image:132.JPG]]<br />
{| border="1"<br />
| Lane 1<br />
| Lane 2<br />
| Lane 3<br />
| Lane 4<br />
<br />
|-<br />
| Ladder <br />
| New S284T PCR - new ligase and the buffer<br />
| 356 R ligation<br />
| S284T PCR + old ligase<br />
|}<br />
<br />
'''30/8/2010'''<br />
* Primers have arrived for fixing the luciferase- removing 6 bases<br />
* Primers have arrived for up and downstream sequences of TnaA region.<br />
* Send Chairman sequence for T7 promoter<br />
* transformed 356R and S284T ligations from Friday<br />
* Used PCR product as a control<br />
* Plated out the transformed cells. Used 900 rather than 100 if possible<br />
<br />
<br />
'''31/8/2010'''<br />
* We have S284T transformants but <br />
** 14 colonies on ligation plate<br />
** 4 colonies on control where we used PCR product instead of ligation<br />
<br />
* For sequencing- <br />
** if sequencing a S284T mutant, use 356R/K reverse primer<br />
** if sequencing 356R mutant, use S284T forward primer.<br />
<br />
'''2/9/2010'''<br />
* minipreped 14 colonies (1-14)<br />
* run the gel with samples digested with EcoRI<br />
* here: samples 1-7<br />
[[Image:135.JPG]]<br />
<br />
'''3/9/2010'''<br />
* S284T 8-14 (from yesterday)<br />
[[Image:136.JPG]]<br />
<br />
'''8/9/2010'''<br />
* wWT of luciferase (left)<br />
* mutation of S284T (right)<br />
<br />
[[Image:168.jpg]]<br />
<br />
'''9/9/2010'''<br />
{| border="1"<br />
| Lane 1<br />
| Lane 2<br />
| Lane 3<br />
| Lane 4<br />
| Lane 5<br />
|-<br />
| Ladder <br />
| WT <br />
| S284T 1<br />
| luxCDE 9:4<br />
| LuxCDE 9:1<br />
|}<br />
<br />
'''20/9/2010'''<br />
<br />
*send for sequencing: 356K, S284T, WT (samples from 16/9: WT- 900, 2; also 16/9 S284T- 900, 4.)<br />
* all in psB1C3 vector. <br />
* Transform 356K and S284T with RBS and promoter (lacZ, LacI, RBS). <br />
* Run gel of 365K in psb1C3 of the digest. --> control: RFP + psb1C3<br />
* then add promoter<br />
<br />
'''23/9/2010'''<br />
<br />
* Added in incubator room liquid culture '''?)''' LacP + RBS + Luciferase for S284T, 356K and WT. <br />
* miniprep- 6 bottles<br />
* GLOWING<br />
<br />
* S248T- <br />
**9.2; 5 <br />
** 9.4; 1 <br />
**5; 4<br />
* 356<br />
** 3.3<br />
* WT<br />
** 3.2; 4 <br />
** 1.4, 5</div>Macbereskahttp://2010.igem.org/Team:Edinburgh/Notebook/Red_light_producerTeam:Edinburgh/Notebook/Red light producer2010-10-26T23:34:37Z<p>Macbereska: /* Red Light Producer */</p>
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<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_producer">green light</a></li><br />
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<span style="color:ivory;">Throughout this wiki there are words in <b>bold</b> that indicate a relevance to <b>human aspects</b>. It will become obvious that <b>human aspects</b> are a part of almost everything in <b>iGEM</b>.</span><br />
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<br />
== Red Light Producer ==<br />
<br><br />
<br />
'''29/6/10'''<br />
<br />
Now preparing primers to make a set of different spectra bioluminescent proteins. Need to order primers for Site directed mutagenesis of the Firely luciferase to make it red (MABEL protocol), and to make click beetle luciferase Rluc8 (which emits light at about 420nm in presence of bisdeoxycoelenterazine - about the same price a coelenterazine though)<br />
<br />
'''8/7/2010'''<br />
* Red luciferase mutagenesis primers arrived- 3 sets:<br />
** S248T<br />
** 356 K<br />
** 356 R<br />
<br />
* Followed the protocol with 34ul water + 0.5 template DNA<br />
* Put the primers and template in green box '''(???)'''<br />
*Ran Gel: 5 µL PCR product, 5 µL water, 2.5 µL buffer<br />
[[Image:151.JPG]]<br />
<br />
{| border="1"<br />
| Marker<br />
| S284T <br />
| 356K <br />
| 356R<br />
|}<br />
<br />
<br />
[[File:07-08.JPG]]<br />
<br />
<br />
*356K and 356R look fine<br />
*going to run S284T at a higher ** temp tomorrow to see if it works better<br />
*all PCR tubes stored in freezer<br />
<br />
<br />
<br />
'''09/7/10'''<br />
<br />
Redo PCR of Red Luciferase<br />
*Everything except template DNA + Kod polymerase kept on ice<br />
*Ran PCR<br />
*Template DNA used PyeaR and P. pyralis luciferase BBa_K216015<br />
*Purification done for 356K and 356R; stored in iGEM box (-20C).<br />
<br />
[[File:07-09straight.JPG]]<br />
<br />
'''9/7/2010'''<br />
*Ligations set up overnight- might have put too much ligase in 356K... Incubated at 15C.<br />
*Oure 356R and 356K are in the freezer- row 10 (G &H)?<br />
<br />
'''13/07/10'''<br />
Goal of the day: <br />
<br />
Transformations:<br />
*356R (2): one from 16 °C and one from room temp(RT)<br />
*356K (2): one from 16 °C and one from room temp(RT)<br />
*S284T (2): one with 25/5 ligase, one with 1/7 ligase<br />
<br />
* I had a burger for the lunch. It was very nice.<br />
* Now we are transforming cells:<br />
** 356R (2)& 356K (2): one form 16C, one from RT<br />
** S248T (2): 1 with 25/5 ligase, other with 1.7.10 ligase/<br />
Empty edinbrick vector for control. <br />
* LuxAB + edinbrick ligation.<br />
<br />
* Using 100 ul transformed cells + 800 ul LB for all. Incubation started half 5. <br />
'''22/07/10'''<br />
<br />
Liquid Cultures (amp 80):<br />
*356R (RT) – 1 with NaNO3 and 1 without<br />
**(16°C) – 2 with NaNO3 and 2 without<br />
*356K (RT) – 1 with NaNO3 and 1 without<br />
**(16°C) – 2 with NaNO3 and 2 without <br />
*S284T (100 µL and 25/5 ligase) – 2 with NaNO3 and 2 without<br />
**(900 µL and 25/5 ligase) – 3 with NaNO3 and 3 without<br />
**(900 µL and 1/7 ligase) – 5 with NaNO3 and 5 without<br />
<br />
NaNO3 = 30mM Sodium Nitrate<br />
<br />
'''23/07/10'''<br />
<br />
Double digest of K098010<br />
<br />
'''28/07/10'''<br />
<br />
Primer design to produce a stable red light luciferase. The mutations for increasing bioluminescence come from [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W9V-4NHM57C-1&_user=809099&_coverDate=07%2F15%2F2007&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1414502247&_rerunOrigin=google&_acct=C000043939&_version=1&_urlVersion=0&_userid=809099&md5=00d9c92f6452bd276c6ea606ed7d3bbf Fujii et al, 2007] and the paper from [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W9V-4X6MSYB-7&_user=809099&_coverDate=01%2F15%2F2010&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1414503636&_rerunOrigin=google&_acct=C000043939&_version=1&_urlVersion=0&_userid=809099&md5=297be52832193b4d026953632a1edf3c Branchini et al, 2010]<br />
<br />
'''29/7/2010'''<br />
<br />
*Double digest with EcoRI HF&PstI in buffer 2 (neb).<br />
* 2 bands: with and without vector?<br />
* did not leave the digest for too long... <br />
[[Image:157.jpg]]<br />
<br />
{| border="1"<br />
| Lane 1<br />
| Lane 2<br />
| Lane 3<br />
| Lane 4<br />
| Lane 5<br />
| Lane 6<br />
|-<br />
| Ladder <br />
| S284T (4) <br />
| S284T (6)<br />
| 356R (6, 900)<br />
| 356R (6)<br />
| 356K (4)<br />
|}<br />
<br />
'''2/8/2010'''<br />
* Red luciferases EcoRI HF&PstI double digests- lanes 2-7 in the second miniprep set. <br />
*Ladder didn't work too well ,though...<br />
[[Image:113.JPG]]<br />
* Transform last years luciferase<br />
* Look at papers for expression in E. coli<br />
* Replace B0034 by stronger RBS?<br />
* Confirm that we order bright luc.<br />
* Single digest of constructs<br />
* Test glowing with positive control<br />
<br />
'''9/8/2010'''<br />
<br />
[[Image:159.jpg]]<br />
<br />
Digest: <br />
*WT1<br />
*356 R: (2)<br />
* 356 K: (4)<br />
* S284T: (6)<br />
* 3 eith EcoRI; 3 with EcoRi + PstI<br />
<br />
<br />
'''10/8/2010'''<br />
* Sequencing of red mutant luciferases. <br />
* Forward primers for S284T and reverse for 356R and K should do. <br />
* Concentration of primers for sequencing: 5uM.<br />
* sequencing tubE: 6 ul liquid: 3ul DNA + 2ul water, 1 ul primer mix.<br />
* when using miniprep DNA- remember to spin down the dirt- 1min, max speed, then take the DNA from the top to avoid disturbing any dirt that is settled. <br />
<br />
<br />
'''12/8/2010'''<br />
* Got the sequences for red luciferases<br />
*see: if we can detect Red Light with the luminometer?<br />
* Red Luciferases 356R and S284T were nNOT MUTANTS- just WT<br />
* set up ;iquid cultures with 30 ul nitrate for: <br />
** 356R - no 3,1 from RT plate, no 5 from 16C plate; no 1 from Master plate<br />
** S284T - 5 from masterplate; 2, 4, 6 from masterplate<br />
** 356K: 4 from masterplate<br />
<br />
'''13/8/2010'''<br />
Big day today... TESTING YESTERDAYS PLAN- LUMINESCENCE<br />
<br />
'''16/8/2010'''<br />
* Sequecning of: <br />
** WR1- 356R -use S284T forward primer<br />
** WR2 -S284T -use 356 R reverse primer<br />
** WR3- LovTap 03<br />
**WR4 -LovTap 09<br />
<br />
* RESULTS: ONLY 356K WAS A REAL MUTANT; S284T AND 356R TURNED OUT TO BE STILL A WT<br />
<br />
'''19/8/2010'''<br />
* star6: 56,869<br />
* star4: 13,471<br />
* 356K: 263,778<br />
* star 2: 3,717<br />
* WT: 1,617832<br />
<br />
*Gel of the ligations: marker (lane 1), 356K (lane 2,3), S284T (lane 3,4), 358Rs (lane 5,6).<br />
<br />
[[Image:163.jpg]]<br />
<br />
'''25/8/2010'''<br />
* Next step:- redo ligations for 356R; redo PCR for S284T<br />
* Ligations<br />
*Did MABEL PCR PPyInc + S284T primers<br />
*used 0.5 ul template DNA<br />
*Gel: marker (Lane1), ligation of PCR product for 356R (Lane2), Ligation of 07 PCR for S284T (lane3), Pure PCR from S284T. <br />
<br />
[[Image:164.jpg]]<br />
<br />
'''26/8/2010'''<br />
* check the Fuji et. al paper, see what sequence they used to start off with. <br />
* check IAV in other luciferase<br />
* Fix scar before RBS and in teh second stop codon. <br />
* compared to WT, luciferase sequence has: <br />
** N 50 P<br />
** N 99 G<br />
** SKL- IAV<br />
<br />
[[Image:165.jpg]]<br />
<br />
'''27/8/2010'''<br />
* Ligations from yesterday out of freezer.<br />
* Running on gel<br />
* Column 3 9with 356R) has less DNA in it- not enough from ligation and messed up loading of gel). <br />
[[Image:132.JPG]]<br />
{| border="1"<br />
| Lane 1<br />
| Lane 2<br />
| Lane 3<br />
| Lane 4<br />
<br />
|-<br />
| Ladder <br />
| New S284T PCR - new ligase and the buffer<br />
| 356 R ligation<br />
| S284T PCR + old ligase<br />
|}<br />
<br />
'''30/8/2010'''<br />
* Primers have arrived for fixing the luciferase- removing 6 bases<br />
* Primers have arrived for up and downstream sequences of TnaA region.<br />
* Send Chairman sequence for T7 promoter<br />
* transformed 356R and S284T ligations from Friday<br />
* Used PCR product as a control<br />
* Plated out the transformed cells. Used 900 rather than 100 if possible<br />
<br />
<br />
'''31/8/2010'''<br />
* We have S284T transformants but <br />
** 14 colonies on ligation plate<br />
** 4 colonies on control where we used PCR product instead of ligation<br />
<br />
* For sequencing- <br />
** if sequencing a S284T mutant, use 356R/K reverse primer<br />
** if sequencing 356R mutant, use S284T forward primer.<br />
<br />
'''2/9/2010'''<br />
* minipreped 14 colonies (1-14)<br />
* run the gel with samples digested with EcoRI<br />
* here: samples 1-7<br />
[[Image:135.JPG]]<br />
<br />
'''3/9/2010'''<br />
* S284T 8-14 (from yesterday)<br />
[[Image:136.JPG]]<br />
<br />
'''8/9/2010'''<br />
* wWT of luciferase (left)<br />
* mutation of S284T (right)<br />
<br />
<br />
'''9/9/2010'''<br />
{| border="1"<br />
| Lane 1<br />
| Lane 2<br />
| Lane 3<br />
| Lane 4<br />
| Lane 5<br />
|-<br />
| Ladder <br />
| WT <br />
| S284T 1<br />
| luxCDE 9:4<br />
| LuxCDE 9:1<br />
|}<br />
<br />
'''20/9/2010'''<br />
<br />
*send for sequencing: 356K, S284T, WT (samples from 16/9: WT- 900, 2; also 16/9 S284T- 900, 4.)<br />
* all in psB1C3 vector. <br />
* Transform 356K and S284T with RBS and promoter (lacZ, LacI, RBS). <br />
* Run gel of 365K in psb1C3 of the digest. --> control: RFP + psb1C3<br />
* then add promoter<br />
<br />
'''23/9/2010'''<br />
<br />
* Added in incubator room liquid culture '''?)''' LacP + RBS + Luciferase for S284T, 356K and WT. <br />
* miniprep- 6 bottles<br />
* GLOWING<br />
<br />
* S248T- <br />
**9.2; 5 <br />
** 9.4; 1 <br />
**5; 4<br />
* 356<br />
** 3.3<br />
* WT<br />
** 3.2; 4 <br />
** 1.4, 5</div>Macbereskahttp://2010.igem.org/Team:Edinburgh/Notebook/Red_light_producerTeam:Edinburgh/Notebook/Red light producer2010-10-26T23:33:15Z<p>Macbereska: /* Red Light Producer */</p>
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<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_producer">red light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_sensor">red sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_producer">blue light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_producer">green light</a></li><br />
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<span style="color:ivory;">Throughout this wiki there are words in <b>bold</b> that indicate a relevance to <b>human aspects</b>. It will become obvious that <b>human aspects</b> are a part of almost everything in <b>iGEM</b>.</span><br />
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<br />
== Red Light Producer ==<br />
<br><br />
<br />
'''29/6/10'''<br />
<br />
Now preparing primers to make a set of different spectra bioluminescent proteins. Need to order primers for Site directed mutagenesis of the Firely luciferase to make it red (MABEL protocol), and to make click beetle luciferase Rluc8 (which emits light at about 420nm in presence of bisdeoxycoelenterazine - about the same price a coelenterazine though)<br />
<br />
'''8/7/2010'''<br />
* Red luciferase mutagenesis primers arrived- 3 sets:<br />
** S248T<br />
** 356 K<br />
** 356 R<br />
<br />
* Followed the protocol with 34ul water + 0.5 template DNA<br />
* Put the primers and template in green box '''(???)'''<br />
*Ran Gel: 5 µL PCR product, 5 µL water, 2.5 µL buffer<br />
[[Image:151.JPG]]<br />
<br />
{| border="1"<br />
| Marker<br />
| S284T <br />
| 356K <br />
| 356R<br />
|}<br />
<br />
<br />
[[File:07-08.JPG]]<br />
<br />
<br />
*356K and 356R look fine<br />
*going to run S284T at a higher ** temp tomorrow to see if it works better<br />
*all PCR tubes stored in freezer<br />
<br />
<br />
<br />
'''09/7/10'''<br />
<br />
Redo PCR of Red Luciferase<br />
*Everything except template DNA + Kod polymerase kept on ice<br />
*Ran PCR<br />
*Template DNA used PyeaR and P. pyralis luciferase BBa_K216015<br />
*Purification done for 356K and 356R; stored in iGEM box (-20C).<br />
<br />
[[File:07-09straight.JPG]]<br />
<br />
'''9/7/2010'''<br />
*Ligations set up overnight- might have put too much ligase in 356K... Incubated at 15C.<br />
*Oure 356R and 356K are in the freezer- row 10 (G &H)?<br />
<br />
'''13/07/10'''<br />
Goal of the day: <br />
<br />
Transformations:<br />
*356R (2): one from 16 °C and one from room temp(RT)<br />
*356K (2): one from 16 °C and one from room temp(RT)<br />
*S284T (2): one with 25/5 ligase, one with 1/7 ligase<br />
<br />
* I had a burger for the lunch. It was very nice.<br />
* Now we are transforming cells:<br />
** 356R (2)& 356K (2): one form 16C, one from RT<br />
** S248T (2): 1 with 25/5 ligase, other with 1.7.10 ligase/<br />
Empty edinbrick vector for control. <br />
* LuxAB + edinbrick ligation.<br />
<br />
* Using 100 ul transformed cells + 800 ul LB for all. Incubation started half 5. <br />
'''22/07/10'''<br />
<br />
Liquid Cultures (amp 80):<br />
*356R (RT) – 1 with NaNO3 and 1 without<br />
**(16°C) – 2 with NaNO3 and 2 without<br />
*356K (RT) – 1 with NaNO3 and 1 without<br />
**(16°C) – 2 with NaNO3 and 2 without <br />
*S284T (100 µL and 25/5 ligase) – 2 with NaNO3 and 2 without<br />
**(900 µL and 25/5 ligase) – 3 with NaNO3 and 3 without<br />
**(900 µL and 1/7 ligase) – 5 with NaNO3 and 5 without<br />
<br />
NaNO3 = 30mM Sodium Nitrate<br />
<br />
'''23/07/10'''<br />
<br />
Double digest of K098010<br />
<br />
'''28/07/10'''<br />
<br />
Primer design to produce a stable red light luciferase. The mutations for increasing bioluminescence come from [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W9V-4NHM57C-1&_user=809099&_coverDate=07%2F15%2F2007&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1414502247&_rerunOrigin=google&_acct=C000043939&_version=1&_urlVersion=0&_userid=809099&md5=00d9c92f6452bd276c6ea606ed7d3bbf Fujii et al, 2007] and the paper from [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W9V-4X6MSYB-7&_user=809099&_coverDate=01%2F15%2F2010&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1414503636&_rerunOrigin=google&_acct=C000043939&_version=1&_urlVersion=0&_userid=809099&md5=297be52832193b4d026953632a1edf3c Branchini et al, 2010]<br />
<br />
'''29/7/2010'''<br />
<br />
*Double digest with EcoRI HF&PstI in buffer 2 (neb).<br />
* 2 bands: with and without vector?<br />
* did not leave the digest for too long... <br />
[[Image:157.jpg]]<br />
<br />
{| border="1"<br />
| Lane 1<br />
| Lane 2<br />
| Lane 3<br />
| Lane 4<br />
| Lane 5<br />
| Lane 6<br />
|-<br />
| Ladder <br />
| S284T (4) <br />
| S284T (6)<br />
| 356R (6, 900)<br />
| 356R (6)<br />
| 356K (4)<br />
|}<br />
<br />
'''2/8/2010'''<br />
* Red luciferases EcoRI HF&PstI double digests- lanes 2-7 in the second miniprep set. <br />
*Ladder didn't work too well ,though...<br />
[[Image:113.JPG]]<br />
* Transform last years luciferase<br />
* Look at papers for expression in E. coli<br />
* Replace B0034 by stronger RBS?<br />
* Confirm that we order bright luc.<br />
* Single digest of constructs<br />
* Test glowing with positive control<br />
<br />
'''9/8/2010'''<br />
<br />
[[Image:159.jpg]]<br />
<br />
Digest: <br />
*WT1<br />
*356 R: (2)<br />
* 356 K: (4)<br />
* S284T: (6)<br />
* 3 eith EcoRI; 3 with EcoRi + PstI<br />
<br />
<br />
'''10/8/2010'''<br />
* Sequencing of red mutant luciferases. <br />
* Forward primers for S284T and reverse for 356R and K should do. <br />
* Concentration of primers for sequencing: 5uM.<br />
* sequencing tubE: 6 ul liquid: 3ul DNA + 2ul water, 1 ul primer mix.<br />
* when using miniprep DNA- remember to spin down the dirt- 1min, max speed, then take the DNA from the top to avoid disturbing any dirt that is settled. <br />
<br />
<br />
'''12/8/2010'''<br />
* Got the sequences for red luciferases<br />
*see: if we can detect Red Light with the luminometer?<br />
* Red Luciferases 356R and S284T were nNOT MUTANTS- just WT<br />
* set up ;iquid cultures with 30 ul nitrate for: <br />
** 356R - no 3,1 from RT plate, no 5 from 16C plate; no 1 from Master plate<br />
** S284T - 5 from masterplate; 2, 4, 6 from masterplate<br />
** 356K: 4 from masterplate<br />
<br />
'''13/8/2010'''<br />
Big day today... TESTING YESTERDAYS PLAN- LUMINESCENCE<br />
<br />
'''16/8/2010'''<br />
* Sequecning of: <br />
** WR1- 356R -use S284T forward primer<br />
** WR2 -S284T -use 356 R reverse primer<br />
** WR3- LovTap 03<br />
**WR4 -LovTap 09<br />
<br />
* RESULTS: ONLY 356K WAS A REAL MUTANT; S284T AND 356R TURNED OUT TO BE STILL A WT<br />
<br />
'''19/8/2010'''<br />
* star6: 56,869<br />
* star4: 13,471<br />
* 356K: 263,778<br />
* star 2: 3,717<br />
* WT: 1,617832<br />
<br />
*Gel of the ligations: marker (lane 1), 356K (lane 2,3), S284T (lane 3,4), 358Rs (lane 5,6).<br />
<br />
[[Image:163.jpg]]<br />
<br />
'''25/8/2010'''<br />
* Next step:- redo ligations for 356R; redo PCR for S284T<br />
* Ligations<br />
*Did MABEL PCR PPyInc + S284T primers<br />
*used 0.5 ul template DNA<br />
*Gel: marker (Lane1), ligation of PCR product for 356R (Lane2), Ligation of 07 PCR for S284T (lane3), Pure PCR from S284T. <br />
<br />
[[Image:164.jpg]]<br />
<br />
'''26/8/2010'''<br />
* check the Fuji et. al paper, see what sequence they used to start off with. <br />
* check IAV in other luciferase<br />
* Fix scar before RBS and in teh second stop codon. <br />
* compared to WT, luciferase sequence has: <br />
** N 50 P<br />
** N 99 G<br />
** SKL- IAV<br />
<br />
'''27/8/2010'''<br />
* Ligations from yesterday out of freezer.<br />
* Running on gel<br />
* Column 3 9with 356R) has less DNA in it- not enough from ligation and messed up loading of gel). <br />
[[Image:132.JPG]]<br />
{| border="1"<br />
| Lane 1<br />
| Lane 2<br />
| Lane 3<br />
| Lane 4<br />
<br />
|-<br />
| Ladder <br />
| New S284T PCR - new ligase and the buffer<br />
| 356 R ligation<br />
| S284T PCR + old ligase<br />
|}<br />
<br />
'''30/8/2010'''<br />
* Primers have arrived for fixing the luciferase- removing 6 bases<br />
* Primers have arrived for up and downstream sequences of TnaA region.<br />
* Send Chairman sequence for T7 promoter<br />
* transformed 356R and S284T ligations from Friday<br />
* Used PCR product as a control<br />
* Plated out the transformed cells. Used 900 rather than 100 if possible<br />
<br />
<br />
'''31/8/2010'''<br />
* We have S284T transformants but <br />
** 14 colonies on ligation plate<br />
** 4 colonies on control where we used PCR product instead of ligation<br />
<br />
* For sequencing- <br />
** if sequencing a S284T mutant, use 356R/K reverse primer<br />
** if sequencing 356R mutant, use S284T forward primer.<br />
<br />
'''2/9/2010'''<br />
* minipreped 14 colonies (1-14)<br />
* run the gel with samples digested with EcoRI<br />
* here: samples 1-7<br />
[[Image:135.JPG]]<br />
<br />
'''3/9/2010'''<br />
* S284T 8-14 (from yesterday)<br />
[[Image:136.JPG]]<br />
<br />
'''8/9/2010'''<br />
* wWT of luciferase (left)<br />
* mutation of S284T (right)<br />
<br />
<br />
'''9/9/2010'''<br />
{| border="1"<br />
| Lane 1<br />
| Lane 2<br />
| Lane 3<br />
| Lane 4<br />
| Lane 5<br />
|-<br />
| Ladder <br />
| WT <br />
| S284T 1<br />
| luxCDE 9:4<br />
| LuxCDE 9:1<br />
|}<br />
<br />
'''20/9/2010'''<br />
<br />
*send for sequencing: 356K, S284T, WT (samples from 16/9: WT- 900, 2; also 16/9 S284T- 900, 4.)<br />
* all in psB1C3 vector. <br />
* Transform 356K and S284T with RBS and promoter (lacZ, LacI, RBS). <br />
* Run gel of 365K in psb1C3 of the digest. --> control: RFP + psb1C3<br />
* then add promoter<br />
<br />
'''23/9/2010'''<br />
<br />
* Added in incubator room liquid culture '''?)''' LacP + RBS + Luciferase for S284T, 356K and WT. <br />
* miniprep- 6 bottles<br />
* GLOWING<br />
<br />
* S248T- <br />
**9.2; 5 <br />
** 9.4; 1 <br />
**5; 4<br />
* 356<br />
** 3.3<br />
* WT<br />
** 3.2; 4 <br />
** 1.4, 5</div>Macbereskahttp://2010.igem.org/Team:Edinburgh/Notebook/Red_light_producerTeam:Edinburgh/Notebook/Red light producer2010-10-26T23:29:56Z<p>Macbereska: /* Red Light Producer */</p>
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<li><a href="https://2010.igem.org/Team:Edinburgh/Team" class="dir">team - illuminati</a><br />
<ul><br />
<li><a href="https://igem.org/Team.cgi?id=322">official</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Team/Supervisors">supervisors</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Team/Advisors">advisors</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Team/Students">students</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Team/Environment">environment</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Team/Gallery" class="dir">gallery</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Project" class="dir">genomic BRIDGEs</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Project/Protocol">the protocol</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/BioBricks#Genomic">submitted parts</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Genomic">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Project/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Project/References">references</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial" class="dir">bacterial BRIDGEs</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Core_repressilator">the repressilator</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_producer">red light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_sensor">red sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_producer">blue light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Green_light_producer">green light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Green_light_sensor">green sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/BioBricks#Bacterial">submitted parts</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Bacterial">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/References">references</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling" class="dir">modelling BRIDGEs</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Kappa">kappa</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Genomic">the genomic model</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Bacterial">the bacterial model</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Signalling">the signalling model</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Tools">tools</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Modelling">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/References">references</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human" class="dir">human BRIDGEs</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Aspects">human aspects</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Communication">communication of science</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Terminology">terminology research</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Wiki">wiki</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Conversations">conversations</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Identity">identity</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Collaboration">collaboration</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Branding">branding research</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/SciFi">science fiction writing</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/FutureApps">future applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/SelfReflection">self-reflection</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Epic">the epic</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Human">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/References">references</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook" class="dir">lab notes&nbsp;&nbsp;&nbsp;</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Collaboration">collaboration</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Attribution">attribution</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/BRIDGE">BRIDGE</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_producer">red light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_sensor">red sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_producer">blue light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_producer">green light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_sensor">green sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Modelling">modelling</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Safety">safety</a></li><br />
<li><a href="http://www.openwetware.org/wiki/French_Lab">protocols</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Acknowledgements" class="dir">acknowledgements</a><br />
</li><br />
<br />
</ul><br />
</div><br />
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<span style="color:ivory;">Throughout this wiki there are words in <b>bold</b> that indicate a relevance to <b>human aspects</b>. It will become obvious that <b>human aspects</b> are a part of almost everything in <b>iGEM</b>.</span><br />
<br />
</div><br />
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</html><br />
<br />
== Red Light Producer ==<br />
<br><br />
<br />
'''29/6/10'''<br />
<br />
Now preparing primers to make a set of different spectra bioluminescent proteins. Need to order primers for Site directed mutagenesis of the Firely luciferase to make it red (MABEL protocol), and to make click beetle luciferase Rluc8 (which emits light at about 420nm in presence of bisdeoxycoelenterazine - about the same price a coelenterazine though)<br />
<br />
'''8/7/2010'''<br />
* Red luciferase mutagenesis primers arrived- 3 sets:<br />
** S248T<br />
** 356 K<br />
** 356 R<br />
<br />
* Followed the protocol with 34ul water + 0.5 template DNA<br />
* Put the primers and template in green box '''(???)'''<br />
*Ran Gel: 5 µL PCR product, 5 µL water, 2.5 µL buffer<br />
[[Image:151.JPG]]<br />
<br />
{| border="1"<br />
| Marker<br />
| S284T <br />
| 356K <br />
| 356R<br />
|}<br />
<br />
<br />
[[File:07-08.JPG]]<br />
<br />
<br />
*356K and 356R look fine<br />
*going to run S284T at a higher ** temp tomorrow to see if it works better<br />
*all PCR tubes stored in freezer<br />
<br />
<br />
<br />
'''09/7/10'''<br />
<br />
Redo PCR of Red Luciferase<br />
*Everything except template DNA + Kod polymerase kept on ice<br />
*Ran PCR<br />
*Template DNA used PyeaR and P. pyralis luciferase BBa_K216015<br />
*Purification done for 356K and 356R; stored in iGEM box (-20C).<br />
<br />
[[File:07-09straight.JPG]]<br />
<br />
'''9/7/2010'''<br />
*Ligations set up overnight- might have put too much ligase in 356K... Incubated at 15C.<br />
*Oure 356R and 356K are in the freezer- row 10 (G &H)?<br />
<br />
'''13/07/10'''<br />
Goal of the day: <br />
<br />
Transformations:<br />
*356R (2): one from 16 °C and one from room temp(RT)<br />
*356K (2): one from 16 °C and one from room temp(RT)<br />
*S284T (2): one with 25/5 ligase, one with 1/7 ligase<br />
<br />
* I had a burger for the lunch. It was very nice.<br />
* Now we are transforming cells:<br />
** 356R (2)& 356K (2): one form 16C, one from RT<br />
** S248T (2): 1 with 25/5 ligase, other with 1.7.10 ligase/<br />
Empty edinbrick vector for control. <br />
* LuxAB + edinbrick ligation.<br />
<br />
* Using 100 ul transformed cells + 800 ul LB for all. Incubation started half 5. <br />
'''22/07/10'''<br />
<br />
Liquid Cultures (amp 80):<br />
*356R (RT) – 1 with NaNO3 and 1 without<br />
**(16°C) – 2 with NaNO3 and 2 without<br />
*356K (RT) – 1 with NaNO3 and 1 without<br />
**(16°C) – 2 with NaNO3 and 2 without <br />
*S284T (100 µL and 25/5 ligase) – 2 with NaNO3 and 2 without<br />
**(900 µL and 25/5 ligase) – 3 with NaNO3 and 3 without<br />
**(900 µL and 1/7 ligase) – 5 with NaNO3 and 5 without<br />
<br />
NaNO3 = 30mM Sodium Nitrate<br />
<br />
'''23/07/10'''<br />
<br />
Double digest of K098010<br />
<br />
'''28/07/10'''<br />
<br />
Primer design to produce a stable red light luciferase. The mutations for increasing bioluminescence come from [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W9V-4NHM57C-1&_user=809099&_coverDate=07%2F15%2F2007&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1414502247&_rerunOrigin=google&_acct=C000043939&_version=1&_urlVersion=0&_userid=809099&md5=00d9c92f6452bd276c6ea606ed7d3bbf Fujii et al, 2007] and the paper from [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W9V-4X6MSYB-7&_user=809099&_coverDate=01%2F15%2F2010&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1414503636&_rerunOrigin=google&_acct=C000043939&_version=1&_urlVersion=0&_userid=809099&md5=297be52832193b4d026953632a1edf3c Branchini et al, 2010]<br />
<br />
'''29/7/2010'''<br />
<br />
*Double digest with EcoRI HF&PstI in buffer 2 (neb).<br />
* 2 bands: with and without vector?<br />
* did not leave the digest for too long... <br />
[[Image:157.jpg]]<br />
<br />
{| border="1"<br />
| Lane 1<br />
| Lane 2<br />
| Lane 3<br />
| Lane 4<br />
| Lane 5<br />
| Lane 6<br />
|-<br />
| Ladder <br />
| S284T (4) <br />
| S284T (6)<br />
| 356R (6, 900)<br />
| 356R (6)<br />
| 356K (4)<br />
|}<br />
<br />
'''2/8/2010'''<br />
* Red luciferases EcoRI HF&PstI double digests- lanes 2-7 in the second miniprep set. <br />
*Ladder didn't work too well ,though...<br />
[[Image:113.JPG]]<br />
* Transform last years luciferase<br />
* Look at papers for expression in E. coli<br />
* Replace B0034 by stronger RBS?<br />
* Confirm that we order bright luc.<br />
* Single digest of constructs<br />
* Test glowing with positive control<br />
<br />
'''9/8/2010'''<br />
<br />
[[Image:159.jpg]]<br />
<br />
Digest: <br />
*WT1<br />
*356 R: (2)<br />
* 356 K: (4)<br />
* S284T: (6)<br />
* 3 eith EcoRI; 3 with EcoRi + PstI<br />
<br />
<br />
'''10/8/2010'''<br />
* Sequencing of red mutant luciferases. <br />
* Forward primers for S284T and reverse for 356R and K should do. <br />
* Concentration of primers for sequencing: 5uM.<br />
* sequencing tubE: 6 ul liquid: 3ul DNA + 2ul water, 1 ul primer mix.<br />
* when using miniprep DNA- remember to spin down the dirt- 1min, max speed, then take the DNA from the top to avoid disturbing any dirt that is settled. <br />
<br />
<br />
'''12/8/2010'''<br />
* Got the sequences for red luciferases<br />
*see: if we can detect Red Light with the luminometer?<br />
* Red Luciferases 356R and S284T were nNOT MUTANTS- just WT<br />
* set up ;iquid cultures with 30 ul nitrate for: <br />
** 356R - no 3,1 from RT plate, no 5 from 16C plate; no 1 from Master plate<br />
** S284T - 5 from masterplate; 2, 4, 6 from masterplate<br />
** 356K: 4 from masterplate<br />
<br />
'''13/8/2010'''<br />
Big day today... TESTING YESTERDAYS PLAN- LUMINESCENCE<br />
<br />
'''16/8/2010'''<br />
* Sequecning of: <br />
** WR1- 356R -use S284T forward primer<br />
** WR2 -S284T -use 356 R reverse primer<br />
** WR3- LovTap 03<br />
**WR4 -LovTap 09<br />
<br />
* RESULTS: ONLY 356K WAS A REAL MUTANT; S284T AND 356R TURNED OUT TO BE STILL A WT<br />
<br />
'''19/8/2010'''<br />
* star6: 56,869<br />
* star4: 13,471<br />
* 356K: 263,778<br />
* star 2: 3,717<br />
* WT: 1,617832<br />
<br />
*Gel of the ligations: marker (lane 1), 356K (lane 2,3), S284T (lane 3,4), 358Rs (lane 5,6).<br />
<br />
[[Image:163.jpg]]<br />
<br />
'''25/8/2010'''<br />
* Next step:- redo ligations for 356R; redo PCR for S284T<br />
* Ligations<br />
*Did MABEL PCR PPyInc + S284T primers<br />
*used 0.5 ul template DNA<br />
<br />
'''26/8/2010'''<br />
* check the Fuji et. al paper, see what sequence they used to start off with. <br />
* check IAV in other luciferase<br />
* Fix scar before RBS and in teh second stop codon. <br />
* compared to WT, luciferase sequence has: <br />
** N 50 P<br />
** N 99 G<br />
** SKL- IAV<br />
<br />
'''27/8/2010'''<br />
* Ligations from yesterday out of freezer.<br />
* Running on gel<br />
* Column 3 9with 356R) has less DNA in it- not enough from ligation and messed up loading of gel). <br />
[[Image:132.JPG]]<br />
{| border="1"<br />
| Lane 1<br />
| Lane 2<br />
| Lane 3<br />
| Lane 4<br />
<br />
|-<br />
| Ladder <br />
| New S284T PCR - new ligase and the buffer<br />
| 356 R ligation<br />
| S284T PCR + old ligase<br />
|}<br />
<br />
'''30/8/2010'''<br />
* Primers have arrived for fixing the luciferase- removing 6 bases<br />
* Primers have arrived for up and downstream sequences of TnaA region.<br />
* Send Chairman sequence for T7 promoter<br />
* transformed 356R and S284T ligations from Friday<br />
* Used PCR product as a control<br />
* Plated out the transformed cells. Used 900 rather than 100 if possible<br />
<br />
<br />
'''31/8/2010'''<br />
* We have S284T transformants but <br />
** 14 colonies on ligation plate<br />
** 4 colonies on control where we used PCR product instead of ligation<br />
<br />
* For sequencing- <br />
** if sequencing a S284T mutant, use 356R/K reverse primer<br />
** if sequencing 356R mutant, use S284T forward primer.<br />
<br />
'''2/9/2010'''<br />
* minipreped 14 colonies (1-14)<br />
* run the gel with samples digested with EcoRI<br />
* here: samples 1-7<br />
[[Image:135.JPG]]<br />
<br />
'''3/9/2010'''<br />
* S284T 8-14 (from yesterday)<br />
[[Image:136.JPG]]<br />
<br />
'''8/9/2010'''<br />
* wWT of luciferase (left)<br />
* mutation of S284T (right)<br />
<br />
<br />
'''9/9/2010'''<br />
{| border="1"<br />
| Lane 1<br />
| Lane 2<br />
| Lane 3<br />
| Lane 4<br />
| Lane 5<br />
|-<br />
| Ladder <br />
| WT <br />
| S284T 1<br />
| luxCDE 9:4<br />
| LuxCDE 9:1<br />
|}<br />
<br />
'''20/9/2010'''<br />
<br />
*send for sequencing: 356K, S284T, WT (samples from 16/9: WT- 900, 2; also 16/9 S284T- 900, 4.)<br />
* all in psB1C3 vector. <br />
* Transform 356K and S284T with RBS and promoter (lacZ, LacI, RBS). <br />
* Run gel of 365K in psb1C3 of the digest. --> control: RFP + psb1C3<br />
* then add promoter<br />
<br />
'''23/9/2010'''<br />
<br />
* Added in incubator room liquid culture '''?)''' LacP + RBS + Luciferase for S284T, 356K and WT. <br />
* miniprep- 6 bottles<br />
* GLOWING<br />
<br />
* S248T- <br />
**9.2; 5 <br />
** 9.4; 1 <br />
**5; 4<br />
* 356<br />
** 3.3<br />
* WT<br />
** 3.2; 4 <br />
** 1.4, 5</div>Macbereskahttp://2010.igem.org/Team:Edinburgh/Notebook/Red_light_producerTeam:Edinburgh/Notebook/Red light producer2010-10-26T23:28:58Z<p>Macbereska: /* Red Light Producer */</p>
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<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Core_repressilator">the repressilator</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_producer">red light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_sensor">red sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_producer">blue light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Green_light_producer">green light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Green_light_sensor">green sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/BioBricks#Bacterial">submitted parts</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Bacterial">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/References">references</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling" class="dir">modelling BRIDGEs</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Kappa">kappa</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Genomic">the genomic model</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Bacterial">the bacterial model</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Signalling">the signalling model</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Tools">tools</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Modelling">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/References">references</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human" class="dir">human BRIDGEs</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Aspects">human aspects</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Communication">communication of science</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Terminology">terminology research</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Wiki">wiki</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Conversations">conversations</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Identity">identity</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Collaboration">collaboration</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Branding">branding research</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/SciFi">science fiction writing</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/FutureApps">future applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/SelfReflection">self-reflection</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Epic">the epic</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Human">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/References">references</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook" class="dir">lab notes&nbsp;&nbsp;&nbsp;</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Collaboration">collaboration</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Attribution">attribution</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/BRIDGE">BRIDGE</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_producer">red light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_sensor">red sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_producer">blue light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_producer">green light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_sensor">green sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Modelling">modelling</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Safety">safety</a></li><br />
<li><a href="http://www.openwetware.org/wiki/French_Lab">protocols</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Acknowledgements" class="dir">acknowledgements</a><br />
</li><br />
<br />
</ul><br />
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<span style="color:ivory;">Throughout this wiki there are words in <b>bold</b> that indicate a relevance to <b>human aspects</b>. It will become obvious that <b>human aspects</b> are a part of almost everything in <b>iGEM</b>.</span><br />
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<br />
== Red Light Producer ==<br />
<br><br />
<br />
'''29/6/10'''<br />
<br />
Now preparing primers to make a set of different spectra bioluminescent proteins. Need to order primers for Site directed mutagenesis of the Firely luciferase to make it red (MABEL protocol), and to make click beetle luciferase Rluc8 (which emits light at about 420nm in presence of bisdeoxycoelenterazine - about the same price a coelenterazine though)<br />
<br />
'''8/7/2010'''<br />
* Red luciferase mutagenesis primers arrived- 3 sets:<br />
** S248T<br />
** 356 K<br />
** 356 R<br />
<br />
* Followed the protocol with 34ul water + 0.5 template DNA<br />
* Put the primers and template in green box '''(???)'''<br />
*Ran Gel: 5 µL PCR product, 5 µL water, 2.5 µL buffer<br />
[[Image:151.JPG]]<br />
<br />
{| border="1"<br />
| Marker<br />
| S284T <br />
| 356K <br />
| 356R<br />
|}<br />
<br />
<br />
[[File:07-08.JPG]]<br />
<br />
<br />
*356K and 356R look fine<br />
*going to run S284T at a higher ** temp tomorrow to see if it works better<br />
*all PCR tubes stored in freezer<br />
<br />
<br />
<br />
'''09/7/10'''<br />
<br />
Redo PCR of Red Luciferase<br />
*Everything except template DNA + Kod polymerase kept on ice<br />
*Ran PCR<br />
*Template DNA used PyeaR and P. pyralis luciferase BBa_K216015<br />
*Purification done for 356K and 356R; stored in iGEM box (-20C).<br />
<br />
[[File:07-09straight.JPG]]<br />
<br />
'''9/7/2010'''<br />
*Ligations set up overnight- might have put too much ligase in 356K... Incubated at 15C.<br />
*Oure 356R and 356K are in the freezer- row 10 (G &H)?<br />
<br />
'''13/07/10'''<br />
Goal of the day: <br />
<br />
Transformations:<br />
*356R (2): one from 16 °C and one from room temp(RT)<br />
*356K (2): one from 16 °C and one from room temp(RT)<br />
*S284T (2): one with 25/5 ligase, one with 1/7 ligase<br />
<br />
* I had a burger for the lunch. It was very nice.<br />
* Now we are transforming cells:<br />
** 356R (2)& 356K (2): one form 16C, one from RT<br />
** S248T (2): 1 with 25/5 ligase, other with 1.7.10 ligase/<br />
Empty edinbrick vector for control. <br />
* LuxAB + edinbrick ligation.<br />
<br />
* Using 100 ul transformed cells + 800 ul LB for all. Incubation started half 5. <br />
'''22/07/10'''<br />
<br />
Liquid Cultures (amp 80):<br />
*356R (RT) – 1 with NaNO3 and 1 without<br />
**(16°C) – 2 with NaNO3 and 2 without<br />
*356K (RT) – 1 with NaNO3 and 1 without<br />
**(16°C) – 2 with NaNO3 and 2 without <br />
*S284T (100 µL and 25/5 ligase) – 2 with NaNO3 and 2 without<br />
**(900 µL and 25/5 ligase) – 3 with NaNO3 and 3 without<br />
**(900 µL and 1/7 ligase) – 5 with NaNO3 and 5 without<br />
<br />
NaNO3 = 30mM Sodium Nitrate<br />
<br />
'''23/07/10'''<br />
<br />
Double digest of K098010<br />
<br />
'''28/07/10'''<br />
<br />
Primer design to produce a stable red light luciferase. The mutations for increasing bioluminescence come from [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W9V-4NHM57C-1&_user=809099&_coverDate=07%2F15%2F2007&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1414502247&_rerunOrigin=google&_acct=C000043939&_version=1&_urlVersion=0&_userid=809099&md5=00d9c92f6452bd276c6ea606ed7d3bbf Fujii et al, 2007] and the paper from [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W9V-4X6MSYB-7&_user=809099&_coverDate=01%2F15%2F2010&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1414503636&_rerunOrigin=google&_acct=C000043939&_version=1&_urlVersion=0&_userid=809099&md5=297be52832193b4d026953632a1edf3c Branchini et al, 2010]<br />
<br />
'''29/7/2010'''<br />
<br />
*Double digest with EcoRI HF&PstI in buffer 2 (neb).<br />
* 2 bands: with and without vector?<br />
* did not leave the digest for too long... <br />
[[Image:157.jpg]]<br />
<br />
{| border="1"<br />
| Lane 1<br />
| Lane 2<br />
| Lane 3<br />
| Lane 4<br />
| Lane 5<br />
| Lane 6<br />
|-<br />
| Ladder <br />
| S284T (4) <br />
| S284T (6)<br />
| 356R (6, 900)<br />
| 356R (6)<br />
| 356K (4)<br />
|}<br />
<br />
'''2/8/2010'''<br />
* Red luciferases EcoRI HF&PstI double digests- lanes 2-7 in the second miniprep set. <br />
*Ladder didn't work too well ,though...<br />
[[Image:113.JPG]]<br />
* Transform last years luciferase<br />
* Look at papers for expression in E. coli<br />
* Replace B0034 by stronger RBS?<br />
* Confirm that we order bright luc.<br />
* Single digest of constructs<br />
* Test glowing with positive control<br />
<br />
'''9/8/2010'''<br />
<br />
[[Image:159.jpg]]<br />
<br />
Digest: <br />
*WT1<br />
*356 R: (2)<br />
* 356 K: (4)<br />
* S284T: (6)<br />
* 3 eith EcoRI; 3 with EcoRi + PstI<br />
<br />
<br />
'''10/8/2010'''<br />
* Sequencing of red mutant luciferases. <br />
* Forward primers for S284T and reverse for 356R and K should do. <br />
* Concentration of primers for sequencing: 5uM.<br />
* sequencing tubE: 6 ul liquid: 3ul DNA + 2ul water, 1 ul primer mix.<br />
* when using miniprep DNA- remember to spin down the dirt- 1min, max speed, then take the DNA from the top to avoid disturbing any dirt that is settled. <br />
<br />
<br />
'''12/8/2010'''<br />
* Got the sequences for red luciferases<br />
*see: if we can detect Red Light with the luminometer?<br />
* Red Luciferases 356R and S284T were nNOT MUTANTS- just WT<br />
* set up ;iquid cultures with 30 ul nitrate for: <br />
** 356R - no 3,1 from RT plate, no 5 from 16C plate; no 1 from Master plate<br />
** S284T - 5 from masterplate; 2, 4, 6 from masterplate<br />
** 356K: 4 from masterplate<br />
<br />
'''13/8/2010'''<br />
Big day today... TESTING YESTERDAYS PLAN- LUMINESCENCE<br />
<br />
'''16/8/2010'''<br />
* Sequecning of: <br />
** WR1- 356R -use S284T forward primer<br />
** WR2 -S284T -use 356 R reverse primer<br />
** WR3- LovTap 03<br />
**WR4 -LovTap 09<br />
<br />
* RESULTS: ONLY 356K WAS A REAL MUTANT; S284T AND 356R TURNED OUT TO BE STILL A WT<br />
<br />
'''19/8/2010'''<br />
* star6: 56,869<br />
* star4: 13,471<br />
* 356K: 263,778<br />
* star 2: 3,717<br />
* WT: 1,617832<br />
<br />
*Gel of the ligations: marker (lane 1), 356K (lane 2,3), S284T (lane 3,4), 358Rs (lane 5,6).<br />
<br />
[Image:163.jpg]]<br />
<br />
'''25/8/2010'''<br />
* Next step:- redo ligations for 356R; redo PCR for S284T<br />
* Ligations<br />
*Did MABEL PCR PPyInc + S284T primers<br />
*used 0.5 ul template DNA<br />
<br />
'''26/8/2010'''<br />
* check the Fuji et. al paper, see what sequence they used to start off with. <br />
* check IAV in other luciferase<br />
* Fix scar before RBS and in teh second stop codon. <br />
* compared to WT, luciferase sequence has: <br />
** N 50 P<br />
** N 99 G<br />
** SKL- IAV<br />
<br />
'''27/8/2010'''<br />
* Ligations from yesterday out of freezer.<br />
* Running on gel<br />
* Column 3 9with 356R) has less DNA in it- not enough from ligation and messed up loading of gel). <br />
[[Image:132.JPG]]<br />
{| border="1"<br />
| Lane 1<br />
| Lane 2<br />
| Lane 3<br />
| Lane 4<br />
<br />
|-<br />
| Ladder <br />
| New S284T PCR - new ligase and the buffer<br />
| 356 R ligation<br />
| S284T PCR + old ligase<br />
|}<br />
<br />
'''30/8/2010'''<br />
* Primers have arrived for fixing the luciferase- removing 6 bases<br />
* Primers have arrived for up and downstream sequences of TnaA region.<br />
* Send Chairman sequence for T7 promoter<br />
* transformed 356R and S284T ligations from Friday<br />
* Used PCR product as a control<br />
* Plated out the transformed cells. Used 900 rather than 100 if possible<br />
<br />
<br />
'''31/8/2010'''<br />
* We have S284T transformants but <br />
** 14 colonies on ligation plate<br />
** 4 colonies on control where we used PCR product instead of ligation<br />
<br />
* For sequencing- <br />
** if sequencing a S284T mutant, use 356R/K reverse primer<br />
** if sequencing 356R mutant, use S284T forward primer.<br />
<br />
'''2/9/2010'''<br />
* minipreped 14 colonies (1-14)<br />
* run the gel with samples digested with EcoRI<br />
* here: samples 1-7<br />
[[Image:135.JPG]]<br />
<br />
'''3/9/2010'''<br />
* S284T 8-14 (from yesterday)<br />
[[Image:136.JPG]]<br />
<br />
'''8/9/2010'''<br />
* wWT of luciferase (left)<br />
* mutation of S284T (right)<br />
<br />
<br />
'''9/9/2010'''<br />
{| border="1"<br />
| Lane 1<br />
| Lane 2<br />
| Lane 3<br />
| Lane 4<br />
| Lane 5<br />
|-<br />
| Ladder <br />
| WT <br />
| S284T 1<br />
| luxCDE 9:4<br />
| LuxCDE 9:1<br />
|}<br />
<br />
'''20/9/2010'''<br />
<br />
*send for sequencing: 356K, S284T, WT (samples from 16/9: WT- 900, 2; also 16/9 S284T- 900, 4.)<br />
* all in psB1C3 vector. <br />
* Transform 356K and S284T with RBS and promoter (lacZ, LacI, RBS). <br />
* Run gel of 365K in psb1C3 of the digest. --> control: RFP + psb1C3<br />
* then add promoter<br />
<br />
'''23/9/2010'''<br />
<br />
* Added in incubator room liquid culture '''?)''' LacP + RBS + Luciferase for S284T, 356K and WT. <br />
* miniprep- 6 bottles<br />
* GLOWING<br />
<br />
* S248T- <br />
**9.2; 5 <br />
** 9.4; 1 <br />
**5; 4<br />
* 356<br />
** 3.3<br />
* WT<br />
** 3.2; 4 <br />
** 1.4, 5</div>Macbereskahttp://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_producerTeam:Edinburgh/Notebook/Blue light producer2010-10-26T23:24:12Z<p>Macbereska: /* Blue Light Producer */</p>
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<li><a href="https://2010.igem.org/Team:Edinburgh/Team" class="dir">team - illuminati</a><br />
<ul><br />
<li><a href="https://igem.org/Team.cgi?id=322">official</a></li><br />
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<li><a href="https://2010.igem.org/Team:Edinburgh/Project" class="dir">genomic BRIDGEs</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Project/Protocol">the protocol</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/BioBricks#Genomic">submitted parts</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Genomic">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Project/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Project/References">references</a></li><br />
</ul><br />
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<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial" class="dir">bacterial BRIDGEs</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Core_repressilator">the repressilator</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_producer">red light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_sensor">red sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_producer">blue light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Green_light_producer">green light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Green_light_sensor">green sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/BioBricks#Bacterial">submitted parts</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Bacterial">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/References">references</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling" class="dir">modelling BRIDGEs</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Kappa">kappa</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Genomic">the genomic model</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Bacterial">the bacterial model</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Signalling">the signalling model</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Tools">tools</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Modelling">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/References">references</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human" class="dir">human BRIDGEs</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Aspects">human aspects</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Communication">communication of science</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Terminology">terminology research</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Wiki">wiki</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Conversations">conversations</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Identity">identity</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Collaboration">collaboration</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Branding">branding research</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/SciFi">science fiction writing</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/FutureApps">future applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/SelfReflection">self-reflection</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Epic">the epic</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Human">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/References">references</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook" class="dir">lab notes&nbsp;&nbsp;&nbsp;</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Collaboration">collaboration</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Attribution">attribution</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/BRIDGE">BRIDGE</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_producer">red light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_sensor">red sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_producer">blue light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_producer">green light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_sensor">green sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Modelling">modelling</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Safety">safety</a></li><br />
<li><a href="http://www.openwetware.org/wiki/French_Lab">protocols</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Acknowledgements" class="dir">acknowledgements</a><br />
</li><br />
<br />
</ul><br />
</div><br />
<br />
<br><br />
<br><br />
<br><br />
<br />
<div id="windowbox" style="border: .2em solid #660000; padding: 5px; position:fixed; top:50%; right:30px; width:8%;"><br />
<span style="color:ivory;">Throughout this wiki there are words in <b>bold</b> that indicate a relevance to <b>human aspects</b>. It will become obvious that <b>human aspects</b> are a part of almost everything in <b>iGEM</b>.</span><br />
<br />
</div><br />
<br />
</html><br />
<br />
== Blue Light Producer ==<br />
<br><br />
'''5/7/2010'''<br />
*LuxAB & LuxCDE operon agarose gel<br />
* Lane1: About2.1.kbp<br />
* Lane 2: The highest band- very faint- about 3.5kbp<br />
[[Image:150.JPG]]<br />
<br />
'''13/7/2010'''<br />
<br />
Empty edinbrick vector for control. <br />
* LuxAB + edinbrick ligation.<br />
<br />
'''28/7/2010'''<br />
<br />
an update on LuxAB: <br />
*The transformations of Edinbrick are fine but all the luxABs failed (grew blue, not white on Xgal and did not glow, as of 27/7)/<br />
*Still have some digested LuxAB-ediI so re-did ligation and transformation. <br />
[[Image:154.JPG]]<br />
<br />
{| border="1"<br />
| Lane 1&15<br />
| Lane 2<br />
| Others<br />
|-<br />
| Ladder <br />
| EdiI <br />
| LuxAB <br />
|}<br />
[[Image:155.JPG]]<br />
{| border="1"<br />
| Lane 1<br />
| Lane 2 +3<br />
| Lanes 3-6<br />
|-<br />
| Ladder <br />
| EdiI <br />
| LuxAB <br />
|}<br />
<br />
'''29/7/2010'''<br />
<br />
[[Image:156.jpg]]<br />
<br />
* The ligation had the same gel characteristics as previous luxAb-ediI ligations, e.x. it isn't visivle- not hopeful of this transformation working<br />
<br />
'''4/8/2010'''<br />
* made minipreps of blue (negative colonies)- double digest and ran in comparison with pure edinbrickI<br />
* LacZ band dissapears but colonies still grow blue (WTF)<br />
* Has bands equaling to luxAB and EdiI but there was no glowing <br />
<br />
[[Image:162.jpg]]<br />
<br />
'''12/8/2010''' <br />
update on LumP:<br />
* still getting pink/red colonies from transformants<br />
* single digests give a band of about 3kb<br />
* double digests give a band of about 2.5 kb -highly confusing<br />
* and also, no sign on RFP despite red/pink growth.<br />
* Vector wa ssequenced and LumP is definately there. <br />
<br />
[[Image:160.jpg]]<br />
<br />
Lanes 2-5: single digests of lumP<br />
Lane 6: double digest of lumP<br />
<br />
'''26/8/2010'''<br />
<br />
* Marker (Lane 1), minipreps 6-10 luxABlumP (lanes 2-6)<br />
[[Image:129.JPG]]<br />
* single digests of LuxABlumP<br />
[[Image:131.JPG]]<br />
<br />
'''7/9/2010'''<br />
*minipreps of luxCDE: samples 1&4 usable<br />
<br />
{| border="1"<br />
| Lane 1<br />
| Lane 2-6<br />
| Lanes 7<br />
|-<br />
| Ladder <br />
| luxCDE00:1-5 minipreps<br />
| Ladder <br />
|}<br />
[[Image:140.JPG]]</div>Macbereskahttp://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_producerTeam:Edinburgh/Notebook/Blue light producer2010-10-26T23:23:20Z<p>Macbereska: /* Blue Light Producer */</p>
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<li><a href="https://2010.igem.org/Team:Edinburgh/Team/Advisors">advisors</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Team/Students">students</a></li><br />
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<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Project/Protocol">the protocol</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/BioBricks#Genomic">submitted parts</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Genomic">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Project/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Project/References">references</a></li><br />
</ul><br />
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<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial" class="dir">bacterial BRIDGEs</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Core_repressilator">the repressilator</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_producer">red light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_sensor">red sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_producer">blue light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Green_light_producer">green light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Green_light_sensor">green sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/BioBricks#Bacterial">submitted parts</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Bacterial">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/References">references</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling" class="dir">modelling BRIDGEs</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Kappa">kappa</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Genomic">the genomic model</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Bacterial">the bacterial model</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Signalling">the signalling model</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Tools">tools</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Modelling">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/References">references</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human" class="dir">human BRIDGEs</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Aspects">human aspects</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Communication">communication of science</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Terminology">terminology research</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Wiki">wiki</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Conversations">conversations</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Identity">identity</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Collaboration">collaboration</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Branding">branding research</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/SciFi">science fiction writing</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/FutureApps">future applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/SelfReflection">self-reflection</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Epic">the epic</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Human">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/References">references</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook" class="dir">lab notes&nbsp;&nbsp;&nbsp;</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Collaboration">collaboration</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Attribution">attribution</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/BRIDGE">BRIDGE</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_producer">red light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_sensor">red sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_producer">blue light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_producer">green light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_sensor">green sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Modelling">modelling</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Safety">safety</a></li><br />
<li><a href="http://www.openwetware.org/wiki/French_Lab">protocols</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Acknowledgements" class="dir">acknowledgements</a><br />
</li><br />
<br />
</ul><br />
</div><br />
<br />
<br><br />
<br><br />
<br><br />
<br />
<div id="windowbox" style="border: .2em solid #660000; padding: 5px; position:fixed; top:50%; right:30px; width:8%;"><br />
<span style="color:ivory;">Throughout this wiki there are words in <b>bold</b> that indicate a relevance to <b>human aspects</b>. It will become obvious that <b>human aspects</b> are a part of almost everything in <b>iGEM</b>.</span><br />
<br />
</div><br />
<br />
</html><br />
<br />
== Blue Light Producer ==<br />
<br><br />
'''5/7/2010'''<br />
*LuxAB & LuxCDE operon agarose gel<br />
* Lane1: About2.1.kbp<br />
* Lane 2: The highest band- very faint- about 3.5kbp<br />
[[Image:150.JPG]]<br />
<br />
'''13/7/2010'''<br />
<br />
Empty edinbrick vector for control. <br />
* LuxAB + edinbrick ligation.<br />
<br />
'''28/7/2010'''<br />
<br />
an update on LuxAB: <br />
*The transformations of Edinbrick are fine but all the luxABs failed (grew blue, not white on Xgal and did not glow, as of 27/7)/<br />
*Still have some digested LuxAB-ediI so re-did ligation and transformation. <br />
[[Image:154.JPG]]<br />
<br />
{| border="1"<br />
| Lane 1&15<br />
| Lane 2<br />
| Others<br />
|-<br />
| Ladder <br />
| EdiI <br />
| LuxAB <br />
|}<br />
[[Image:155.JPG]]<br />
{| border="1"<br />
| Lane 1<br />
| Lane 2 +3<br />
| Lanes 3-6<br />
|-<br />
| Ladder <br />
| EdiI <br />
| LuxAB <br />
|}<br />
<br />
'''29/7/2010'''<br />
<br />
[[Image:156.jpg]]<br />
<br />
* The ligation had the same gel characteristics as previous luxAb-ediI ligations, e.x. it isn't visivle- not hopeful of this transformation working<br />
<br />
'''4/8/2010'''<br />
* made minipreps of blue (negative colonies)- double digest and ran in comparison with pure edinbrickI#*LacZ band dissapears but colonies still grow blue (WTF)<br />
* Hos bands equaling to luxAB and EdiI but there was no glowing <br />
<br />
[[Image:162.jpg]]<br />
<br />
'''12/8/2010''' <br />
update on LumP:<br />
* still getting pink/red colonies from transformants<br />
* single digests give a band of about 3kb<br />
* double digests give a band of about 2.5 kb -highly confusing<br />
* and also, no sign on RFP despite red/pink growth.<br />
* Vector wa ssequenced and LumP is definately there. <br />
<br />
[[Image:160.jpg]]<br />
<br />
Lanes 2-5: single digests of lumP<br />
Lane 6: double digest of lumP<br />
<br />
<br />
<br />
'''26/8/2010'''<br />
<br />
* Marker (Lane 1), minipreps 6-10 luxABlumP (lanes 2-6)<br />
[[Image:129.JPG]]<br />
* single digests of LuxABlumP<br />
[[Image:131.JPG]]<br />
<br />
'''7/9/2010'''<br />
*minipreps of luxCDE: samples 1&4 usable<br />
<br />
{| border="1"<br />
| Lane 1<br />
| Lane 2-6<br />
| Lanes 7<br />
|-<br />
| Ladder <br />
| luxCDE00:1-5 minipreps<br />
| Ladder <br />
|}<br />
[[Image:140.JPG]]</div>Macbereskahttp://2010.igem.org/File:160.jpgFile:160.jpg2010-10-26T23:18:32Z<p>Macbereska: </p>
<hr />
<div></div>Macbereskahttp://2010.igem.org/Team:Edinburgh/Notebook/Red_light_sensorTeam:Edinburgh/Notebook/Red light sensor2010-10-26T23:17:57Z<p>Macbereska: /* Red Light Sensor */</p>
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<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Project/Protocol">the protocol</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/BioBricks#Genomic">submitted parts</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Genomic">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Project/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Project/References">references</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial" class="dir">bacterial BRIDGEs</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Core_repressilator">the repressilator</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_producer">red light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_sensor">red sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_producer">blue light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Green_light_producer">green light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Green_light_sensor">green sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/BioBricks#Bacterial">submitted parts</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Bacterial">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/References">references</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling" class="dir">modelling BRIDGEs</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Kappa">kappa</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Genomic">the genomic model</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Bacterial">the bacterial model</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Signalling">the signalling model</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Tools">tools</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Modelling">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/References">references</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human" class="dir">human BRIDGEs</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Aspects">human aspects</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Communication">communication of science</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Terminology">terminology research</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Wiki">wiki</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Conversations">conversations</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Identity">identity</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Collaboration">collaboration</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Branding">branding research</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/SciFi">science fiction writing</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/FutureApps">future applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/SelfReflection">self-reflection</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Epic">the epic</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Human">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/References">references</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook" class="dir">lab notes&nbsp;&nbsp;&nbsp;</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Collaboration">collaboration</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Attribution">attribution</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/BRIDGE">BRIDGE</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_producer">red light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_sensor">red sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_producer">blue light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_producer">green light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_sensor">green sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Modelling">modelling</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Safety">safety</a></li><br />
<li><a href="http://www.openwetware.org/wiki/French_Lab">protocols</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Acknowledgements" class="dir">acknowledgements</a><br />
</li><br />
<br />
</ul><br />
</div><br />
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<div id="windowbox" style="border: .2em solid #660000; padding: 5px; position:fixed; top:50%; right:30px; width:8%;"><br />
<span style="color:ivory;">Throughout this wiki there are words in <b>bold</b> that indicate a relevance to <b>human aspects</b>. It will become obvious that <b>human aspects</b> are a part of almost everything in <b>iGEM</b>.</span><br />
<br />
</div><br />
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</html><br />
<br />
== Red Light Sensor ==<br />
<br><br />
<br />
'''22/6/10'''<br />
<br />
Transformation of BBa_M30109; well 14 H, distribution plate 3 (2010)--> red light sensor <br />
<br />
'''24/6/10'''<br />
<br />
Subcultured transformants from ampicillin plates to new plates<br />
<br />
<br />
'''25/6/10'''<br />
<br />
1) DNA mini-preps were prepared from the transformants<br />
2) DNA mini-preps were cut with EcoRI enzyme to check for the presence of the plasmid (and DNA); agarose gel was prepared for DNA electrophoresis after cutting.<br />
3) Results from cutting showed no presence of the sensor.<br />
Troubleshooting: was the plate number correct? Was procedure performed correct?<br />
by:Maria, Will, Marta <br />
<br />
<br />
'''28/6/10'''<br />
<br />
Tried to determine what was wrong with the red sensor that failed to appear in the gel. We definitely had the correct plate and well. We checked the seqeunce of the part and found that the sequence given does not match any given in BLAST. We suspect either that part of the plasmid was added to the distribution/registry or that the plasmid contains an illegal EcoR1 site, but are re-transforming cells just in case there was an experimental error.<br />
Due to failure of the previous transformation competent cells were retransformed with the two plasmids, as before. Two Chloramphenicol spread plates for each plasmid were made one of each pair containing 100micro-l of cells the other containing the 900micro-l remainder spun down and resuspended in 100micro-l of sterile LB.<br />
by: Richard, Lu<br />
<br />
<br />
'''29/6/10'''<br />
<br />
Streaking the colonies from the chloramphenicol plates on the new agar + chloramphenicol plates.<br />
<br />
<br />
'''30/6/10'''<br />
<br />
No growth on plates.<br />
<br />
<br />
'''5/7/10'''<br />
<br />
Made kanomycin 32mg/ml, IPTG 90mg/ml plates. Transformed cells with individual red light sensor parts (BBa_I15008 - 2010 distribution Plate 2 Well 13J; BBa-I15009 - distribution plate 1 Well 20F; BBa_I15010 - distribution Plate 3 Well 20D; & K098010 - distribution Plate 3 11N). Plated cells onto kanomycin plates and left to incubate overnight. <br />
by: Hannah and Richard<br />
<br />
<br />
'''6/7/10'''<br />
<br />
Richard/Hannah - made subcultures of red light sensor part transformants.<br />
<br />
*Agarose Gel Eleckrophoresis of composite red sensor M30109<br />
*The bands are not the right size<br />
*Sequencing done by the registry starts in the middle of the sequence (for forward sequence)<br />
*So it seems the plasmid doesn’t contain the right sequence<br />
*We will try to assemble it from individual parts<br />
<br />
[[Image:149.JPG]]<br />
<br />
'''8/7/10'''<br />
<br />
Plasmid minipreps of I15009 (the only red sensor part transformant that grew in liquid culture)<br />
by: Richard and Hannah<br />
<br />
<br />
'''9/7/10'''<br />
<br />
Gel run with I15009. <br />
<br />
{| border="1"<br />
| Lane 1 <br />
| Lane 2 <br />
| Lane 3 <br />
| Lane 4 <br />
| Lane 5 <br />
| Lane 6 <br />
| Lane 7 <br />
| Lane 8<br />
|-<br />
| Ladder <br />
| Pure SacB <br />
| Pure SacB & EcoRI <br />
| sacB & catR PCR <br />
| I15009 digest <br />
| S248T PCR Product <br />
| Purified 356K <br />
| Purified 356R<br />
|}<br />
<br />
*Insert Picture of Gel*<br />
<br />
'''13/7/2010'''<br />
NOTHING WORKS. No idea why...<br />
<br />
'''16/7/10'''<br />
<br />
Because the red light sensing parts were transformed with 50 µL or water instead of 15 µL during the first transformation, another attempted was tried.<br />
Transforming:<br />
*I15008 (all red light sensor parts)<br />
*I15009<br />
*I15010<br />
used 200 µL of cells, 800 µL of broth. Kan resistance.<br />
<br />
<br />
'''23/07/10'''<br />
<br />
Double digest of K098010. Ran gel. Lane was empty:<br />
<br />
[[Image:153.JPG]]<br />
*Not going to bother saying what is in each lane. <br />
<br />
'''30/7/10'''<br />
<br />
Primers were ordered in order to PCR out the Red Light Sensor Parts from the [http://partsregistry.org/wiki/index.php?title=Part:BBa_M30109 Bio Brick BBa_M30109]<br />
<br />
Forward Primer for PBad promoter [http://partsregistry.org/wiki/index.php?title=Part:BBa_I13453 Bio Brick BBa_I13453]<br><br />
Reverse Primer for Heme Oxygenase (ho1) [http://partsregistry.org/wiki/index.php?title=Part:BBa_I15008 Bio Brick BBa_I15008]<br><br />
Reverse Primer for Phycocyanobilin:Ferredoxin Oxidoreductase (PcyA) [http://partsregistry.org/wiki/index.php?title=Part:BBa_I15009 Bio Brick BBa_I15009]<br><br />
Forward Primer for TetR repressible promoter [http://partsregistry.org/wiki/index.php?title=Part:BBa_R0040 Bio Brick BBa_R0040]<br><br />
Reverse Primer for cph8 (Cph1/EnvZ fusion)Chimeric Cph1 light receptor/EnvZ protein [http://partsregistry.org/wiki/index.php?title=Part:BBa_I15010 Bio Brick BBa_I15010]<br><br />
<br />
<br />
'''3/8/10'''<br />
<br />
A PCR was run with the above primers in 3 distinct mixtures in an attempt to amplify out the Red Light Sensor parts using Bio Brick BBa_M30109 as a template.<br />
PCR 1 Contained The PBad promoter Forward Primer & the PcyA Reverse Primer<br><br />
PCR 2 Contained The TetR repressible promoter Forward Primer & the cph8 Reverse Primer <br><br />
PCR 3 Contained The PBad promoter Forward Primer & the cph8 Reverse Primer<br />
<br />
And a gel was run of the PCR products: Marker(Lane 1&6), only visible product- suggested PCR I15010 with RBS & promoter (Lane 3). <br />
[[Image:116.JPG]]<br />
<br />
Due to the successful amplification of the I15010 cph8 Red light sensor part, the DNA was ligated into plasmid psb1C3.<br />
<br />
<br />
'''4/8/10'''<br />
<br />
Competent cells were transformed with the ligated psb1C3 plasmid containing I15010 and plated out on Cml 40 + IPTG 90 plates and incubated O/N at 37 degrees Celsius.<br />
<br />
A PCR was run using the following primers:<br />
PCR 1 Contained The BBa_B0034 RBS Forward Primer & the ho1 Reverse Primer<br><br />
PCR 2 Contained The BBa_B0034 RBS Forward Primer & the PcyA Reverse Primer<br><br />
PCR 3 Contained The PBad promoter Forward Primer & the ho1 Reverse Primer<br><br />
PCR 4 Contained The PBad promoter Forward Primer & the PcyA Reverse Primer<br><br />
<br />
And a gel was run of the PCR products.<br />
<br />
*Insert picture of Gel*<br />
<br />
No successful amplification was seen.<br />
<br />
'''11/8/10'''<br />
<br />
Update from Maria. <br />
The transformations and PCR amplification of the HO1-pcyA fusion construct have nearly all failed. <br />
This weekend the kanamycin resistant version of the construct (K089010 in pSB3K3) was transformed and cultures grew.<br />
We already have a PCR product of the fused cph8-envZ sensing part. We now need to put the newly transformed K089010 in front of the sensor in order to rebuild the original sensor, which is well and truly fucked.<br />
<br />
[[Image:161.jpg]]<br />
<br />
'''16/8/10'''<br />
<br />
Successful PCR of K098010 using primers for EcoRI & I15009 reverse primer.<br />
[[Image:119.JPG]]<br />
<br />
<br />
'''17/8/10'''<br />
<br />
* The PCR product of the Harvard K098010 part was purified and then digested with both XbaI & SpeI as was the PCR product of cph8.<br />
* These digestions were then ligated together ON using in two orientations.<br />
<br />
'''18 &19/8/2010'''<br />
<br />
* Fusion PCR C + H with R004081 & I15009-1 <br />
* also running H + C same primers as negative control<br />
* run gel of PCR prod & 5 ul of ligation<br />
* luxAB -SpeI; lumP -XbaI, ;uxCDE -XbaI<br />
* lumP = P1024<br />
* luxAB + lumP and luxAB + lyxCDE ligation<br />
[[Image:120.JPG]]<br />
<br />
'''20/8/2010'''<br />
<br />
* Fusion PCR with ?????<br />
* get: <br />
<br />
'''24/8/2010'''<br />
* Digest cph8 (XbaI- yello) & Harvard (SpeI -brown)<br />
* Ligate Harvard and cph8 = harvard + cph*8<br />
* mutagenic PCR of lux CDE to mutate out site in LuxD<br />
* subculture luxAB lumP transformants<br />
* miniprep ON'S '''(?)''' set up<br />
<br />
<br />
'''25/8/2010'''<br />
<br />
* Minipreps of subcultures luxAB lumP,<br />
* digest & run gel<br />
* Purify luxCDE PCR product- from gel and froem the product; run the gel of purifications --> do MABEL self ligation- set 16C ON<br />
* Fusion PCR harv + cph8- run gel of product<br />
<br />
'''26/8/2010'''<br />
<br />
* Gel1 - minipreps 1-5, gluxCDE, PluxCDE<br />
* Gel2 - Minipreps 6-10, H + C ligation<br />
<br />
*Lane 7: Harvard + Cph8 ligation<br />
[[Image:129.JPG]]<br />
<br />
'''27/8/2010'''<br />
<br />
Transform cells with self ligated MABEL mutated luxCDE from gel and PCR product<br />
<br />
<br />
'''30/8/2010'''<br />
<br />
* Lu wants a broad sward (?).<br />
* Re-transform MABEL mutated<br />
* luxCDE from gel- PCR products<br />
* luxAB and luxABlumP double digest with EcoRi/PstI<br />
* Fusion PCR if H + cph8 using H promoter forward and cph8 reverse primer<br />
<br />
'''31/8/2010'''<br />
* Lane 1- marker, Lane 2- H+ Cph8 fPCR<br />
[[Image:133.JPG]]<br />
'''6/9/2010'''<br />
<br />
* ligated H+ C ON in psB1C3<br />
* Double digest luxCDE with EcoRI and PstI - run gel<br />
<br />
'''7/9/2010'''<br />
* Run digest luxCDE with XbaI --> gel<br />
* Gel of H + C ligation<br />
* transform cells with<br />
<br />
'''8/9/2010'''<br />
<br />
* PCR with biobrick pre/suff - primers luxCDE 1 and 4<br />
<br />
'''9/9/2010'''<br />
<br />
* Purify luxCDE PCR<br />
* Subculture transformants H + C<br />
<br />
'''10/9/2010'''<br />
<br />
* Add promoter to luxAB and luxABlumP<br />
* Liquid ON'S of H + C subculture transformants <br />
<br />
* Lab meeting: small--> <br />
**sacB - some preeliminary sucrose sensitivity characterisation data.<br />
** promoter: luxAB, luxAb-lumP- eission spectra, testing thing<br />
** red light luciferase: S184T, 356 K<br />
[[Image:143.JPG]]<br />
{| border="1"<br />
| Lane 1 <br />
| Lane 2 <br />
| Lane 3 <br />
| Lane 4<br />
|-<br />
| Ladder <br />
| Tna up<br />
| Cph8- fixing prefix <br />
| Cph8 - fixing PstI site <br />
|}<br />
<br />
'''13/9/2010'''<br />
* H + C + pSB1C3 minipreps<br />
[[Image:144.JPG]]<br />
<br />
'''14/9/2010'''<br />
* Digest H+ C minipreps with EcoRi. 100: 1,4,5. 900: 3,4,6<br />
[[Image:145.JPG]] <br />
<br />
'''???'''<br />
'''20/9/2010'''<br />
<br />
* Test luxCDE<br />
* luxCDE digest with XBaI and PstI (buffer H)--> purify and ligate to luxAB digested with SPEI PstI in buffer B after purification.<br />
* conclusion: purify digestions, ligate luxCDE into luxAB + promoter<br />
<br />
<br />
'''21/9/2010'''<br />
<br />
* Transform cells with luxAB + luxCDE ligation, plated on CML 40, IPTG 90<br />
* Made up 50 ml liquid cultures of luxAB lumP and luxAB with Cml40, IPTG 90<br />
<br />
<br><br><br />
<b>04/10/10</b><br />
<br><br />
Chris has managed to construct what we hope is a working red light sensor. It is being sent in for sequencing today. <br><br />
Minor characterisation experiment:<br><br />
-Red light sensor constructed with blue-white lacZ reporter<br><br />
-If activated, the red light sensor transformants should produce blue colonies<br><br />
-Red light sensor containing-colonies from transformant plate streaked onto two plates<br><br />
-One plate left in the light, the other left in the dark<br><br />
-If the one in the light grows blue and the one in the dark doesn't, it is a good indication that the construct is working<br><br><br />
<br />
'''12/10/10'''<br />
<br />
*Transform ''envZ'' mutant strain with YFP<br />
*Plate out onto 40mg/ml Cml<br />
<br />
'''13/10/10'''<br />
<br />
*Subcultured ''envZ'' transformants onto Cml 40 plates<br />
<br />
'''15/10/10'''<br />
<br />
*Liquid Cultures made of transformants with Cml 40 & grown ON<br />
<br />
'''16/10/10'''<br />
<br />
*Liquid ONs viewed under blue light, yellow fluorescence seen showing successful transformation.<br />
<br />
'''20/10/10'''<br />
<br />
*RLS characterisation begins.<br />
*Liquid ON's of<br />
<ul><br />
<li>JM109 – RLS.lacZ.YFP</li><br />
<li>envZ – RLS.lacZ.YFP</li><br />
<li>JM109 – YFP Control</li><br />
<li>envZ – YFP Control</li><br />
</ul><br />
were made in 5ml LB + Cml 40 and grown at 37 degrees with shaking in the dark.<br />
<br />
<p>100μl of ONs were used to inoculate two sterile LB + 40mg/ml Cml making up a total volume of 4ml in 5ml flasks which were then incubated in both light and dark, at 37C with shaking, as follows:</p><br />
<br />
<ul><br />
<li>JM109 – RLS.lacZ.YFP (Light / Dark)</li><br />
<li>envZ – RLS.lacZ.YFP (Light / Dark)</li><br />
<li>JM109 – YFP Control (Light / Dark)</li><br />
<li>envZ – YFP Control (Light / Dark)</li><br />
</ul><br />
<br />
<p>At 50 minute time intervals 200μl of each sample was taken and mixed with 800μl of sterile water in plastic cuvettes. The optical density of each sample was taken after the spectrophotometer was set with a sample of 200μl of sterile LB and 800μl water as a control, as was the luminescence, with readings for the background luminescence being taken for the sample of LB and water.</p><br />
<br />
<p>Two readings for each sample at each time interval were taken and then an average was calculated for each time interval for both optical density and luminescence.</p><br />
<br />
<p>For each sample at each time interval the average luminescence (normalised by the background luminescence) was divided by the optical density and plotted on a graph, shown below as Figure 2.</p><br />
<br />
<br />
[[Image:Ed10-RedLightSensorCharData.png]]<br />
<br />
<br />
'''25/10/10'''<br />
<br />
*Liquid ON's made up of <br />
<li>JM109 – RLS.lacZ.YFP</li><br />
<li>envZ – RLS.lacZ.YFP</li><br />
<li>JM109 – RLS.lacZ</li><br />
<li>JM109 - lacZ Control</li><br />
in LB + 40 Cml</div>Macbereskahttp://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_producerTeam:Edinburgh/Notebook/Blue light producer2010-10-26T23:17:19Z<p>Macbereska: /* Blue Light Producer */</p>
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<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/References">references</a></li><br />
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</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human" class="dir">human BRIDGEs</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Aspects">human aspects</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Communication">communication of science</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Terminology">terminology research</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Wiki">wiki</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Conversations">conversations</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Identity">identity</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Collaboration">collaboration</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Branding">branding research</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/SciFi">science fiction writing</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/FutureApps">future applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/SelfReflection">self-reflection</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Epic">the epic</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Human">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/References">references</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook" class="dir">lab notes&nbsp;&nbsp;&nbsp;</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Collaboration">collaboration</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Attribution">attribution</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/BRIDGE">BRIDGE</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_producer">red light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_sensor">red sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_producer">blue light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_producer">green light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_sensor">green sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Modelling">modelling</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Safety">safety</a></li><br />
<li><a href="http://www.openwetware.org/wiki/French_Lab">protocols</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Acknowledgements" class="dir">acknowledgements</a><br />
</li><br />
<br />
</ul><br />
</div><br />
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<br />
<div id="windowbox" style="border: .2em solid #660000; padding: 5px; position:fixed; top:50%; right:30px; width:8%;"><br />
<span style="color:ivory;">Throughout this wiki there are words in <b>bold</b> that indicate a relevance to <b>human aspects</b>. It will become obvious that <b>human aspects</b> are a part of almost everything in <b>iGEM</b>.</span><br />
<br />
</div><br />
<br />
</html><br />
<br />
== Blue Light Producer ==<br />
<br><br />
'''5/7/2010'''<br />
*LuxAB & LuxCDE operon agarose gel<br />
* Lane1: About2.1.kbp<br />
* Lane 2: The highest band- very faint- about 3.5kbp<br />
[[Image:150.JPG]]<br />
<br />
'''13/7/2010'''<br />
<br />
Empty edinbrick vector for control. <br />
* LuxAB + edinbrick ligation.<br />
<br />
'''28/7/2010'''<br />
<br />
an update on LuxAB: <br />
*The transformations of Edinbrick are fine but all the luxABs failed (grew blue, not white on Xgal and did not glow, as of 27/7)/<br />
*Still have some digested LuxAB-ediI so re-did ligation and transformation. <br />
[[Image:154.JPG]]<br />
<br />
{| border="1"<br />
| Lane 1&15<br />
| Lane 2<br />
| Others<br />
|-<br />
| Ladder <br />
| EdiI <br />
| LuxAB <br />
|}<br />
[[Image:155.JPG]]<br />
{| border="1"<br />
| Lane 1<br />
| Lane 2 +3<br />
| Lanes 3-6<br />
|-<br />
| Ladder <br />
| EdiI <br />
| LuxAB <br />
|}<br />
<br />
<br />
<br />
'''29/7/2010'''<br />
<br />
[[Image:156.jpg]]<br />
<br />
* The ligation had the same gel characteristics as previous luxAb-ediI ligations, e.x. it isn't visivle- not hopeful of this transformation working<br />
<br />
'''12/8/2010''' <br />
update on LumP:<br />
* still getting pink/red colonies from transformants<br />
* single digests give a band of about 3kb<br />
* double digests give a band of about 2.5 kb -highly confusing<br />
* and also, no sign on RFP despite red/pink growth.<br />
* Vector wa ssequenced and LumP is definately there. <br />
<br />
[[Image:160.jpg]]<br />
<br />
Lanes 2-5: single digests of lumP<br />
Lane 6: double digest of lumP<br />
<br />
<br />
<br />
'''26/8/2010'''<br />
<br />
* Marker (Lane 1), minipreps 6-10 luxABlumP (lanes 2-6)<br />
[[Image:129.JPG]]<br />
* single digests of LuxABlumP<br />
[[Image:131.JPG]]<br />
<br />
'''7/9/2010'''<br />
*minipreps of luxCDE: samples 1&4 usable<br />
<br />
{| border="1"<br />
| Lane 1<br />
| Lane 2-6<br />
| Lanes 7<br />
|-<br />
| Ladder <br />
| luxCDE00:1-5 minipreps<br />
| Ladder <br />
|}<br />
[[Image:140.JPG]]</div>Macbereskahttp://2010.igem.org/Team:Edinburgh/Notebook/Red_light_producerTeam:Edinburgh/Notebook/Red light producer2010-10-26T23:15:21Z<p>Macbereska: /* Red Light Producer */</p>
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<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Green_light_producer">green light</a></li><br />
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<li><a href="https://2010.igem.org/Team:Edinburgh/Human" class="dir">human BRIDGEs</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Aspects">human aspects</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Communication">communication of science</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Terminology">terminology research</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Wiki">wiki</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Conversations">conversations</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Identity">identity</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Collaboration">collaboration</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Branding">branding research</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/SciFi">science fiction writing</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/FutureApps">future applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/SelfReflection">self-reflection</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Epic">the epic</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Human">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/References">references</a></li><br />
</ul><br />
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<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook" class="dir">lab notes&nbsp;&nbsp;&nbsp;</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Collaboration">collaboration</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Attribution">attribution</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/BRIDGE">BRIDGE</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_producer">red light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_sensor">red sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_producer">blue light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_producer">green light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_sensor">green sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Modelling">modelling</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Safety">safety</a></li><br />
<li><a href="http://www.openwetware.org/wiki/French_Lab">protocols</a></li><br />
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<li><a href="https://2010.igem.org/Team:Edinburgh/Acknowledgements" class="dir">acknowledgements</a><br />
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<br />
<div id="windowbox" style="border: .2em solid #660000; padding: 5px; position:fixed; top:50%; right:30px; width:8%;"><br />
<span style="color:ivory;">Throughout this wiki there are words in <b>bold</b> that indicate a relevance to <b>human aspects</b>. It will become obvious that <b>human aspects</b> are a part of almost everything in <b>iGEM</b>.</span><br />
<br />
</div><br />
<br />
</html><br />
<br />
== Red Light Producer ==<br />
<br><br />
<br />
'''29/6/10'''<br />
<br />
Now preparing primers to make a set of different spectra bioluminescent proteins. Need to order primers for Site directed mutagenesis of the Firely luciferase to make it red (MABEL protocol), and to make click beetle luciferase Rluc8 (which emits light at about 420nm in presence of bisdeoxycoelenterazine - about the same price a coelenterazine though)<br />
<br />
'''8/7/2010'''<br />
* Red luciferase mutagenesis primers arrived- 3 sets:<br />
** S248T<br />
** 356 K<br />
** 356 R<br />
<br />
* Followed the protocol with 34ul water + 0.5 template DNA<br />
* Put the primers and template in green box '''(???)'''<br />
*Ran Gel: 5 µL PCR product, 5 µL water, 2.5 µL buffer<br />
[[Image:151.JPG]]<br />
<br />
{| border="1"<br />
| Marker<br />
| S284T <br />
| 356K <br />
| 356R<br />
|}<br />
<br />
<br />
[[File:07-08.JPG]]<br />
<br />
<br />
*356K and 356R look fine<br />
*going to run S284T at a higher ** temp tomorrow to see if it works better<br />
*all PCR tubes stored in freezer<br />
<br />
<br />
<br />
'''09/7/10'''<br />
<br />
Redo PCR of Red Luciferase<br />
*Everything except template DNA + Kod polymerase kept on ice<br />
*Ran PCR<br />
*Template DNA used PyeaR and P. pyralis luciferase BBa_K216015<br />
*Purification done for 356K and 356R; stored in iGEM box (-20C).<br />
<br />
[[File:07-09straight.JPG]]<br />
<br />
'''9/7/2010'''<br />
*Ligations set up overnight- might have put too much ligase in 356K... Incubated at 15C.<br />
*Oure 356R and 356K are in the freezer- row 10 (G &H)?<br />
<br />
'''13/07/10'''<br />
Goal of the day: <br />
<br />
Transformations:<br />
*356R (2): one from 16 °C and one from room temp(RT)<br />
*356K (2): one from 16 °C and one from room temp(RT)<br />
*S284T (2): one with 25/5 ligase, one with 1/7 ligase<br />
<br />
* I had a burger for the lunch. It was very nice.<br />
* Now we are transforming cells:<br />
** 356R (2)& 356K (2): one form 16C, one from RT<br />
** S248T (2): 1 with 25/5 ligase, other with 1.7.10 ligase/<br />
Empty edinbrick vector for control. <br />
* LuxAB + edinbrick ligation.<br />
<br />
* Using 100 ul transformed cells + 800 ul LB for all. Incubation started half 5. <br />
'''22/07/10'''<br />
<br />
Liquid Cultures (amp 80):<br />
*356R (RT) – 1 with NaNO3 and 1 without<br />
**(16°C) – 2 with NaNO3 and 2 without<br />
*356K (RT) – 1 with NaNO3 and 1 without<br />
**(16°C) – 2 with NaNO3 and 2 without <br />
*S284T (100 µL and 25/5 ligase) – 2 with NaNO3 and 2 without<br />
**(900 µL and 25/5 ligase) – 3 with NaNO3 and 3 without<br />
**(900 µL and 1/7 ligase) – 5 with NaNO3 and 5 without<br />
<br />
NaNO3 = 30mM Sodium Nitrate<br />
<br />
'''23/07/10'''<br />
<br />
Double digest of K098010<br />
<br />
'''28/07/10'''<br />
<br />
Primer design to produce a stable red light luciferase. The mutations for increasing bioluminescence come from [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W9V-4NHM57C-1&_user=809099&_coverDate=07%2F15%2F2007&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1414502247&_rerunOrigin=google&_acct=C000043939&_version=1&_urlVersion=0&_userid=809099&md5=00d9c92f6452bd276c6ea606ed7d3bbf Fujii et al, 2007] and the paper from [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W9V-4X6MSYB-7&_user=809099&_coverDate=01%2F15%2F2010&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1414503636&_rerunOrigin=google&_acct=C000043939&_version=1&_urlVersion=0&_userid=809099&md5=297be52832193b4d026953632a1edf3c Branchini et al, 2010]<br />
<br />
'''29/7/2010'''<br />
<br />
*Double digest with EcoRI HF&PstI in buffer 2 (neb).<br />
* 2 bands: with and without vector?<br />
* did not leave the digest for too long... <br />
[[Image:157.jpg]]<br />
<br />
{| border="1"<br />
| Lane 1<br />
| Lane 2<br />
| Lane 3<br />
| Lane 4<br />
| Lane 5<br />
| Lane 6<br />
|-<br />
| Ladder <br />
| S284T (4) <br />
| S284T (6)<br />
| 356R (6, 900)<br />
| 356R (6)<br />
| 356K (4)<br />
|}<br />
<br />
'''2/8/2010'''<br />
* Red luciferases EcoRI HF&PstI double digests- lanes 2-7 in the second miniprep set. <br />
*Ladder didn't work too well ,though...<br />
[[Image:113.JPG]]<br />
* Transform last years luciferase<br />
* Look at papers for expression in E. coli<br />
* Replace B0034 by stronger RBS?<br />
* Confirm that we order bright luc.<br />
* Single digest of constructs<br />
* Test glowing with positive control<br />
<br />
'''9/8/2010'''<br />
<br />
[[Image:159.jpg]]<br />
<br />
Digest: <br />
*WT1<br />
*356 R: (2)<br />
* 356 K: (4)<br />
* S284T: (6)<br />
* 3 eith EcoRI; 3 with EcoRi + PstI<br />
<br />
<br />
'''10/8/2010'''<br />
* Sequencing of red mutant luciferases. <br />
* Forward primers for S284T and reverse for 356R and K should do. <br />
* Concentration of primers for sequencing: 5uM.<br />
* sequencing tubE: 6 ul liquid: 3ul DNA + 2ul water, 1 ul primer mix.<br />
* when using miniprep DNA- remember to spin down the dirt- 1min, max speed, then take the DNA from the top to avoid disturbing any dirt that is settled. <br />
<br />
<br />
'''12/8/2010'''<br />
* Got the sequences for red luciferases<br />
*see: if we can detect Red Light with the luminometer?<br />
* Red Luciferases 356R and S284T were nNOT MUTANTS- just WT<br />
* set up ;iquid cultures with 30 ul nitrate for: <br />
** 356R - no 3,1 from RT plate, no 5 from 16C plate; no 1 from Master plate<br />
** S284T - 5 from masterplate; 2, 4, 6 from masterplate<br />
** 356K: 4 from masterplate<br />
<br />
'''13/8/2010'''<br />
Big day today... TESTING YESTERDAYS PLAN- LUMINESCENCE<br />
<br />
'''16/8/2010'''<br />
* Sequecning of: <br />
** WR1- 356R -use S284T forward primer<br />
** WR2 -S284T -use 356 R reverse primer<br />
** WR3- LovTap 03<br />
**WR4 -LovTap 09<br />
<br />
* RESULTS: ONLY 356K WAS A REAL MUTANT; S284T AND 356R TURNED OUT TO BE STILL A WT<br />
<br />
'''25/8/2010'''<br />
* Next step:- redo ligations for 356R; redo PCR for S284T<br />
* Ligations<br />
*Did MABEL PCR PPyInc + S284T primers<br />
*used 0.5 ul template DNA<br />
<br />
'''26/8/2010'''<br />
* check the Fuji et. al paper, see what sequence they used to start off with. <br />
* check IAV in other luciferase<br />
* Fix scar before RBS and in teh second stop codon. <br />
* compared to WT, luciferase sequence has: <br />
** N 50 P<br />
** N 99 G<br />
** SKL- IAV<br />
<br />
'''27/8/2010'''<br />
* Ligations from yesterday out of freezer.<br />
* Running on gel<br />
* Column 3 9with 356R) has less DNA in it- not enough from ligation and messed up loading of gel). <br />
[[Image:132.JPG]]<br />
{| border="1"<br />
| Lane 1<br />
| Lane 2<br />
| Lane 3<br />
| Lane 4<br />
<br />
|-<br />
| Ladder <br />
| New S284T PCR - new ligase and the buffer<br />
| 356 R ligation<br />
| S284T PCR + old ligase<br />
|}<br />
<br />
'''30/8/2010'''<br />
* Primers have arrived for fixing the luciferase- removing 6 bases<br />
* Primers have arrived for up and downstream sequences of TnaA region.<br />
* Send Chairman sequence for T7 promoter<br />
* transformed 356R and S284T ligations from Friday<br />
* Used PCR product as a control<br />
* Plated out the transformed cells. Used 900 rather than 100 if possible<br />
<br />
<br />
'''31/8/2010'''<br />
* We have S284T transformants but <br />
** 14 colonies on ligation plate<br />
** 4 colonies on control where we used PCR product instead of ligation<br />
<br />
* For sequencing- <br />
** if sequencing a S284T mutant, use 356R/K reverse primer<br />
** if sequencing 356R mutant, use S284T forward primer.<br />
<br />
'''2/9/2010'''<br />
* minipreped 14 colonies (1-14)<br />
* run the gel with samples digested with EcoRI<br />
* here: samples 1-7<br />
[[Image:135.JPG]]<br />
<br />
'''3/9/2010'''<br />
* S284T 8-14 (from yesterday)<br />
[[Image:136.JPG]]<br />
<br />
'''8/9/2010'''<br />
* wWT of luciferase (left)<br />
* mutation of S284T (right)<br />
<br />
<br />
'''9/9/2010'''<br />
{| border="1"<br />
| Lane 1<br />
| Lane 2<br />
| Lane 3<br />
| Lane 4<br />
| Lane 5<br />
|-<br />
| Ladder <br />
| WT <br />
| S284T 1<br />
| luxCDE 9:4<br />
| LuxCDE 9:1<br />
|}<br />
<br />
'''20/9/2010'''<br />
<br />
*send for sequencing: 356K, S284T, WT (samples from 16/9: WT- 900, 2; also 16/9 S284T- 900, 4.)<br />
* all in psB1C3 vector. <br />
* Transform 356K and S284T with RBS and promoter (lacZ, LacI, RBS). <br />
* Run gel of 365K in psb1C3 of the digest. --> control: RFP + psb1C3<br />
* then add promoter<br />
<br />
'''23/9/2010'''<br />
<br />
* Added in incubator room liquid culture '''?)''' LacP + RBS + Luciferase for S284T, 356K and WT. <br />
* miniprep- 6 bottles<br />
* GLOWING<br />
<br />
* S248T- <br />
**9.2; 5 <br />
** 9.4; 1 <br />
**5; 4<br />
* 356<br />
** 3.3<br />
* WT<br />
** 3.2; 4 <br />
** 1.4, 5</div>Macbereskahttp://2010.igem.org/Team:Edinburgh/Notebook/Red_light_producerTeam:Edinburgh/Notebook/Red light producer2010-10-26T23:14:33Z<p>Macbereska: /* Red Light Producer */</p>
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<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/BRIDGE">BRIDGE</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_producer">red light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_sensor">red sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_producer">blue light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_producer">green light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_sensor">green sensor</a></li><br />
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<span style="color:ivory;">Throughout this wiki there are words in <b>bold</b> that indicate a relevance to <b>human aspects</b>. It will become obvious that <b>human aspects</b> are a part of almost everything in <b>iGEM</b>.</span><br />
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<br />
== Red Light Producer ==<br />
<br><br />
<br />
'''29/6/10'''<br />
<br />
Now preparing primers to make a set of different spectra bioluminescent proteins. Need to order primers for Site directed mutagenesis of the Firely luciferase to make it red (MABEL protocol), and to make click beetle luciferase Rluc8 (which emits light at about 420nm in presence of bisdeoxycoelenterazine - about the same price a coelenterazine though)<br />
<br />
'''8/7/2010'''<br />
* Red luciferase mutagenesis primers arrived- 3 sets:<br />
** S248T<br />
** 356 K<br />
** 356 R<br />
<br />
* Followed the protocol with 34ul water + 0.5 template DNA<br />
* Put the primers and template in green box '''(???)'''<br />
*Ran Gel: 5 µL PCR product, 5 µL water, 2.5 µL buffer<br />
[[Image:151.JPG]]<br />
<br />
{| border="1"<br />
| Marker<br />
| S284T <br />
| 356K <br />
| 356R<br />
|}<br />
<br />
<br />
[[File:07-08.JPG]]<br />
<br />
<br />
*356K and 356R look fine<br />
*going to run S284T at a higher ** temp tomorrow to see if it works better<br />
*all PCR tubes stored in freezer<br />
<br />
<br />
<br />
'''09/7/10'''<br />
<br />
Redo PCR of Red Luciferase<br />
*Everything except template DNA + Kod polymerase kept on ice<br />
*Ran PCR<br />
*Template DNA used PyeaR and P. pyralis luciferase BBa_K216015<br />
*Purification done for 356K and 356R; stored in iGEM box (-20C).<br />
<br />
[[File:07-09straight.JPG]]<br />
<br />
'''9/7/2010'''<br />
*Ligations set up overnight- might have put too much ligase in 356K... Incubated at 15C.<br />
*Oure 356R and 356K are in the freezer- row 10 (G &H)?<br />
<br />
'''13/07/10'''<br />
Goal of the day: <br />
<br />
Transformations:<br />
*356R (2): one from 16 °C and one from room temp(RT)<br />
*356K (2): one from 16 °C and one from room temp(RT)<br />
*S284T (2): one with 25/5 ligase, one with 1/7 ligase<br />
<br />
* I had a burger for the lunch. It was very nice.<br />
* Now we are transforming cells:<br />
** 356R (2)& 356K (2): one form 16C, one from RT<br />
** S248T (2): 1 with 25/5 ligase, other with 1.7.10 ligase/<br />
Empty edinbrick vector for control. <br />
* LuxAB + edinbrick ligation.<br />
<br />
* Using 100 ul transformed cells + 800 ul LB for all. Incubation started half 5. <br />
'''22/07/10'''<br />
<br />
Liquid Cultures (amp 80):<br />
*356R (RT) – 1 with NaNO3 and 1 without<br />
**(16°C) – 2 with NaNO3 and 2 without<br />
*356K (RT) – 1 with NaNO3 and 1 without<br />
**(16°C) – 2 with NaNO3 and 2 without <br />
*S284T (100 µL and 25/5 ligase) – 2 with NaNO3 and 2 without<br />
**(900 µL and 25/5 ligase) – 3 with NaNO3 and 3 without<br />
**(900 µL and 1/7 ligase) – 5 with NaNO3 and 5 without<br />
<br />
NaNO3 = 30mM Sodium Nitrate<br />
<br />
'''23/07/10'''<br />
<br />
Double digest of K098010<br />
<br />
'''28/07/10'''<br />
<br />
Primer design to produce a stable red light luciferase. The mutations for increasing bioluminescence come from [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W9V-4NHM57C-1&_user=809099&_coverDate=07%2F15%2F2007&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1414502247&_rerunOrigin=google&_acct=C000043939&_version=1&_urlVersion=0&_userid=809099&md5=00d9c92f6452bd276c6ea606ed7d3bbf Fujii et al, 2007] and the paper from [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W9V-4X6MSYB-7&_user=809099&_coverDate=01%2F15%2F2010&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1414503636&_rerunOrigin=google&_acct=C000043939&_version=1&_urlVersion=0&_userid=809099&md5=297be52832193b4d026953632a1edf3c Branchini et al, 2010]<br />
<br />
'''29/7/2010'''<br />
<br />
*Double digest with EcoRI HF&PstI in buffer 2 (neb).<br />
* 2 bands: with and without vector?<br />
* did not leave the digest for too long... <br />
[[Image:157.jpg]]<br />
<br />
{| border="1"<br />
| Lane 1<br />
| Lane 2<br />
| Lane 3<br />
| Lane 4<br />
| Lane 5<br />
| Lane 6<br />
|-<br />
| Ladder <br />
| S284T (4) <br />
| S284T (6)<br />
| 356R (6, 900)<br />
| 356R (6)<br />
| 356K (4)<br />
|}<br />
<br />
'''2/8/2010'''<br />
* Red luciferases EcoRI HF&PstI double digests- lanes 2-7 in the second miniprep set. <br />
*Ladder didn't work too well ,though...<br />
[[Image:113.JPG]]<br />
* Transform last years luciferase<br />
* Look at papers for expression in E. coli<br />
* Replace B0034 by stronger RBS?<br />
* Confirm that we order bright luc.<br />
* Single digest of constructs<br />
* Test glowing with positive control<br />
<br />
'''9/8/2010'''<br />
[[Image:159.jpg]]<br />
<br />
Digest: <br />
*WT1<br />
*356 R: (2)<br />
* 356 K: (4)<br />
* S284T: (6)<br />
* 3 eith EcoRI; 3 with EcoRi + PstI<br />
<br />
<br />
'''10/8/2010'''<br />
* Sequencing of red mutant luciferases. <br />
* Forward primers for S284T and reverse for 356R and K should do. <br />
* Concentration of primers for sequencing: 5uM.<br />
* sequencing tubE: 6 ul liquid: 3ul DNA + 2ul water, 1 ul primer mix.<br />
* when using miniprep DNA- remember to spin down the dirt- 1min, max speed, then take the DNA from the top to avoid disturbing any dirt that is settled. <br />
<br />
<br />
'''12/8/2010'''<br />
* Got the sequences for red luciferases<br />
*see: if we can detect Red Light with the luminometer?<br />
* Red Luciferases 356R and S284T were nNOT MUTANTS- just WT<br />
* set up ;iquid cultures with 30 ul nitrate for: <br />
** 356R - no 3,1 from RT plate, no 5 from 16C plate; no 1 from Master plate<br />
** S284T - 5 from masterplate; 2, 4, 6 from masterplate<br />
** 356K: 4 from masterplate<br />
<br />
'''13/8/2010'''<br />
Big day today... TESTING YESTERDAYS PLAN- LUMINESCENCE<br />
<br />
'''16/8/2010'''<br />
* Sequecning of: <br />
** WR1- 356R -use S284T forward primer<br />
** WR2 -S284T -use 356 R reverse primer<br />
** WR3- LovTap 03<br />
**WR4 -LovTap 09<br />
<br />
* RESULTS: ONLY 356K WAS A REAL MUTANT; S284T AND 356R TURNED OUT TO BE STILL A WT<br />
<br />
'''25/8/2010'''<br />
* Next step:- redo ligations for 356R; redo PCR for S284T<br />
* Ligations<br />
*Did MABEL PCR PPyInc + S284T primers<br />
*used 0.5 ul template DNA<br />
<br />
'''26/8/2010'''<br />
* check the Fuji et. al paper, see what sequence they used to start off with. <br />
* check IAV in other luciferase<br />
* Fix scar before RBS and in teh second stop codon. <br />
* compared to WT, luciferase sequence has: <br />
** N 50 P<br />
** N 99 G<br />
** SKL- IAV<br />
<br />
'''27/8/2010'''<br />
* Ligations from yesterday out of freezer.<br />
* Running on gel<br />
* Column 3 9with 356R) has less DNA in it- not enough from ligation and messed up loading of gel). <br />
[[Image:132.JPG]]<br />
{| border="1"<br />
| Lane 1<br />
| Lane 2<br />
| Lane 3<br />
| Lane 4<br />
<br />
|-<br />
| Ladder <br />
| New S284T PCR - new ligase and the buffer<br />
| 356 R ligation<br />
| S284T PCR + old ligase<br />
|}<br />
<br />
'''30/8/2010'''<br />
* Primers have arrived for fixing the luciferase- removing 6 bases<br />
* Primers have arrived for up and downstream sequences of TnaA region.<br />
* Send Chairman sequence for T7 promoter<br />
* transformed 356R and S284T ligations from Friday<br />
* Used PCR product as a control<br />
* Plated out the transformed cells. Used 900 rather than 100 if possible<br />
<br />
<br />
'''31/8/2010'''<br />
* We have S284T transformants but <br />
** 14 colonies on ligation plate<br />
** 4 colonies on control where we used PCR product instead of ligation<br />
<br />
* For sequencing- <br />
** if sequencing a S284T mutant, use 356R/K reverse primer<br />
** if sequencing 356R mutant, use S284T forward primer.<br />
<br />
'''2/9/2010'''<br />
* minipreped 14 colonies (1-14)<br />
* run the gel with samples digested with EcoRI<br />
* here: samples 1-7<br />
[[Image:135.JPG]]<br />
<br />
'''3/9/2010'''<br />
* S284T 8-14 (from yesterday)<br />
[[Image:136.JPG]]<br />
<br />
'''8/9/2010'''<br />
* wWT of luciferase (left)<br />
* mutation of S284T (right)<br />
<br />
<br />
'''9/9/2010'''<br />
{| border="1"<br />
| Lane 1<br />
| Lane 2<br />
| Lane 3<br />
| Lane 4<br />
| Lane 5<br />
|-<br />
| Ladder <br />
| WT <br />
| S284T 1<br />
| luxCDE 9:4<br />
| LuxCDE 9:1<br />
|}<br />
<br />
'''20/9/2010'''<br />
<br />
*send for sequencing: 356K, S284T, WT (samples from 16/9: WT- 900, 2; also 16/9 S284T- 900, 4.)<br />
* all in psB1C3 vector. <br />
* Transform 356K and S284T with RBS and promoter (lacZ, LacI, RBS). <br />
* Run gel of 365K in psb1C3 of the digest. --> control: RFP + psb1C3<br />
* then add promoter<br />
<br />
'''23/9/2010'''<br />
<br />
* Added in incubator room liquid culture '''?)''' LacP + RBS + Luciferase for S284T, 356K and WT. <br />
* miniprep- 6 bottles<br />
* GLOWING<br />
<br />
* S248T- <br />
**9.2; 5 <br />
** 9.4; 1 <br />
**5; 4<br />
* 356<br />
** 3.3<br />
* WT<br />
** 3.2; 4 <br />
** 1.4, 5</div>Macbereskahttp://2010.igem.org/Team:Edinburgh/Notebook/Red_light_producerTeam:Edinburgh/Notebook/Red light producer2010-10-26T23:12:29Z<p>Macbereska: /* Red Light Producer */</p>
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<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Core_repressilator">the repressilator</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_producer">red light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_sensor">red sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_producer">blue light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Green_light_producer">green light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Green_light_sensor">green sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/BioBricks#Bacterial">submitted parts</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Bacterial">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/References">references</a></li><br />
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<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling" class="dir">modelling BRIDGEs</a><br />
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<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Kappa">kappa</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Genomic">the genomic model</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Bacterial">the bacterial model</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Signalling">the signalling model</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Tools">tools</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Modelling">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/References">references</a></li><br />
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<li><a href="https://2010.igem.org/Team:Edinburgh/Human" class="dir">human BRIDGEs</a><br />
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<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Aspects">human aspects</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Communication">communication of science</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Terminology">terminology research</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Wiki">wiki</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Conversations">conversations</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Identity">identity</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Collaboration">collaboration</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Branding">branding research</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/SciFi">science fiction writing</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/FutureApps">future applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/SelfReflection">self-reflection</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Epic">the epic</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Human">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/References">references</a></li><br />
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<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook" class="dir">lab notes&nbsp;&nbsp;&nbsp;</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Collaboration">collaboration</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Attribution">attribution</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/BRIDGE">BRIDGE</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_producer">red light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_sensor">red sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_producer">blue light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_producer">green light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_sensor">green sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Modelling">modelling</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Safety">safety</a></li><br />
<li><a href="http://www.openwetware.org/wiki/French_Lab">protocols</a></li><br />
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<li><a href="https://2010.igem.org/Team:Edinburgh/Acknowledgements" class="dir">acknowledgements</a><br />
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<span style="color:ivory;">Throughout this wiki there are words in <b>bold</b> that indicate a relevance to <b>human aspects</b>. It will become obvious that <b>human aspects</b> are a part of almost everything in <b>iGEM</b>.</span><br />
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<br />
== Red Light Producer ==<br />
<br><br />
<br />
'''29/6/10'''<br />
<br />
Now preparing primers to make a set of different spectra bioluminescent proteins. Need to order primers for Site directed mutagenesis of the Firely luciferase to make it red (MABEL protocol), and to make click beetle luciferase Rluc8 (which emits light at about 420nm in presence of bisdeoxycoelenterazine - about the same price a coelenterazine though)<br />
<br />
'''8/7/2010'''<br />
* Red luciferase mutagenesis primers arrived- 3 sets:<br />
** S248T<br />
** 356 K<br />
** 356 R<br />
<br />
* Followed the protocol with 34ul water + 0.5 template DNA<br />
* Put the primers and template in green box '''(???)'''<br />
*Ran Gel: 5 µL PCR product, 5 µL water, 2.5 µL buffer<br />
[[Image:151.JPG]]<br />
<br />
{| border="1"<br />
| Marker<br />
| S284T <br />
| 356K <br />
| 356R<br />
|}<br />
<br />
<br />
[[File:07-08.JPG]]<br />
<br />
<br />
*356K and 356R look fine<br />
*going to run S284T at a higher ** temp tomorrow to see if it works better<br />
*all PCR tubes stored in freezer<br />
<br />
<br />
<br />
'''09/7/10'''<br />
<br />
Redo PCR of Red Luciferase<br />
*Everything except template DNA + Kod polymerase kept on ice<br />
*Ran PCR<br />
*Template DNA used PyeaR and P. pyralis luciferase BBa_K216015<br />
*Purification done for 356K and 356R; stored in iGEM box (-20C).<br />
<br />
[[File:07-09straight.JPG]]<br />
<br />
'''9/7/2010'''<br />
*Ligations set up overnight- might have put too much ligase in 356K... Incubated at 15C.<br />
*Oure 356R and 356K are in the freezer- row 10 (G &H)?<br />
<br />
'''13/07/10'''<br />
Goal of the day: <br />
<br />
Transformations:<br />
*356R (2): one from 16 °C and one from room temp(RT)<br />
*356K (2): one from 16 °C and one from room temp(RT)<br />
*S284T (2): one with 25/5 ligase, one with 1/7 ligase<br />
<br />
* I had a burger for the lunch. It was very nice.<br />
* Now we are transforming cells:<br />
** 356R (2)& 356K (2): one form 16C, one from RT<br />
** S248T (2): 1 with 25/5 ligase, other with 1.7.10 ligase/<br />
Empty edinbrick vector for control. <br />
* LuxAB + edinbrick ligation.<br />
<br />
* Using 100 ul transformed cells + 800 ul LB for all. Incubation started half 5. <br />
'''22/07/10'''<br />
<br />
Liquid Cultures (amp 80):<br />
*356R (RT) – 1 with NaNO3 and 1 without<br />
**(16°C) – 2 with NaNO3 and 2 without<br />
*356K (RT) – 1 with NaNO3 and 1 without<br />
**(16°C) – 2 with NaNO3 and 2 without <br />
*S284T (100 µL and 25/5 ligase) – 2 with NaNO3 and 2 without<br />
**(900 µL and 25/5 ligase) – 3 with NaNO3 and 3 without<br />
**(900 µL and 1/7 ligase) – 5 with NaNO3 and 5 without<br />
<br />
NaNO3 = 30mM Sodium Nitrate<br />
<br />
'''23/07/10'''<br />
<br />
Double digest of K098010<br />
<br />
'''28/07/10'''<br />
<br />
Primer design to produce a stable red light luciferase. The mutations for increasing bioluminescence come from [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W9V-4NHM57C-1&_user=809099&_coverDate=07%2F15%2F2007&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1414502247&_rerunOrigin=google&_acct=C000043939&_version=1&_urlVersion=0&_userid=809099&md5=00d9c92f6452bd276c6ea606ed7d3bbf Fujii et al, 2007] and the paper from [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W9V-4X6MSYB-7&_user=809099&_coverDate=01%2F15%2F2010&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1414503636&_rerunOrigin=google&_acct=C000043939&_version=1&_urlVersion=0&_userid=809099&md5=297be52832193b4d026953632a1edf3c Branchini et al, 2010]<br />
<br />
'''29/7/2010'''<br />
<br />
*Double digest with EcoRI HF&PstI in buffer 2 (neb).<br />
* 2 bands: with and without vector?<br />
* did not leave the digest for too long... <br />
[[Image:157.jpg]]<br />
<br />
{| border="1"<br />
| Lane 1<br />
| Lane 2<br />
| Lane 3<br />
| Lane 4<br />
| Lane 5<br />
| Lane 6<br />
|-<br />
| Ladder <br />
| S284T (4) <br />
| S284T (6)<br />
| 356R (6, 900)<br />
| 356R (6)<br />
| 356K (4)<br />
|}<br />
<br />
'''2/8/2010'''<br />
* Red luciferases EcoRI HF&PstI double digests- lanes 2-7 in the second miniprep set. <br />
*Ladder didn't work too well ,though...<br />
[[Image:113.JPG]]<br />
* Transform last years luciferase<br />
* Look at papers for expression in E. coli<br />
* Replace B0034 by stronger RBS?<br />
* Confirm that we order bright luc.<br />
* Single digest of constructs<br />
* Test glowing with positive control<br />
<br />
'''9/8/2010'''<br />
Digest: <br />
*WT1<br />
*356 R: (2)<br />
* 356 K: (4)<br />
* S284T: (6)<br />
* '''(?)'''<br />
* 3 eith EcoRI; 3 with EcoRi + PstI<br />
<br />
<br />
'''10/8/2010'''<br />
* Sequencing of red mutant luciferases. <br />
* Forward primers for S284T and reverse for 356R and K should do. <br />
* Concentration of primers for sequencing: 5uM.<br />
* sequencing tubE: 6 ul liquid: 3ul DNA + 2ul water, 1 ul primer mix.<br />
* when using miniprep DNA- remember to spin down the dirt- 1min, max speed, then take the DNA from the top to avoid disturbing any dirt that is settled. <br />
<br />
<br />
'''12/8/2010'''<br />
* Got the sequences for red luciferases<br />
*see: if we can detect Red Light with the luminometer?<br />
* Red Luciferases 356R and S284T were nNOT MUTANTS- just WT<br />
* set up ;iquid cultures with 30 ul nitrate for: <br />
** 356R - no 3,1 from RT plate, no 5 from 16C plate; no 1 from Master plate<br />
** S284T - 5 from masterplate; 2, 4, 6 from masterplate<br />
** 356K: 4 from masterplate<br />
<br />
'''13/8/2010'''<br />
Big day today... TESTING YESTERDAYS PLAN- LUMINESCENCE<br />
<br />
'''16/8/2010'''<br />
* Sequecning of: <br />
** WR1- 356R -use S284T forward primer<br />
** WR2 -S284T -use 356 R reverse primer<br />
** WR3- LovTap 03<br />
**WR4 -LovTap 09<br />
<br />
* RESULTS: ONLY 356K WAS A REAL MUTANT; S284T AND 356R TURNED OUT TO BE STILL A WT<br />
<br />
'''25/8/2010'''<br />
* Next step:- redo ligations for 356R; redo PCR for S284T<br />
* Ligations<br />
*Did MABEL PCR PPyInc + S284T primers<br />
*used 0.5 ul template DNA<br />
<br />
'''26/8/2010'''<br />
* check the Fuji et. al paper, see what sequence they used to start off with. <br />
* check IAV in other luciferase<br />
* Fix scar before RBS and in teh second stop codon. <br />
* compared to WT, luciferase sequence has: <br />
** N 50 P<br />
** N 99 G<br />
** SKL- IAV<br />
<br />
'''27/8/2010'''<br />
* Ligations from yesterday out of freezer.<br />
* Running on gel<br />
* Column 3 9with 356R) has less DNA in it- not enough from ligation and messed up loading of gel). <br />
[[Image:132.JPG]]<br />
{| border="1"<br />
| Lane 1<br />
| Lane 2<br />
| Lane 3<br />
| Lane 4<br />
<br />
|-<br />
| Ladder <br />
| New S284T PCR - new ligase and the buffer<br />
| 356 R ligation<br />
| S284T PCR + old ligase<br />
|}<br />
<br />
'''30/8/2010'''<br />
* Primers have arrived for fixing the luciferase- removing 6 bases<br />
* Primers have arrived for up and downstream sequences of TnaA region.<br />
* Send Chairman sequence for T7 promoter<br />
* transformed 356R and S284T ligations from Friday<br />
* Used PCR product as a control<br />
* Plated out the transformed cells. Used 900 rather than 100 if possible<br />
<br />
<br />
'''31/8/2010'''<br />
* We have S284T transformants but <br />
** 14 colonies on ligation plate<br />
** 4 colonies on control where we used PCR product instead of ligation<br />
<br />
* For sequencing- <br />
** if sequencing a S284T mutant, use 356R/K reverse primer<br />
** if sequencing 356R mutant, use S284T forward primer.<br />
<br />
'''2/9/2010'''<br />
* minipreped 14 colonies (1-14)<br />
* run the gel with samples digested with EcoRI<br />
* here: samples 1-7<br />
[[Image:135.JPG]]<br />
<br />
'''3/9/2010'''<br />
* S284T 8-14 (from yesterday)<br />
[[Image:136.JPG]]<br />
<br />
'''8/9/2010'''<br />
* wWT of luciferase (left)<br />
* mutation of S284T (right)<br />
<br />
<br />
'''9/9/2010'''<br />
{| border="1"<br />
| Lane 1<br />
| Lane 2<br />
| Lane 3<br />
| Lane 4<br />
| Lane 5<br />
|-<br />
| Ladder <br />
| WT <br />
| S284T 1<br />
| luxCDE 9:4<br />
| LuxCDE 9:1<br />
|}<br />
<br />
'''20/9/2010'''<br />
<br />
*send for sequencing: 356K, S284T, WT (samples from 16/9: WT- 900, 2; also 16/9 S284T- 900, 4.)<br />
* all in psB1C3 vector. <br />
* Transform 356K and S284T with RBS and promoter (lacZ, LacI, RBS). <br />
* Run gel of 365K in psb1C3 of the digest. --> control: RFP + psb1C3<br />
* then add promoter<br />
<br />
'''23/9/2010'''<br />
<br />
* Added in incubator room liquid culture '''?)''' LacP + RBS + Luciferase for S284T, 356K and WT. <br />
* miniprep- 6 bottles<br />
* GLOWING<br />
<br />
* S248T- <br />
**9.2; 5 <br />
** 9.4; 1 <br />
**5; 4<br />
* 356<br />
** 3.3<br />
* WT<br />
** 3.2; 4 <br />
** 1.4, 5</div>Macbereskahttp://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_producerTeam:Edinburgh/Notebook/Blue light producer2010-10-26T23:11:27Z<p>Macbereska: /* Blue Light Producer */</p>
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<li><a href="https://2010.igem.org/Team:Edinburgh/Project" class="dir">genomic BRIDGEs</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Project/Protocol">the protocol</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/BioBricks#Genomic">submitted parts</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Genomic">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Project/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Project/References">references</a></li><br />
</ul><br />
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<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial" class="dir">bacterial BRIDGEs</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Core_repressilator">the repressilator</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_producer">red light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_sensor">red sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_producer">blue light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Green_light_producer">green light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Green_light_sensor">green sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/BioBricks#Bacterial">submitted parts</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Bacterial">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/References">references</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling" class="dir">modelling BRIDGEs</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Kappa">kappa</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Genomic">the genomic model</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Bacterial">the bacterial model</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Signalling">the signalling model</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Tools">tools</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Modelling">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/References">references</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human" class="dir">human BRIDGEs</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Aspects">human aspects</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Communication">communication of science</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Terminology">terminology research</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Wiki">wiki</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Conversations">conversations</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Identity">identity</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Collaboration">collaboration</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Branding">branding research</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/SciFi">science fiction writing</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/FutureApps">future applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/SelfReflection">self-reflection</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Epic">the epic</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Human">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/References">references</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook" class="dir">lab notes&nbsp;&nbsp;&nbsp;</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Collaboration">collaboration</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Attribution">attribution</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/BRIDGE">BRIDGE</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_producer">red light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_sensor">red sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_producer">blue light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_producer">green light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_sensor">green sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Modelling">modelling</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Safety">safety</a></li><br />
<li><a href="http://www.openwetware.org/wiki/French_Lab">protocols</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Acknowledgements" class="dir">acknowledgements</a><br />
</li><br />
<br />
</ul><br />
</div><br />
<br />
<br><br />
<br><br />
<br><br />
<br />
<div id="windowbox" style="border: .2em solid #660000; padding: 5px; position:fixed; top:50%; right:30px; width:8%;"><br />
<span style="color:ivory;">Throughout this wiki there are words in <b>bold</b> that indicate a relevance to <b>human aspects</b>. It will become obvious that <b>human aspects</b> are a part of almost everything in <b>iGEM</b>.</span><br />
<br />
</div><br />
<br />
</html><br />
<br />
== Blue Light Producer ==<br />
<br><br />
'''5/7/2010'''<br />
*LuxAB & LuxCDE operon agarose gel<br />
* Lane1: About2.1.kbp<br />
* Lane 2: The highest band- very faint- about 3.5kbp<br />
[[Image:150.JPG]]<br />
<br />
'''13/7/2010'''<br />
<br />
Empty edinbrick vector for control. <br />
* LuxAB + edinbrick ligation.<br />
<br />
'''28/7/2010'''<br />
<br />
an update on LuxAB: <br />
*The transformations of Edinbrick are fine but all the luxABs failed (grew blue, not white on Xgal and did not glow, as of 27/7)/<br />
*Still have some digested LuxAB-ediI so re-did ligation and transformation. <br />
[[Image:154.JPG]]<br />
<br />
{| border="1"<br />
| Lane 1&15<br />
| Lane 2<br />
| Others<br />
|-<br />
| Ladder <br />
| EdiI <br />
| LuxAB <br />
|}<br />
[[Image:155.JPG]]<br />
{| border="1"<br />
| Lane 1<br />
| Lane 2 +3<br />
| Lanes 3-6<br />
|-<br />
| Ladder <br />
| EdiI <br />
| LuxAB <br />
|}<br />
<br />
<br />
<br />
'''29/7/2010'''<br />
<br />
[[Image:156.jpg]]<br />
<br />
* The ligation had the same gel characteristics as previous luxAb-ediI ligations, e.x. it isn't visivle- not hopeful of this transformation working<br />
<br />
'''12/8/2010''' <br />
update on LumP:<br />
* still getting pink/red colonies from transformants<br />
* single digests give a band of about 3kb<br />
* double digests give a band of about 2.5 kb -highly confusing<br />
* and also, no sign on RFP despite red/pink growth.<br />
* Vector wa ssequenced and LumP is definately there. <br />
<br />
'''gel pohot no x and y''' + description.<br />
<br />
'''26/8/2010'''<br />
<br />
* Marker (Lane 1), minipreps 6-10 luxABlumP (lanes 2-6)<br />
[[Image:129.JPG]]<br />
* single digests of LuxABlumP<br />
[[Image:131.JPG]]<br />
<br />
'''7/9/2010'''<br />
*minipreps of luxCDE: samples 1&4 usable<br />
<br />
{| border="1"<br />
| Lane 1<br />
| Lane 2-6<br />
| Lanes 7<br />
|-<br />
| Ladder <br />
| luxCDE00:1-5 minipreps<br />
| Ladder <br />
|}<br />
[[Image:140.JPG]]</div>Macbereskahttp://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_producerTeam:Edinburgh/Notebook/Blue light producer2010-10-26T23:10:58Z<p>Macbereska: /* Blue Light Producer */</p>
<hr />
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<div id="banner"><a href="https://2010.igem.org/Team:Edinburgh"><img src="https://static.igem.org/mediawiki/2010/5/5f/Ed10-Banner.jpg" /></a></div><br />
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<li><a href="https://2010.igem.org/Team:Edinburgh" class="dir">home</a><br />
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<li><a href="https://2010.igem.org/Team:Edinburgh/Team" class="dir">team - illuminati</a><br />
<ul><br />
<li><a href="https://igem.org/Team.cgi?id=322">official</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Team/Supervisors">supervisors</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Team/Advisors">advisors</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Team/Students">students</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Team/Environment">environment</a></li><br />
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<li><a href="https://2010.igem.org/Team:Edinburgh/Project" class="dir">genomic BRIDGEs</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Project/Protocol">the protocol</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/BioBricks#Genomic">submitted parts</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Genomic">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Project/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Project/References">references</a></li><br />
</ul><br />
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<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial" class="dir">bacterial BRIDGEs</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Core_repressilator">the repressilator</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_producer">red light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_sensor">red sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_producer">blue light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Green_light_producer">green light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Green_light_sensor">green sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/BioBricks#Bacterial">submitted parts</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Bacterial">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/References">references</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling" class="dir">modelling BRIDGEs</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Kappa">kappa</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Genomic">the genomic model</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Bacterial">the bacterial model</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Signalling">the signalling model</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Tools">tools</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Modelling">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/References">references</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human" class="dir">human BRIDGEs</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Aspects">human aspects</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Communication">communication of science</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Terminology">terminology research</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Wiki">wiki</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Conversations">conversations</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Identity">identity</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Collaboration">collaboration</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Branding">branding research</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/SciFi">science fiction writing</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/FutureApps">future applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/SelfReflection">self-reflection</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Epic">the epic</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Results#Human">results</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/Future">future work</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Human/References">references</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook" class="dir">lab notes&nbsp;&nbsp;&nbsp;</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Collaboration">collaboration</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Attribution">attribution</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/BRIDGE">BRIDGE</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_producer">red light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_sensor">red sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_producer">blue light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_sensor">blue sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_producer">green light</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_sensor">green sensor</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Modelling">modelling</a></li><br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Safety">safety</a></li><br />
<li><a href="http://www.openwetware.org/wiki/French_Lab">protocols</a></li><br />
</ul><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Edinburgh/Acknowledgements" class="dir">acknowledgements</a><br />
</li><br />
<br />
</ul><br />
</div><br />
<br />
<br><br />
<br><br />
<br><br />
<br />
<div id="windowbox" style="border: .2em solid #660000; padding: 5px; position:fixed; top:50%; right:30px; width:8%;"><br />
<span style="color:ivory;">Throughout this wiki there are words in <b>bold</b> that indicate a relevance to <b>human aspects</b>. It will become obvious that <b>human aspects</b> are a part of almost everything in <b>iGEM</b>.</span><br />
<br />
</div><br />
<br />
</html><br />
<br />
== Blue Light Producer ==<br />
<br><br />
'''5/7/2010'''<br />
*LuxAB & LuxCDE operon agarose gel<br />
* Lane1: About2.1.kbp<br />
* Lane 2: The highest band- very faint- about 3.5kbp<br />
[[Image:150.JPG]]<br />
<br />
'''13/7/2010'''<br />
<br />
Empty edinbrick vector for control. <br />
* LuxAB + edinbrick ligation.<br />
<br />
'''28/7/2010'''<br />
<br />
an update on LuxAB: <br />
*The transformations of Edinbrick are fine but all the luxABs failed (grew blue, not white on Xgal and did not glow, as of 27/7)/<br />
*Still have some digested LuxAB-ediI so re-did ligation and transformation. <br />
[[Image:154.JPG]]<br />
<br />
{| border="1"<br />
| Lane 1&15<br />
| Lane 2<br />
| Others<br />
|-<br />
| Ladder <br />
| EdiI <br />
| LuxAB <br />
|}<br />
[[Image:155.JPG]]<br />
{| border="1"<br />
| Lane 1<br />
| Lane 2 +3<br />
| Lanes 3-6<br />
|-<br />
| Ladder <br />
| EdiI <br />
| LuxAB <br />
|}<br />
<br />
<br />
<br />
'''29/7/2010'''<br />
[[Image:156.jpg]]<br />
* The ligation had the same gel characteristics as previous luxAb-ediI ligations, e.x. it isn't visivle- not hopeful of this transformation working<br />
<br />
'''12/8/2010''' <br />
update on LumP:<br />
* still getting pink/red colonies from transformants<br />
* single digests give a band of about 3kb<br />
* double digests give a band of about 2.5 kb -highly confusing<br />
* and also, no sign on RFP despite red/pink growth.<br />
* Vector wa ssequenced and LumP is definately there. <br />
<br />
'''gel pohot no x and y''' + description.<br />
<br />
'''26/8/2010'''<br />
<br />
* Marker (Lane 1), minipreps 6-10 luxABlumP (lanes 2-6)<br />
[[Image:129.JPG]]<br />
* single digests of LuxABlumP<br />
[[Image:131.JPG]]<br />
<br />
'''7/9/2010'''<br />
*minipreps of luxCDE: samples 1&4 usable<br />
<br />
{| border="1"<br />
| Lane 1<br />
| Lane 2-6<br />
| Lanes 7<br />
|-<br />
| Ladder <br />
| luxCDE00:1-5 minipreps<br />
| Ladder <br />
|}<br />
[[Image:140.JPG]]</div>Macbereska