http://2010.igem.org/wiki/index.php?title=Experimental_Layout&feed=atom&action=historyExperimental Layout - Revision history2024-03-28T19:50:41ZRevision history for this page on the wikiMediaWiki 1.16.5http://2010.igem.org/wiki/index.php?title=Experimental_Layout&diff=194589&oldid=prevKrystal at 20:05, 27 October 20102010-10-27T20:05:09Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h1>Troubleshooting the CUP1p-[MS2-CFP] construct</h1></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h1>Troubleshooting the CUP1p-[MS2-CFP] construct</h1></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Aim</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Aim</h3></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>Following the analysis of the two promoters using CUP1p-GFP and GAL1p-GFP the next experiments planned would start to characterise the interactions of both constructs (CUP1p-[MS2-CFP] and GAL1p-[Npep-GFP]) and more specifically the mutual repression that would be taking place. Experiments using FACS analysis were set up in order to measure various levels of CFP and GFP depending on different conditions (i.e. different ranges of inducers, different ratios of pRS415 to pRS414 etc.). However the FACS machine was unable to detect any GFP or CFP in our calibration samples.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>Following the analysis of the two promoters using CUP1p-GFP and GAL1p-GFP the next experiments planned would start to characterise the interactions of both constructs (CUP1p-[MS2-CFP] and GAL1p-[Npep-GFP]) and more specifically the mutual repression that would be taking place. Experiments using <ins class="diffchange diffchange-inline"><a href="https://2010.igem.org/FACS_analysis_of_fluorescent_proteins"><i></ins>FACS<ins class="diffchange diffchange-inline"></i></a></> </ins>analysis were set up in order to measure various levels of CFP and GFP depending on different conditions (i.e. different ranges of inducers, different ratios of pRS415 to pRS414 etc.). However the FACS machine was unable to detect any GFP or CFP in our calibration samples.</p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Hypothesis</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Hypothesis</h3></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>Our initial hypothesis was that the lack of GFP and CFP expression was due to experimental error during the set up of the cultures (this proved to be correct as far as pRS415 was concerned where the lack of expression was traced back to a faulty stock of inducing agent).<br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>Our initial hypothesis was that the lack of GFP and CFP expression was due to experimental error during the set up of the cultures (this proved to be correct as far as pRS415 was concerned where the lack of expression was traced back to a faulty stock of inducing agent).<br><br></div></td></tr>
</table>Krystalhttp://2010.igem.org/wiki/index.php?title=Experimental_Layout&diff=158451&oldid=prevPorter at 11:53, 26 October 20102010-10-26T11:53:30Z<p></p>
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</table>Porterhttp://2010.igem.org/wiki/index.php?title=Experimental_Layout&diff=158213&oldid=prevPorter at 11:34, 26 October 20102010-10-26T11:34:15Z<p></p>
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</table>Porterhttp://2010.igem.org/wiki/index.php?title=Experimental_Layout&diff=130592&oldid=prevSlam at 10:06, 24 October 20102010-10-24T10:06:46Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><a href="https://2010.igem.org/1._Confirmation_using_microscope_and_fluorometer_analysis_that_the_pRS414_construct_was_not_expressing_CFP">1. Confirmation using microscope and fluorometer analysis that the CUP1p-[MS2-CFP] construct was not expressing CFP</a><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><a href="https://2010.igem.org/1._Confirmation_using_microscope_and_fluorometer_analysis_that_the_pRS414_construct_was_not_expressing_CFP">1. Confirmation using microscope and fluorometer analysis that the CUP1p-[MS2-CFP] construct was not expressing CFP</a><br></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">[[</del>2. <del class="diffchange diffchange-inline"> </del>Confirming <del class="diffchange diffchange-inline"> the </del>CFP <del class="diffchange diffchange-inline">sequence is functional]] </del> <br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">[[</del>3. <del class="diffchange diffchange-inline"> N</del>-<del class="diffchange diffchange-inline">GFP Swap Experiment</del>]]<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><a href="https://2010.igem.org/</ins>3.<ins class="diffchange diffchange-inline">_N</ins>-<ins class="diffchange diffchange-inline">GFP_Swap_Experiment"> 3. Replacement of MS2-[CFP</ins>] <ins class="diffchange diffchange-inline">in CUP1p - [MS2-CFP] with Npep-GFP from GAL1p-[Npep-GFP</ins>] <ins class="diffchange diffchange-inline">to Determine Functionality of Promoter/5'leader/Binding Stem Loop Sequence </a></ins><br></div></td></tr>
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</table>Slamhttp://2010.igem.org/wiki/index.php?title=Experimental_Layout&diff=106717&oldid=prevI.stansfield at 16:55, 17 October 20102010-10-17T16:55:16Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Protocol</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Protocol</h3></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>A series of experiments were set up in order to determine the functionality of various parts of CUP1p-[MS2-CFP] with a view to repair the construct.<br><br<del class="diffchange diffchange-inline">></html</del>></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>A series of experiments were set up in order to determine the functionality of various parts of CUP1p-[MS2-CFP] with a view to repair the construct.<br><br></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">[[</del>1. <del class="diffchange diffchange-inline"> </del>Confirmation using microscope and fluorometer analysis that the CUP1p-[MS2-CFP] construct was not expressing CFP<del class="diffchange diffchange-inline">]]</del><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><a href="https://2010.igem.org/</ins>1.<ins class="diffchange diffchange-inline">_Confirmation_using_microscope_and_fluorometer_analysis_that_the_pRS414_construct_was_not_expressing_CFP">1. </ins>Confirmation using microscope and fluorometer analysis that the CUP1p-[MS2-CFP] construct was not expressing CFP<ins class="diffchange diffchange-inline"></a></ins><br></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[2. Confirming the CFP sequence is functional]] <br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[2. Confirming the CFP sequence is functional]] <br></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Conclusion</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Conclusion</h3></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>The first experiment did not detect any CFP fluorescent whilst the second experiment indicated that the CFP sequence used was accurate and in another vector resulted in fluorescence. The third experiment revealed that a fault lies with either the CUP1 promoter or/and the Bbox stem loop however the fourth experiment showed that the promoter did not seem to be contributing to the lack of expression.<br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>The first experiment did not detect any CFP fluorescent whilst the second experiment indicated that the CFP sequence used was accurate and in another vector resulted in fluorescence. The third experiment revealed that a fault lies with either the CUP1 promoter or/and the Bbox stem loop however the fourth experiment showed that the promoter did not seem to be contributing to the lack of expression.<br><br></div></td></tr>
</table>I.stansfieldhttp://2010.igem.org/wiki/index.php?title=Experimental_Layout&diff=106691&oldid=prevI.stansfield at 16:48, 17 October 20102010-10-17T16:48:41Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>A series of experiments were set up in order to determine the functionality of various parts of CUP1p-[MS2-CFP] with a view to repair the construct.<br><br></html></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>A series of experiments were set up in order to determine the functionality of various parts of CUP1p-[MS2-CFP] with a view to repair the construct.<br><br></html></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[1. Confirmation using microscope and fluorometer analysis that the CUP1p-[MS2-CFP] construct was not expressing CFP]]<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[1. Confirmation using microscope and fluorometer analysis that the CUP1p-[MS2-CFP] construct was not expressing CFP]]<br></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[2. Confirming the CFP sequence is functional]] <br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[2. Confirming the CFP sequence is functional]] <br></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[3. N-GFP Swap Experiment]]<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[3. N-GFP Swap Experiment]]<br></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[4. Replacing the CUP1 promoter in CUP1p-[MS2-CFP] with the CUP1-2 promoter from the CUP1p-GFP construct]]<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[4. Replacing the CUP1 promoter in CUP1p-[MS2-CFP] with the CUP1-2 promoter from the CUP1p-GFP construct]]<br></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Conclusion</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Conclusion</h3></div></td></tr>
</table>I.stansfieldhttp://2010.igem.org/wiki/index.php?title=Experimental_Layout&diff=106685&oldid=prevI.stansfield at 16:46, 17 October 20102010-10-17T16:46:02Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:Aberdeen_Scotland/Title}}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:Aberdeen_Scotland/Title}}</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><html></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><html></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><h1>Troubleshooting <del class="diffchange diffchange-inline">pRS414</del></h1></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><h1>Troubleshooting <ins class="diffchange diffchange-inline">the CUP1p-[MS2-CFP] construct</ins></h1></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Aim</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Aim</h3></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>Following the analysis of the two promoters using <del class="diffchange diffchange-inline">N4 </del>and <del class="diffchange diffchange-inline">N25 </del>the next experiments planned would start to characterise the interactions of both constructs (<del class="diffchange diffchange-inline">pRS414 </del>and <del class="diffchange diffchange-inline">pRS415</del>) and more specifically the mutual repression that would be taking place. Experiments using FACS analysis were set up in order to measure various levels of CFP and GFP depending on different conditions (i.e. different ranges of inducers, different ratios of pRS415 to pRS414 etc.). However the FACS machine was unable to detect any GFP or CFP in our calibration samples.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>Following the analysis of the two promoters using <ins class="diffchange diffchange-inline">CUP1p-GFP </ins>and <ins class="diffchange diffchange-inline">GAL1p-GFP </ins>the next experiments planned would start to characterise the interactions of both constructs (<ins class="diffchange diffchange-inline">CUP1p-[MS2-CFP] </ins>and <ins class="diffchange diffchange-inline">GAL1p-[Npep-GFP]</ins>) and more specifically the mutual repression that would be taking place. Experiments using FACS analysis were set up in order to measure various levels of CFP and GFP depending on different conditions (i.e. different ranges of inducers, different ratios of pRS415 to pRS414 etc.). However the FACS machine was unable to detect any GFP or CFP in our calibration samples.</p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Hypothesis</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Hypothesis</h3></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>Our initial hypothesis was that the lack of GFP and CFP expression was due to experimental error during the set up of the cultures (this proved to be correct as far as pRS415 was concerned where the lack of expression was traced back to a faulty stock of inducing agent).<br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>Our initial hypothesis was that the lack of GFP and CFP expression was due to experimental error during the set up of the cultures (this proved to be correct as far as pRS415 was concerned where the lack of expression was traced back to a faulty stock of inducing agent).<br><br></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Protocol</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Protocol</h3></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>A series of experiments were set up in order to determine the functionality of various parts of <del class="diffchange diffchange-inline">pRS414 </del>with a view to repair the construct.<br><br></html></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>A series of experiments were set up in order to determine the functionality of various parts of <ins class="diffchange diffchange-inline">CUP1p-[MS2-CFP] </ins>with a view to repair the construct.<br><br></html></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[1. Confirmation using microscope and fluorometer analysis that the <del class="diffchange diffchange-inline">pRS414 </del>construct was not expressing CFP]]<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[1. Confirmation using microscope and fluorometer analysis that the <ins class="diffchange diffchange-inline">CUP1p-[MS2-CFP] </ins>construct was not expressing CFP]]<br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[2. Confirming the CFP sequence is functional]] <br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[2. Confirming the CFP sequence is functional]] <br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[3. N-GFP Swap Experiment]]<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[3. N-GFP Swap Experiment]]<br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[4. Replacing the CUP1 promoter in <del class="diffchange diffchange-inline">pRS414 </del>with the CUP1-2 promoter from the <del class="diffchange diffchange-inline">N4 </del>construct]]<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[4. Replacing the CUP1 promoter in <ins class="diffchange diffchange-inline">CUP1p-[MS2-CFP] </ins>with the CUP1-2 promoter from the <ins class="diffchange diffchange-inline">CUP1p-GFP </ins>construct]]<br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><html></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><html></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Conclusion</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Conclusion</h3></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>The first experiment did not detect any CFP fluorescent whilst the second experiment indicated that the CFP sequence used was accurate and in another vector resulted in fluorescence. The third experiment revealed that a fault lies with either the CUP1 promoter or/and the Bbox stem loop however the fourth experiment showed that the promoter did not seem to be contributing to the lack of expression.<br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>The first experiment did not detect any CFP fluorescent whilst the second experiment indicated that the CFP sequence used was accurate and in another vector resulted in fluorescence. The third experiment revealed that a fault lies with either the CUP1 promoter or/and the Bbox stem loop however the fourth experiment showed that the promoter did not seem to be contributing to the lack of expression.<br><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Our data suggests that the reason why <del class="diffchange diffchange-inline">pRS414 </del>is not producing any CFP is due to the presence of the Bbox Stem loop in the 5’UTR. Although Bbox stem loops have already be used in conjunction with the λ-N peptide as markers for mRNA, it is possible that it has never been placed in 5’UTR but is rather located to a 3’UTR. As a result we cannot be sure that the loop can be melted and could therefore be preventing the expression of the following proteins.<br><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Our data suggests that the reason why <ins class="diffchange diffchange-inline">CUP1p-[MS2-CFP] </ins>is not producing any CFP is due to the presence of the Bbox Stem loop in the 5’UTR. Although Bbox stem loops have already be used in conjunction with the λ-N peptide as markers for mRNA, it is possible that it has never been placed in 5’UTR but is rather located to a 3’UTR. As a result we cannot be sure that the loop can be melted and could therefore be preventing the expression of the following proteins.<br><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Our experiments however, do not rule out the possibility that the fusion of MS2 to CFP is defective and is resulting in an unstable protein that is being turned-over rapidly. It is also possible that the fusion is preventing the CFP portion from fluorescencing due to improper folding.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Our experiments however, do not rule out the possibility that the fusion of MS2 to CFP is defective and is resulting in an unstable protein that is being turned-over rapidly. It is also possible that the fusion is preventing the CFP portion from fluorescencing due to improper folding.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></p></div></td></tr>
</table>I.stansfieldhttp://2010.igem.org/wiki/index.php?title=Experimental_Layout&diff=93510&oldid=prevSlam at 17:22, 9 October 20102010-10-09T17:22:13Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h1>Troubleshooting pRS414</h1></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h1>Troubleshooting pRS414</h1></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Aim</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Aim</h3></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>Following the analysis of the two promoters using N4 and N25 the next experiments planned would start to characterise the interactions of both constructs (pRS414 and pRS415) and more specifically the mutual repression that would be taking place. Experiments using FACS analysis were set up in order to measure various levels of CFP and GFP depending on different conditions (i.e. different ranges of inducers, different ratios of pRS415 to pRS414 etc.). However the FACS machine was unable to detect any GFP or <del class="diffchange diffchange-inline">any </del>CFP in our calibration samples.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>Following the analysis of the two promoters using N4 and N25 the next experiments planned would start to characterise the interactions of both constructs (pRS414 and pRS415) and more specifically the mutual repression that would be taking place. Experiments using FACS analysis were set up in order to measure various levels of CFP and GFP depending on different conditions (i.e. different ranges of inducers, different ratios of pRS415 to pRS414 etc.). However the FACS machine was unable to detect any GFP or CFP in our calibration samples.</p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Hypothesis</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Hypothesis</h3></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>Our initial hypothesis was that the lack of GFP and CFP expression was due to experimental error during the set up of the cultures (this proved to be correct as far as pRS415 was concerned where the lack of expression was traced back to a faulty stock of inducing agent).</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>Our initial hypothesis was that the lack of GFP and CFP expression was due to experimental error during the set up of the cultures (this proved to be correct as far as pRS415 was concerned where the lack of expression was traced back to a faulty stock of inducing agent).<ins class="diffchange diffchange-inline"><br><br></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The second hypothesis put forward to explain the lack of CFP expression was that one of the parts that made up pRS414 was defective.<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The second hypothesis put forward to explain the lack of CFP expression was that one of the parts that made up pRS414 was defective.<br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Protocol</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Protocol</h3></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>A series of experiments were set up in order to determine the functionality of various parts of pRS414 <del class="diffchange diffchange-inline">in </del>view to repair the construct.<br><br></html></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>A series of experiments were set up in order to determine the functionality of various parts of pRS414 <ins class="diffchange diffchange-inline">with a </ins>view to repair the construct.<br><br></html></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[1. Confirmation using microscope and fluorometer analysis that the pRS414 construct was not expressing CFP]]<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[1. Confirmation using microscope and fluorometer analysis that the pRS414 construct was not expressing CFP]]<br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[2. Confirming the CFP sequence is functional]] <br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[2. Confirming the CFP sequence is functional]] <br></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Conclusion</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Conclusion</h3></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>The second experiment indicated that the CFP sequence used was accurate and in another vector resulted in fluorescence. The third experiment revealed that a fault lies with either the <del class="diffchange diffchange-inline">Cup1 </del>promoter or/and the Bbox stem loop however the fourth experiment showed that the promoter did not seem to be contributing to the lack of expression.<br><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>The <ins class="diffchange diffchange-inline">first experiment did not detect any CFP fluorescent whilst the </ins>second experiment indicated that the CFP sequence used was accurate and in another vector resulted in fluorescence. The third experiment revealed that a fault lies with either the <ins class="diffchange diffchange-inline">CUP1 </ins>promoter or/and the Bbox stem loop however the fourth experiment showed that the promoter did not seem to be contributing to the lack of expression.<br><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Our data suggests that the reason why pRS414 is not producing any CFP is due to the presence of the Bbox Stem loop in the 5’UTR. Although Bbox stem loops have already be used in <del class="diffchange diffchange-inline">conjuncture </del>with the λ-N peptide as markers for mRNA, it is possible that it has never been placed in 5’UTR but is rather located to a 3’UTR <del class="diffchange diffchange-inline">as </del>a result we cannot be sure that the loop can be melted and could therefore be preventing the expression of the following proteins.<br><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Our data suggests that the reason why pRS414 is not producing any CFP is due to the presence of the Bbox Stem loop in the 5’UTR. Although Bbox stem loops have already be used in <ins class="diffchange diffchange-inline">conjunction </ins>with the λ-N peptide as markers for mRNA, it is possible that it has never been placed in 5’UTR but is rather located to a 3’UTR<ins class="diffchange diffchange-inline">. As </ins>a result we cannot be sure that the loop can be melted and could therefore be preventing the expression of the following proteins.<br><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Our experiments do not rule out <del class="diffchange diffchange-inline">however </del>the possibility that the fusion of MS2 to CFP is defective and is resulting in an unstable protein that is being turned-over rapidly. It is also possible that the fusion is preventing the CFP portion <del class="diffchange diffchange-inline">to fluorescence </del>due to improper folding.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Our experiments <ins class="diffchange diffchange-inline">however, </ins>do not rule out the possibility that the fusion of MS2 to CFP is defective and is resulting in an unstable protein that is being turned-over rapidly. It is also possible that the fusion is preventing the CFP portion <ins class="diffchange diffchange-inline">from fluorescencing </ins>due to improper folding.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></p></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><b>[[https://2010.igem.org/Team:Aberdeen_Scotland/Results Return to Results Main page]]</b></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><b>[[https://2010.igem.org/Team:Aberdeen_Scotland/Results Return to Results Main page]]</b></div></td></tr>
</table>Slamhttp://2010.igem.org/wiki/index.php?title=Experimental_Layout&diff=90925&oldid=prevJoseph at 15:34, 8 October 20102010-10-08T15:34:27Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Hypothesis</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Hypothesis</h3></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>Our initial hypothesis was that the lack of GFP and CFP expression was due to experimental error during the set up of the cultures (this proved to be correct as far as pRS415 was concerned where the lack of expression was traced back to a faulty stock of inducing agent).</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>Our initial hypothesis was that the lack of GFP and CFP expression was due to experimental error during the set up of the cultures (this proved to be correct as far as pRS415 was concerned where the lack of expression was traced back to a faulty stock of inducing agent).</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>The second hypothesis put forward to explain the lack of CFP expression was that one of the parts that made up pRS414 was defective.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The second hypothesis put forward to explain the lack of CFP expression was that one of the parts that made up pRS414 was defective.<ins class="diffchange diffchange-inline"><br></ins></div></td></tr>
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<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><img src="https://static.igem.org/mediawiki/2010/2/2c/Diagram_of_pRS414.jpg"/></ins></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Protocol</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Protocol</h3></div></td></tr>
</table>Josephhttp://2010.igem.org/wiki/index.php?title=Experimental_Layout&diff=90842&oldid=prevJoseph at 14:39, 8 October 20102010-10-08T14:39:32Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Conclusion</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Conclusion</h3></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>The second experiment indicated that the CFP sequence used was accurate and in another vector resulted in fluorescence. The third experiment revealed that a fault lies with either the Cup1 promoter or/and the Bbox stem loop however the fourth experiment showed that the promoter did not seem to be contributing to the lack of expression.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>The second experiment indicated that the CFP sequence used was accurate and in another vector resulted in fluorescence. The third experiment revealed that a fault lies with either the Cup1 promoter or/and the Bbox stem loop however the fourth experiment showed that the promoter did not seem to be contributing to the lack of expression.<ins class="diffchange diffchange-inline"><br><br></ins></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Our data suggests that the reason why pRS414 is not producing any CFP is due to the presence of the Bbox Stem loop in the 5’UTR. Although Bbox stem loops have already be used in conjuncture with the λ-N peptide as markers for mRNA, it is possible that it has never been placed in 5’UTR but is rather located to a 3’UTR as a result we cannot be sure that the loop can be melted and could therefore be preventing the expression of the following proteins.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Our data suggests that the reason why pRS414 is not producing any CFP is due to the presence of the Bbox Stem loop in the 5’UTR. Although Bbox stem loops have already be used in conjuncture with the λ-N peptide as markers for mRNA, it is possible that it has never been placed in 5’UTR but is rather located to a 3’UTR as a result we cannot be sure that the loop can be melted and could therefore be preventing the expression of the following proteins.<ins class="diffchange diffchange-inline"><br><br></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Our experiments do not rule out however the possibility that the fusion of MS2 to CFP is defective and is resulting in an unstable protein that is being turned-over rapidly. It is also possible that the fusion is preventing the CFP portion to fluorescence due to improper folding.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Our experiments do not rule out however the possibility that the fusion of MS2 to CFP is defective and is resulting in an unstable protein that is being turned-over rapidly. It is also possible that the fusion is preventing the CFP portion to fluorescence due to improper folding.</div></td></tr>
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</table>Joseph