http://2010.igem.org/wiki/index.php?title=Team:Aberdeen_Scotland/General_Overview&feed=atom&action=historyTeam:Aberdeen Scotland/General Overview - Revision history2024-03-28T10:14:32ZRevision history for this page on the wikiMediaWiki 1.16.5http://2010.igem.org/wiki/index.php?title=Team:Aberdeen_Scotland/General_Overview&diff=164408&oldid=prevSlam at 18:57, 26 October 20102010-10-26T18:57:00Z<p></p>
<table style="background-color: white; color:black;">
<col class='diff-marker' />
<col class='diff-content' />
<col class='diff-marker' />
<col class='diff-content' />
<tr valign='top'>
<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 18:57, 26 October 2010</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 19:</td>
<td colspan="2" class="diff-lineno">Line 19:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><LI>Would this switch exhibit ‘bi-stability’ (i.e. could it switch from one state to another and back depending on the inducer present).</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><LI>Would this switch exhibit ‘bi-stability’ (i.e. could it switch from one state to another and back depending on the inducer present).</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><LI>Would this bi-stability switching be dose dependent.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><LI>Would this bi-stability switching be dose dependent.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><LI>What would <del class="diffchange diffchange-inline">happens </del>if both inducers were present in equal concentration and length of time.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><LI>What would <ins class="diffchange diffchange-inline">happen </ins>if both inducers were present in equal concentration and length of time.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><LI>Could the bi-stability sensitivity be ‘fine-tuned’ using different mRNA stem loops and binding proteins.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><LI>Could the bi-stability sensitivity be ‘fine-tuned’ using different mRNA stem loops and binding proteins.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></ol></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></ol></div></td></tr>
<tr><td colspan="2" class="diff-lineno">Line 30:</td>
<td colspan="2" class="diff-lineno">Line 30:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></p><p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></p><p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">In the remainder of the project</del>, as described in the Switch troubleshooting section, we carried out a series of carefully designed cassette replacement experiments to identify the component in the CUP1p-[MS2-CFP] construct that was preventing CFP expression. This involved microscopy and fluorimetry (see <a href="https://2010.igem.org/Team:Aberdeen_Scotland/Results"><i> Troubleshooting CUP1p-[MS2-CFP]</i></a> Troubleshooting CUP1p-[MS2-CFP]). From these experiments, we concluded that the Bbox stem loop or fusion of MS2 coat binding protein to CFP may have prevented the expression of CFP. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">Following this</ins>, as described in the Switch troubleshooting section, we carried out a series of carefully designed cassette replacement experiments to identify the component in the CUP1p-[MS2-CFP] construct that was preventing CFP expression. This involved microscopy and fluorimetry (see <a href="https://2010.igem.org/Team:Aberdeen_Scotland/Results"><i> Troubleshooting CUP1p-[MS2-CFP]</i></a> Troubleshooting CUP1p-[MS2-CFP]). From these experiments, we concluded that the Bbox stem loop or fusion of MS2 coat binding protein to CFP may have prevented the expression of CFP. </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><<del class="diffchange diffchange-inline">br</del>><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><<ins class="diffchange diffchange-inline">/p</ins>></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div></p><p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><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 class="diffchange diffchange-inline"><p></ins></div></td></tr>
<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">However, we were able to show using the construct MET17p - [MS2], which was available from the host lab, that translational inhibition of GFP expression via the binding of MS2 coat protein to its corresponding stem loop was possible (see <a href="https://2010.igem.org/MS2_Coat-Protein_Effect_on_Expression_of_GFP_in_pRS415"><i> Characterising the translational repression of GAL1p-[Npeptide-GFP] by trans expression of the MS2 protein </i></a>). This suggests that the toggle switch is a viable design.</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></p></div></td></tr>
<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"><br></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Finally, we characterised a pre-existing bioBrick using one of our constructs, by replacing GFP in the functioning expression construct, GAL1p-[Npeptide-GFP], with mOrange (E2050) using homologous recombination and transformation into yeast. The results from this experiment showed that no mOrange fluorescence was observed from this modified construct. <a href="http://partsregistry.org/Part:BBa_E2020:Design"><i> (see mOrange part entry)</i></a></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Finally, we characterised a pre-existing bioBrick using one of our constructs, by replacing GFP in the functioning expression construct, GAL1p-[Npeptide-GFP], with mOrange (E2050) using homologous recombination and transformation into yeast. The results from this experiment showed that no mOrange fluorescence was observed from this modified construct. <a href="http://partsregistry.org/Part:BBa_E2020:Design"><i> (see mOrange part entry)</i></a></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br></div></td></tr>
</table>Slamhttp://2010.igem.org/wiki/index.php?title=Team:Aberdeen_Scotland/General_Overview&diff=149930&oldid=prevPorter at 21:47, 25 October 20102010-10-25T21:47:38Z<p></p>
<table style="background-color: white; color:black;">
<col class='diff-marker' />
<col class='diff-content' />
<col class='diff-marker' />
<col class='diff-content' />
<tr valign='top'>
<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 21:47, 25 October 2010</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 36:</td>
<td colspan="2" class="diff-lineno">Line 36:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br></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>
<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;"></html></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></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 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;"><html></ins></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;"><hr></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><table class="nav"></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><table class="nav"></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><tr></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><tr></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><td></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><td></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><a href="https://2010.igem.org/Team:Aberdeen_Scotland/<del class="diffchange diffchange-inline">Advisors</del>"><img src="https://static.igem.org/mediawiki/2010/8/8e/Left_arrow.png">&nbsp;&nbsp;Return to <del class="diffchange diffchange-inline">Team Advisors</del></a></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><a href="https://2010.igem.org/Team:Aberdeen_Scotland/<ins class="diffchange diffchange-inline">LabPics</ins>"><img src="https://static.igem.org/mediawiki/2010/8/8e/Left_arrow.png">&nbsp;&nbsp;Return to <ins class="diffchange diffchange-inline">iGEM at Work</ins></a></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></td></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></td></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><td align="right"></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><td align="right"></div></td></tr>
</table>Porterhttp://2010.igem.org/wiki/index.php?title=Team:Aberdeen_Scotland/General_Overview&diff=142999&oldid=prevSlam at 10:45, 25 October 20102010-10-25T10:45:13Z<p></p>
<table style="background-color: white; color:black;">
<col class='diff-marker' />
<col class='diff-content' />
<col class='diff-marker' />
<col class='diff-content' />
<tr valign='top'>
<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 10:45, 25 October 2010</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 27:</td>
<td colspan="2" class="diff-lineno">Line 27:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></p><p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></p><p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Following this, we set out to test the two central constructs of our project (see Fig 1 above). As explained in the Results section, only one of these (GAL1p-[Npeptide-GFP]) was successfully expressed <del class="diffchange diffchange-inline">(see </del>Switch characterisation<del class="diffchange diffchange-inline">)</del>. As a result we characterised the dose - and time-dependent induction of GAL1p-[Npeptide-GFP] using FACS analysis and fluorimetry. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Following this, we set out to test the two central constructs of our project (see Fig 1 above). As explained in the Results section, only one of these (GAL1p-[Npeptide-GFP]) was successfully expressed <ins class="diffchange diffchange-inline"><a href="https://2010.igem.org/Team:Aberdeen_Scotland/Results"><i> </ins>Switch characterisation <ins class="diffchange diffchange-inline"></i></a></ins>. As a result we characterised the dose - and time-dependent induction of GAL1p-[Npeptide-GFP] using FACS analysis and fluorimetry. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></p><p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></p><p></div></td></tr>
</table>Slamhttp://2010.igem.org/wiki/index.php?title=Team:Aberdeen_Scotland/General_Overview&diff=138042&oldid=prevPorter at 22:05, 24 October 20102010-10-24T22:05:38Z<p></p>
<table style="background-color: white; color:black;">
<col class='diff-marker' />
<col class='diff-content' />
<col class='diff-marker' />
<col class='diff-content' />
<tr valign='top'>
<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 22:05, 24 October 2010</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 49:</td>
<td colspan="2" class="diff-lineno">Line 49:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></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 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;">{{:Team:Aberdeen_Scotland/Footer}}</ins></div></td></tr>
</table>Porterhttp://2010.igem.org/wiki/index.php?title=Team:Aberdeen_Scotland/General_Overview&diff=135105&oldid=prevPorter at 17:32, 24 October 20102010-10-24T17:32:01Z<p></p>
<table style="background-color: white; color:black;">
<col class='diff-marker' />
<col class='diff-content' />
<col class='diff-marker' />
<col class='diff-content' />
<tr valign='top'>
<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 17:32, 24 October 2010</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 40:</td>
<td colspan="2" class="diff-lineno">Line 40:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><tr></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><tr></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><td></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><td></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><a href="https://2010.igem.org/Team:Aberdeen_Scotland/Advisors"><img src="https://static.igem.org/mediawiki/2010/8/8e/Left_arrow.png">&nbsp;&nbsp;<del class="diffchange diffchange-inline">Back </del>to Team Advisors</a></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><a href="https://2010.igem.org/Team:Aberdeen_Scotland/Advisors"><img src="https://static.igem.org/mediawiki/2010/8/8e/Left_arrow.png">&nbsp;&nbsp;<ins class="diffchange diffchange-inline">Return </ins>to Team Advisors</a></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></td></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></td></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><td align="right"></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><td align="right"></div></td></tr>
</table>Porterhttp://2010.igem.org/wiki/index.php?title=Team:Aberdeen_Scotland/General_Overview&diff=133936&oldid=prevPorter at 16:11, 24 October 20102010-10-24T16:11:05Z<p></p>
<table style="background-color: white; color:black;">
<col class='diff-marker' />
<col class='diff-content' />
<col class='diff-marker' />
<col class='diff-content' />
<tr valign='top'>
<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 16:11, 24 October 2010</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 2:</td>
<td colspan="2" class="diff-lineno">Line 2:</td></tr>
<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>Biology <del class="diffchange diffchange-inline">Synopsis</del></h1></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><h1>Biology <ins class="diffchange diffchange-inline">Summary</ins></h1></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>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For iGEM2010, our team decided that the biology project would aim to produce a genetic toggle switch, which operated by controlling gene expression at the translational level. To implement the switch, two gene expression constructs were established, each consisting of an RNA-binding protein fused to either Green (GFP) or Cyan (CFP) fluorescent protein. When co-expressed in yeast, these translational fusions would be mutually inhibitory at the translational level, thereby forming a biological, ‘Toggle Switch’ system as shown by diagram 1. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For iGEM2010, our team decided that the biology project would aim to produce a genetic toggle switch, which operated by controlling gene expression at the translational level. To implement the switch, two gene expression constructs were established, each consisting of an RNA-binding protein fused to either Green (GFP) or Cyan (CFP) fluorescent protein. When co-expressed in yeast, these translational fusions would be mutually inhibitory at the translational level, thereby forming a biological, ‘Toggle Switch’ system as shown by diagram 1. </div></td></tr>
</table>Porterhttp://2010.igem.org/wiki/index.php?title=Team:Aberdeen_Scotland/General_Overview&diff=133759&oldid=prevPorter at 15:57, 24 October 20102010-10-24T15:57:28Z<p></p>
<table style="background-color: white; color:black;">
<col class='diff-marker' />
<col class='diff-content' />
<col class='diff-marker' />
<col class='diff-content' />
<tr valign='top'>
<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 15:57, 24 October 2010</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 2:</td>
<td colspan="2" class="diff-lineno">Line 2:</td></tr>
<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>Biology <del class="diffchange diffchange-inline">Overview</del></h1></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><h1>Biology <ins class="diffchange diffchange-inline">Synopsis</ins></h1></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>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For iGEM2010, our team decided that the biology project would aim to produce a genetic toggle switch, which operated by controlling gene expression at the translational level. To implement the switch, two gene expression constructs were established, each consisting of an RNA-binding protein fused to either Green (GFP) or Cyan (CFP) fluorescent protein. When co-expressed in yeast, these translational fusions would be mutually inhibitory at the translational level, thereby forming a biological, ‘Toggle Switch’ system as shown by diagram 1. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For iGEM2010, our team decided that the biology project would aim to produce a genetic toggle switch, which operated by controlling gene expression at the translational level. To implement the switch, two gene expression constructs were established, each consisting of an RNA-binding protein fused to either Green (GFP) or Cyan (CFP) fluorescent protein. When co-expressed in yeast, these translational fusions would be mutually inhibitory at the translational level, thereby forming a biological, ‘Toggle Switch’ system as shown by diagram 1. </div></td></tr>
</table>Porterhttp://2010.igem.org/wiki/index.php?title=Team:Aberdeen_Scotland/General_Overview&diff=132755&oldid=prevPorter at 14:41, 24 October 20102010-10-24T14:41:22Z<p></p>
<table style="background-color: white; color:black;">
<col class='diff-marker' />
<col class='diff-content' />
<col class='diff-marker' />
<col class='diff-content' />
<tr valign='top'>
<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 14:41, 24 October 2010</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 2:</td>
<td colspan="2" class="diff-lineno">Line 2:</td></tr>
<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><<del class="diffchange diffchange-inline">h2</del>><del class="diffchange diffchange-inline">Project overview</del></<del class="diffchange diffchange-inline">h2</del>></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><<ins class="diffchange diffchange-inline">h1</ins>><ins class="diffchange diffchange-inline">Biology Overview</ins></<ins class="diffchange diffchange-inline">h1</ins>></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><p></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For iGEM2010, our team decided that the biology project would aim to produce a genetic toggle switch, which operated by controlling gene expression at the translational level. To implement the switch, two gene expression constructs were established, each consisting of an RNA-binding protein fused to either Green (GFP) or Cyan (CFP) fluorescent protein. When co-expressed in yeast, these translational fusions would be mutually inhibitory at the translational level, thereby forming a biological, ‘Toggle Switch’ system as shown by diagram 1. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For iGEM2010, our team decided that the biology project would aim to produce a genetic toggle switch, which operated by controlling gene expression at the translational level. To implement the switch, two gene expression constructs were established, each consisting of an RNA-binding protein fused to either Green (GFP) or Cyan (CFP) fluorescent protein. When co-expressed in yeast, these translational fusions would be mutually inhibitory at the translational level, thereby forming a biological, ‘Toggle Switch’ system as shown by diagram 1. </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"></p></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><img src="https://static.igem.org/mediawiki/2010/f/ff/Toggle_switch.jpg"/></center></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><img src="https://static.igem.org/mediawiki/2010/f/ff/Toggle_switch.jpg"/></center></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><p></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We have called this the ‘AyeSwitch’ and it is regulated by controlling the two constructs via different inducible yeast promoters, either GAL1 or CUP1 via the presence or absence of galactose and Cu2+ ions respectively. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We have called this the ‘AyeSwitch’ and it is regulated by controlling the two constructs via different inducible yeast promoters, either GAL1 or CUP1 via the presence or absence of galactose and Cu2+ ions respectively. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"></p><p></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Some of the important questions regarding our AyeSwitch were:</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Some of the important questions regarding our AyeSwitch were:</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">(i) </del>Would this switch exhibit ‘bi-stability’ (i.e. could it switch from one state to another and back depending on the inducer present)<<del class="diffchange diffchange-inline">br</del>></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"></p></ins></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">(ii) </del>Would this bi-stability switching be dose dependent <<del class="diffchange diffchange-inline">br</del>></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><ol></ins></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">(iii) </del>What would happens if both inducers were present in equal concentration and length of time <<del class="diffchange diffchange-inline">br</del>></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><LI></ins>Would this switch exhibit ‘bi-stability’ (i.e. could it switch from one state to another and back depending on the inducer present)<ins class="diffchange diffchange-inline">.</ins></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">(iv) </del>Could the bi-stability sensitivity be ‘fine-tuned’ using different mRNA stem loops and binding proteins</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><<ins class="diffchange diffchange-inline">LI</ins>>Would this bi-stability switching be dose dependent<ins class="diffchange diffchange-inline">.</ins></div></td></tr>
<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">LI</ins>>What would happens if both inducers were present in equal concentration and length of time<ins class="diffchange diffchange-inline">.</ins></div></td></tr>
<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">LI</ins>>Could the bi-stability sensitivity be ‘fine-tuned’ using different mRNA stem loops and binding proteins<ins class="diffchange diffchange-inline">.</ins></div></td></tr>
<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"></ol></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"></p><p></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Since this translationally regulated toggle switch is a novel design, the project started with pilot experiments to determine the promoter responses to their respective inducing agents. This was successfully achieved using the genomically integrated constructs in which GFP gene was placed under the control of GAL1 or CUP1 promoter. The results are described in the <a href="https://2010.igem.org/Team:Aberdeen_Scotland/Results"><i> promoter characterisation section results</i></a>.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Since this translationally regulated toggle switch is a novel design, the project started with pilot experiments to determine the promoter responses to their respective inducing agents. This was successfully achieved using the genomically integrated constructs in which GFP gene was placed under the control of GAL1 or CUP1 promoter. The results are described in the <a href="https://2010.igem.org/Team:Aberdeen_Scotland/Results"><i> promoter characterisation section results</i></a>.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><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;"></p><p></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Following this, we set out to test the two central constructs of our project (see Fig 1 above). As explained in the Results section, only one of these (GAL1p-[Npeptide-GFP]) was successfully expressed (see Switch characterisation). As a result we characterised the dose - and time-dependent induction of GAL1p-[Npeptide-GFP] using FACS analysis and fluorimetry. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Following this, we set out to test the two central constructs of our project (see Fig 1 above). As explained in the Results section, only one of these (GAL1p-[Npeptide-GFP]) was successfully expressed (see Switch characterisation). As a result we characterised the dose - and time-dependent induction of GAL1p-[Npeptide-GFP] using FACS analysis and fluorimetry. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><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;"></p><p></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In the remainder of the project, as described in the Switch troubleshooting section, we carried out a series of carefully designed cassette replacement experiments to identify the component in the CUP1p-[MS2-CFP] construct that was preventing CFP expression. This involved microscopy and fluorimetry (see <a href="https://2010.igem.org/Team:Aberdeen_Scotland/Results"><i> Troubleshooting CUP1p-[MS2-CFP]</i></a> Troubleshooting CUP1p-[MS2-CFP]). From these experiments, we concluded that the Bbox stem loop or fusion of MS2 coat binding protein to CFP may have prevented the expression of CFP. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In the remainder of the project, as described in the Switch troubleshooting section, we carried out a series of carefully designed cassette replacement experiments to identify the component in the CUP1p-[MS2-CFP] construct that was preventing CFP expression. This involved microscopy and fluorimetry (see <a href="https://2010.igem.org/Team:Aberdeen_Scotland/Results"><i> Troubleshooting CUP1p-[MS2-CFP]</i></a> Troubleshooting CUP1p-[MS2-CFP]). From these experiments, we concluded that the Bbox stem loop or fusion of MS2 coat binding protein to CFP may have prevented the expression of CFP. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><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;"></p><p></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Finally, we characterised a pre-existing bioBrick using one of our constructs, by replacing GFP in the functioning expression construct, GAL1p-[Npeptide-GFP], with mOrange (E2050) using homologous recombination and transformation into yeast. The results from this experiment showed that no mOrange fluorescence was observed from this modified construct. <a href="http://partsregistry.org/Part:BBa_E2020:Design"><i> (see mOrange part entry)</i></a></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Finally, we characterised a pre-existing bioBrick using one of our constructs, by replacing GFP in the functioning expression construct, GAL1p-[Npeptide-GFP], with mOrange (E2050) using homologous recombination and transformation into yeast. The results from this experiment showed that no mOrange fluorescence was observed from this modified construct. <a href="http://partsregistry.org/Part:BBa_E2020:Design"><i> (see mOrange part entry)</i></a></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><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;"></p></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></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;"><table class="nav"></ins></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;"><tr></ins></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;"><td></ins></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;"><a href="https://2010.igem.org/Team:Aberdeen_Scotland/Advisors"><img src="https://static.igem.org/mediawiki/2010/8/8e/Left_arrow.png">&nbsp;&nbsp;Back to Team Advisors</a></ins></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;"></td></ins></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;"><td align="right"></ins></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;"><a href="https://2010.igem.org/Team:Aberdeen_Scotland/Constructs">Continue to DNA Constructs&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2010/3/36/Right_arrow.png"></a></ins></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;"></td></ins></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;"></tr></ins></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;"></table></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"><html></del></div></td><td colspan="2"> </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>
</table>Porterhttp://2010.igem.org/wiki/index.php?title=Team:Aberdeen_Scotland/General_Overview&diff=106304&oldid=prevSlam at 14:20, 17 October 20102010-10-17T14:20:57Z<p></p>
<table style="background-color: white; color:black;">
<col class='diff-marker' />
<col class='diff-content' />
<col class='diff-marker' />
<col class='diff-content' />
<tr valign='top'>
<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 14:20, 17 October 2010</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 7:</td>
<td colspan="2" class="diff-lineno">Line 7:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><img src="https://static.igem.org/mediawiki/2010/f/ff/Toggle_switch.jpg"/></center></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><center></ins><img src="https://static.igem.org/mediawiki/2010/f/ff/Toggle_switch.jpg"/></center></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td colspan="2" class="diff-lineno">Line 23:</td>
<td colspan="2" class="diff-lineno">Line 23:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Since this translationally regulated toggle switch is a novel design, the project started with pilot experiments to determine the promoter responses to their respective inducing agents. This was successfully achieved using the genomically integrated constructs in which GFP gene was placed under the control of GAL1 or CUP1 promoter. The results are described in the <a href="https://2010.igem.org/Team:Aberdeen_Scotland/Results"><i> promoter characterisation section results</i></a>.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Since this translationally regulated toggle switch is a novel design, the project started with pilot experiments to determine the promoter responses to their respective inducing agents. This was successfully achieved using the genomically integrated constructs in which GFP gene was placed under the control of GAL1 or CUP1 promoter. The results are described in the <a href="https://2010.igem.org/Team:Aberdeen_Scotland/Results"><i> promoter characterisation section results</i></a>.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Following this, we set out to test the two central constructs of our project (see Fig 1 above). As explained in the Results section, only one of these (GAL1p-[Npeptide-GFP]) was successfully expressed (see Switch characterisation). As a result we <del class="diffchange diffchange-inline"> </del>characterised the dose- and time-dependent induction of GAL1p-[Npeptide-GFP] using FACS analysis and fluorimetry. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Following this, we set out to test the two central constructs of our project (see Fig 1 above). As explained in the Results section, only one of these (GAL1p-[Npeptide-GFP]) was successfully expressed (see Switch characterisation). As a result we characterised the dose - and time-dependent induction of GAL1p-[Npeptide-GFP] using FACS analysis and fluorimetry. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In the remainder of the project, as described in the Switch troubleshooting section, we carried out a series of carefully designed cassette replacement experiments to identify the component in the CUP1p-[MS2-CFP] construct that was preventing CFP expression. This involved microscopy and fluorimetry (see <a href="https://2010.igem.org/Team:Aberdeen_Scotland/Results"><i> Troubleshooting CUP1p-[MS2-CFP]</i></a> Troubleshooting CUP1p-[MS2-CFP]). From these experiments, we concluded that the Bbox stem loop or fusion of MS2 coat binding protein to CFP may have prevented the expression of CFP. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In the remainder of the project, as described in the Switch troubleshooting section, we carried out a series of carefully designed cassette replacement experiments to identify the component in the CUP1p-[MS2-CFP] construct that was preventing CFP expression. This involved microscopy and fluorimetry (see <a href="https://2010.igem.org/Team:Aberdeen_Scotland/Results"><i> Troubleshooting CUP1p-[MS2-CFP]</i></a> Troubleshooting CUP1p-[MS2-CFP]). From these experiments, we concluded that the Bbox stem loop or fusion of MS2 coat binding protein to CFP may have prevented the expression of CFP. </div></td></tr>
</table>Slamhttp://2010.igem.org/wiki/index.php?title=Team:Aberdeen_Scotland/General_Overview&diff=106090&oldid=prevI.stansfield at 10:50, 17 October 20102010-10-17T10:50:54Z<p></p>
<table style="background-color: white; color:black;">
<col class='diff-marker' />
<col class='diff-content' />
<col class='diff-marker' />
<col class='diff-content' />
<tr valign='top'>
<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 10:50, 17 October 2010</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 25:</td>
<td colspan="2" class="diff-lineno">Line 25:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Following this, we set out to test the two central constructs of our project (see Fig 1 above). As explained in the Results section, only one of these (GAL1p-[Npeptide-GFP]) was successfully expressed (see Switch characterisation). As a result we characterised the dose- and time-dependent induction of GAL1p-[Npeptide-GFP] using FACS analysis and fluorimetry. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Following this, we set out to test the two central constructs of our project (see Fig 1 above). As explained in the Results section, only one of these (GAL1p-[Npeptide-GFP]) was successfully expressed (see Switch characterisation). As a result we characterised the dose- and time-dependent induction of GAL1p-[Npeptide-GFP] using FACS analysis and fluorimetry. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>In the remainder of the project, as described in the Switch troubleshooting section, we carried out a series of carefully designed cassette replacement experiments to identify the component in the CUP1p-[MS2-CFP] construct that was preventing CFP expression. This involved microscopy and fluorimetry (see Troubleshooting CUP1p-[MS2-CFP]). From these experiments, we concluded that the Bbox stem loop or fusion of MS2 coat binding protein to CFP may have prevented the expression of CFP. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>In the remainder of the project, as described in the Switch troubleshooting section, we carried out a series of carefully designed cassette replacement experiments to identify the component in the CUP1p-[MS2-CFP] construct that was preventing CFP expression. This involved microscopy and fluorimetry (see <ins class="diffchange diffchange-inline"><a href="https://2010.igem.org/Team:Aberdeen_Scotland/Results"><i> Troubleshooting CUP1p-[MS2-CFP]</i></a> </ins>Troubleshooting CUP1p-[MS2-CFP]). From these experiments, we concluded that the Bbox stem loop or fusion of MS2 coat binding protein to CFP may have prevented the expression of CFP. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Finally, we characterised a pre-existing bioBrick using one of our constructs, by replacing GFP in the functioning expression construct, GAL1p-[Npeptide-GFP], with mOrange (E2050) using homologous recombination and transformation into yeast. The results from this experiment showed that no mOrange fluorescence was observed from this modified construct. <a href="http://partsregistry.org/Part:BBa_E2020:Design"><i> (see mOrange part entry)</i></a></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Finally, we characterised a pre-existing bioBrick using one of our constructs, by replacing GFP in the functioning expression construct, GAL1p-[Npeptide-GFP], with mOrange (E2050) using homologous recombination and transformation into yeast. The results from this experiment showed that no mOrange fluorescence was observed from this modified construct. <a href="http://partsregistry.org/Part:BBa_E2020:Design"><i> (see mOrange part entry)</i></a></div></td></tr>
</table>I.stansfield