http://2009.igem.org/wiki/index.php?title=Team:Imperial_College_London/M3&feed=atom&action=historyTeam:Imperial College London/M3 - Revision history2024-03-28T18:39:59ZRevision history for this page on the wikiMediaWiki 1.16.5http://2009.igem.org/wiki/index.php?title=Team:Imperial_College_London/M3&diff=169043&oldid=prevCharlesFracchia at 03:54, 22 October 20092009-10-22T03:54:45Z<p></p>
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</table>CharlesFracchiahttp://2009.igem.org/wiki/index.php?title=Team:Imperial_College_London/M3&diff=168971&oldid=prevCharlesFracchia at 03:52, 22 October 20092009-10-22T03:52:17Z<p></p>
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</table>CharlesFracchiahttp://2009.igem.org/wiki/index.php?title=Team:Imperial_College_London/M3&diff=168511&oldid=prevCharlesFracchia at 03:41, 22 October 20092009-10-22T03:41:55Z<p></p>
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</table>CharlesFracchiahttp://2009.igem.org/wiki/index.php?title=Team:Imperial_College_London/M3&diff=168484&oldid=prevCharlesFracchia: /* Module 3: Genome Deletion */2009-10-22T03:41:20Z<p><span class="autocomment">Module 3: Genome Deletion</span></p>
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</table>CharlesFracchiahttp://2009.igem.org/wiki/index.php?title=Team:Imperial_College_London/M3&diff=168449&oldid=prevCharlesFracchia: /* Module 3: Genome Deletion */2009-10-22T03:40:14Z<p><span class="autocomment">Module 3: Genome Deletion</span></p>
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</table>CharlesFracchiahttp://2009.igem.org/wiki/index.php?title=Team:Imperial_College_London/M3&diff=167652&oldid=prevCharlesFracchia: /* Module 3: Genome Deletion */2009-10-22T03:16:24Z<p><span class="autocomment">Module 3: Genome Deletion</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Rationale==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Rationale==</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Module 3 acts as a <b>reusable</b> module for <b>removal of genetic material</b> without toxic effects.<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Module 3 acts as a <b>reusable</b> module for <b>removal of genetic material</b> without toxic effects.<br></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Theory==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Theory==</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Due to the possible <b>pathogenicity and health concerns</b>, cell death must occur before the pill is ready for consumption. Therefore, the method chosen needs to be foolproof and have <b>failsafe mechanism</b>. <br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Due to the possible <b>pathogenicity and health concerns</b>, cell death must occur before the pill is ready for consumption. Therefore, the method chosen needs to be foolproof and have <b>failsafe mechanism</b>. <br></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><html><a href="https://2009.igem.org/Team:Imperial_College_London/M3/DamMethylation</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><html><a href="https://2009.igem.org/Team:Imperial_College_London/M3/DamMethylation</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>"><img style="vertical-align:bottom;"width=50px align="left" src="http://i691.photobucket.com/albums/vv271/dk806/II09Learnmore.png"></a></html><b>&nbsp; About Methylation</b></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>"><img style="vertical-align:bottom;"width=50px align="left" src="http://i691.photobucket.com/albums/vv271/dk806/II09Learnmore.png"></a></html><b>&nbsp; About Methylation</b></div></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;"><br></del></div></td><td colspan="2"> </td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Results==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Results==</div></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;"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Wet Lab===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Wet Lab===</div></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;"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:II09 DpnII Digest.png|right|400px]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:II09 DpnII Digest.png|right|400px]]</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The activity of the restriction enzymes is critical to module 3. We have tested this using a genomic digest assay. <br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The activity of the restriction enzymes is critical to module 3. We have tested this using a genomic digest assay. <br></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Dry Lab===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Dry Lab===</div></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;"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We have also attempted to link our restriction enzymes with cell death using a model.<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We have also attempted to link our restriction enzymes with cell death using a model.<br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Results summary===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Results summary===</div></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;"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We have shown that cells can be protected from low concentrations of the restriction enzymes DpnII and TaqI by Dam methylation, and how the cell population rapidly decreases with thermoinduction of restriction enzymes. <br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We have shown that cells can be protected from low concentrations of the restriction enzymes DpnII and TaqI by Dam methylation, and how the cell population rapidly decreases with thermoinduction of restriction enzymes. <br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></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;"></del></div></td><td colspan="2"> </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;"><!--</del></div></td><td colspan="2"> </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;"><b>The system</b></del></div></td><td colspan="2"> </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;"><br></del></div></td><td colspan="2"> </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;">Under control of a thermoinducible promoter, when the temperature is raised to 42°C, the restriction enzymes DpnII and TaqI are produced. These restriction enzymes cut frequently in the genome, digesting the entirity of the genetic material. Without its genetic material, cell death is also naturally induced. However, restriction enzymes are highly toxic to the cell, where even minute amounts can induce destruction of the genome. Therefore, the native Dam methylation system is used to prevent leaky levels of restriction enzymes from killing the cell prematurely. The restriction enzyme cleavage properties and Dam methylation protection form an interesting system for further study. <br></del></div></td><td colspan="2"> </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;"></del></div></td><td colspan="2"> </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;"><br></del></div></td><td colspan="2"> </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;">--></del></div></td><td colspan="2"> </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;"></del></div></td><td colspan="2"> </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;"><!--This prevents any possible pathogenic effects, and also allays health concerns of eating genetically modified organisms. --></del></div></td><td colspan="2"> </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;"><!--</del></div></td><td colspan="2"> </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><a href="https://2009.igem.org/Team:Imperial_College_London/Stomach"><img style="vertical-align:bottom;" width=90px align="left" src="http://i691.photobucket.com/albums/vv271/dk806/II09Learnmore.png"></a></html><br><br>&nbsp; About the ethical implications of live organisms.</del></div></td><td colspan="2"> </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;">--></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><center></html></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><html><center></html></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>
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<td colspan="2" class="diff-lineno">Line 105:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></tr></table></html></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></tr></table></html></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;"><!--</del></div></td><td colspan="2"> </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;"><br></del></div></td><td colspan="2"> </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;">{{Imperial/09/Division}}</del></div></td><td colspan="2"> </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;">--></del></div></td><td colspan="2"> </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;"><!--</del></div></td><td colspan="2"> </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;"></del></div></td><td colspan="2"> </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;">==Restriction Enzymes==</del></div></td><td colspan="2"> </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;"></del></div></td><td colspan="2"> </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;"><!--After Module 2 has been completed, genome deletion is triggered by raising the temperature. <br></del></div></td><td colspan="2"> </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;">see [[Team:Imperial_College_London/Temporal_Control/Thermoinduction| thermoinduction under temporal control]])<br></del></div></td><td colspan="2"> </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;"><br></del></div></td><td colspan="2"> </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;">The cell then produces the [[Team:Imperial_College_London/M1/RE| restriction enzymes]] DpnII and TaqI. </del></div></td><td colspan="2"> </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;"></del></div></td><td colspan="2"> </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;">Restriction enzymes DpnII and TaqI specifically target and cut short 4 base DNA sequences. With a high frequency of cutting, the genetic material contained within the cell will all be destroyed, including any inserted DNA. <br></del></div></td><td colspan="2"> </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;">[[Image: II09_cut dna.jpg| center]]</del></div></td><td colspan="2"> </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;"></del></div></td><td colspan="2"> </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;">A distinct advantage of using restriction enzymes for our 'killing' mechanism is that the cell membrane is left intact afterwards, and the protein of interest will still be protected by the encapsulated cell. This renders the bacterium no more than an inanimate shell containing our protein drug of choice. <br></del></div></td><td colspan="2"> </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;">[[Image: inanimate shell.jpg| center]]</del></div></td><td colspan="2"> </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;"><br></del></div></td><td colspan="2"> </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><a href="https://2009.igem.org/Team:Imperial_College_London/Restriction_Enzymes"><img style="vertical-align:bottom;" width=90px align="left" src="http://i691.photobucket.com/albums/vv271/dk806/II09_Learnmore.png"></a></html><br><br>&nbsp; about the restriction enzymes TaqI and DpnII.</del></div></td><td colspan="2"> </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;"></del></div></td><td colspan="2"> </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;"></del></div></td><td colspan="2"> </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;"><!--{{Imperial/09/Division}}</del></div></td><td colspan="2"> </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;"></del></div></td><td colspan="2"> </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;"></del></div></td><td colspan="2"> </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;">==Dam methylation==</del></div></td><td colspan="2"> </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;"></del></div></td><td colspan="2"> </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;">To protect against DNA destruction due to basal levels of restriction enzyme production, we have made use of the native E. coli [[Team:Imperial_College_London/M1/Dam| Dam methylase protection system]]. This methylates DNA. Therefore, only high levels of restriction enzyme (ie. after thermal triggering) will cleave the DNA.<br></del></div></td><td colspan="2"> </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;"></del></div></td><td colspan="2"> </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><a href="https://2009.igem.org/Team:Imperial_College_London/Dam"><img style="vertical-align:bottom;" width=90px align="left" src="http://i691.photobucket.com/albums/vv271/dk806/II09_Learnmore.png"></a></html><br><br>&nbsp; about the Dam methylation Defense Mechanism --></del></div></td><td colspan="2"> </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;"></del></div></td><td colspan="2"> </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;"><!--</del></div></td><td colspan="2"> </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;"></del></div></td><td colspan="2"> </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;">==Thermoinduction==</del></div></td><td colspan="2"> </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;"></del></div></td><td colspan="2"> </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;">[[Image: II09_temp increase.jpg| right]]</del></div></td><td colspan="2"> </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;">After Module 2 has been completed, genome deletion is triggered by raising the temperature. This is especially suitable since it is difficult for normal chemical induction to penetrate the colanic acid coating. <br></del></div></td><td colspan="2"> </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;"><br></del></div></td><td colspan="2"> </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;">The pLambda promoter and cI protein form a thermoregulatable system. <br></del></div></td><td colspan="2"> </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;">see [[Team:Imperial_College_London/Temporal_Control/Thermoinduction| thermoinduction under temporal control]])<br></del></div></td><td colspan="2"> </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;"></del></div></td><td colspan="2"> </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;">[[Image:II09_DNApic.png |right|250px]]</del></div></td><td colspan="2"> </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;"><br><br><br><b>Module 3</b> is the final module of our system. It is designed so that the cell is programmed to destroy it's genetic material after encapsulation has finished. <br><br></del></div></td><td colspan="2"> </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;">After <b>Module 2</b> has been completed, <i>The E.ncapsulator</i> is programmed to 'genetically self-destruct.' The cell produces enzymes which specifically target and cut DNA sequences, in order to destroy all genetic material contained within the cell. One advantage of using these enzymes for our 'killing' mechanism is that the cell membrane is left intact afterwards, and the protein of interest will still be protected by the encapsulated cell.</del></div></td><td colspan="2"> </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;"><br><br><br><br><br></del></div></td><td colspan="2"> </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;"></del></div></td><td colspan="2"> </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;">More detail can be found [[Imperial_College_London/M3/Detail |here]].</del></div></td><td colspan="2"> </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;"></del></div></td><td colspan="2"> </td></tr>
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<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><center><a href="https://2009.igem.org/Team:Imperial_College_London/M2"><img width=150px src="https://static.igem.org/mediawiki/2009/1/11/II09_M3ArrowLeft.png"></a></del></div></td><td colspan="2"> </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: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </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;">[[Image:Mod3.jpg| 70px]]</del></div></td><td colspan="2"> </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;">==Why==</del></div></td><td colspan="2"> </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;"></del></div></td><td colspan="2"> </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;">The <i>E.ncapsulator</i> requires the E. coli to be dead upon ingestion. This will prevent any transfer of genetic material between the bacterium and any gut microflora present, thereby avoiding any unexpected pathogenic effects. This is also especially important if the <i>E.ncapsulator</i> is to attain public acceptance, due to concerns over genetically modified organisms.</del></div></td><td colspan="2"> </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;"></del></div></td><td colspan="2"> </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;">==When==</del></div></td><td colspan="2"> </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;"></del></div></td><td colspan="2"> </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;">The thermally induced killing mechanism will only be triggered once encapsulation is complete.</del></div></td><td colspan="2"> </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;"></del></div></td><td colspan="2"> </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;">==How==</del></div></td><td colspan="2"> </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;"></del></div></td><td colspan="2"> </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;">Restriction enzymes found within bacteria and act as defense mechanisms against invading viruses. They work by recognising a certain DNA sequence of a few bases, and then cleaving the DNA strand. The <i>E.ncapsulator</i> is engineered to manufacture the restriction enzymes DpnII and TaqI when triggered, and these will cleave the genetic material within into fragments - thereby killing the cell.</del></div></td><td colspan="2"> </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;"></del></div></td><td colspan="2"> </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;"></del></div></td><td colspan="2"> </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;">As a protective mechanism against DNA destruction due to basal levels of restriction enzyme production, we have made use of the native E. coli Dam methylase protection system. This methylates DNA, which means that only high levels of restriction enzyme (ie. upon trigger) will cleave the DNA. </del></div></td><td colspan="2"> </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;">is induced, rendering the bacterium no more than an inanimate shell containing our protein drug of choice.</del></div></td><td colspan="2"> </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;">--></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{Imperial/09/TemplateBottom}}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{Imperial/09/TemplateBottom}}</div></td></tr>
</table>CharlesFracchiahttp://2009.igem.org/wiki/index.php?title=Team:Imperial_College_London/M3&diff=165147&oldid=prevCharlesFracchia: /* Module 3: Genome Deletion */2009-10-22T02:20:36Z<p><span class="autocomment">Module 3: Genome Deletion</span></p>
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<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>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><img src="<del class="diffchange diffchange-inline">http</del>://<del class="diffchange diffchange-inline">i691</del>.<del class="diffchange diffchange-inline">photobucket</del>.<del class="diffchange diffchange-inline">com</del>/<del class="diffchange diffchange-inline">albums</del>/<del class="diffchange diffchange-inline">vv271</del>/<del class="diffchange diffchange-inline">dk806</del>/<del class="diffchange diffchange-inline">MainOverviewPicM3</del>.<del class="diffchange diffchange-inline">jpg</del>" usemap="#MAP91791" border="0" width="75%" title="Overview picture of the E.ncapsulator"></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><img src="<ins class="diffchange diffchange-inline">https</ins>://<ins class="diffchange diffchange-inline">static</ins>.<ins class="diffchange diffchange-inline">igem</ins>.<ins class="diffchange diffchange-inline">org</ins>/<ins class="diffchange diffchange-inline">mediawiki</ins>/<ins class="diffchange diffchange-inline">2009</ins>/<ins class="diffchange diffchange-inline">8</ins>/<ins class="diffchange diffchange-inline">8d/II09_MapIndicator_Module3</ins>.<ins class="diffchange diffchange-inline">png</ins>" usemap="#MAP91791" border="0" width="75%" title="Overview picture of the E.ncapsulator"></div></td></tr>
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</table>CharlesFracchiahttp://2009.igem.org/wiki/index.php?title=Team:Imperial_College_London/M3&diff=160686&oldid=prevTywang: /* Engineering cell death */2009-10-22T00:00:45Z<p><span class="autocomment">Engineering cell death</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Dam methylase ([http://partsregistry.org/Part:BBa_K200001 <del class="diffchange diffchange-inline">| </del>K200001]) is constitutively produced at a low amount. This prevents leaky expression of restriction enzymes from damaging the genome prematurely. Consequently, a balance exists between Dam methylation and restriction enzyme activity. <br><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Dam methylase ([http://partsregistry.org/Part:BBa_K200001 K200001]) is constitutively produced at a low amount. This prevents leaky expression of restriction enzymes from damaging the genome prematurely. Consequently, a balance exists between Dam methylation and restriction enzyme activity. <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><html><a href="https://2009.igem.org/Team:Imperial_College_London/M3/DamMethylation</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><html><a href="https://2009.igem.org/Team:Imperial_College_London/M3/DamMethylation</div></td></tr>
</table>Tywanghttp://2009.igem.org/wiki/index.php?title=Team:Imperial_College_London/M3&diff=160678&oldid=prevTywang: /* Engineering cell death */2009-10-22T00:00:20Z<p><span class="autocomment">Engineering cell death</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Under the control of a thermoinducible promoter system[http://partsregistry.org/Part:BBa_K098995 K098995]), when the temperature is raised, the promoter is activated and restriction enzymes are produced. There is a safeguard here as the temperature of the human body is around 37°C, so that even if the bacteria are not killed by the heat pulse, they will be killed after they enter the human body. <br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Under the control of a thermoinducible promoter system <ins class="diffchange diffchange-inline">(</ins>[http://partsregistry.org/Part:BBa_K098995 K098995]), when the temperature is raised, the promoter is activated and restriction enzymes are produced. There is a safeguard here as the temperature of the human body is around 37°C, so that even if the bacteria are not killed by the heat pulse, they will be killed after they enter the human body. <br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The restriction enzymes DpnII ([http://partsregistry.org/Part:BBa_K200009 K200009]) and TaqI ([http://partsregistry.org/Part:BBa_K200010 K200010]) are produced. This duplicity of restriction enzymes ensures that even when one enzyme becomes mutated and dysfunctional, the other restriction enzyme still works well by itself. Therefore, by using two restriction enzymes, we can be more certain that our DNA has been digested. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The restriction enzymes DpnII ([http://partsregistry.org/Part:BBa_K200009 K200009]) and TaqI ([http://partsregistry.org/Part:BBa_K200010 K200010]) are produced. This duplicity of restriction enzymes ensures that even when one enzyme becomes mutated and dysfunctional, the other restriction enzyme still works well by itself. Therefore, by using two restriction enzymes, we can be more certain that our DNA has been digested. </div></td></tr>
</table>Tywanghttp://2009.igem.org/wiki/index.php?title=Team:Imperial_College_London/M3&diff=160660&oldid=prevTywang: /* Engineering cell death */2009-10-21T23:59:43Z<p><span class="autocomment">Engineering cell death</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Under the control of a thermoinducible promoter system<del class="diffchange diffchange-inline">(</del>[http://partsregistry.org/Part:BBa_K098995 <del class="diffchange diffchange-inline">| </del>K098995]), when the temperature is raised, the promoter is activated and restriction enzymes are produced. There is a safeguard here as the temperature of the human body is around 37°C, so that even if the bacteria are not killed by the heat pulse, they will be killed after they enter the human body. <br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Under the control of a thermoinducible promoter system[http://partsregistry.org/Part:BBa_K098995 K098995]), when the temperature is raised, the promoter is activated and restriction enzymes are produced. There is a safeguard here as the temperature of the human body is around 37°C, so that even if the bacteria are not killed by the heat pulse, they will be killed after they enter the human body. <br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>The restriction enzymes DpnII ([http://partsregistry.org/Part:BBa_K200009 <del class="diffchange diffchange-inline">| </del>K200009]) and TaqI (http://partsregistry.org/Part:BBa_K200010 <del class="diffchange diffchange-inline">| </del>K200010]) are produced. This duplicity of restriction enzymes ensures that even when one enzyme becomes mutated and dysfunctional, the other restriction enzyme still works well by itself. Therefore, by using two restriction enzymes, we can be more certain that our DNA has been digested. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The restriction enzymes DpnII ([http://partsregistry.org/Part:BBa_K200009 K200009]) and TaqI (<ins class="diffchange diffchange-inline">[</ins>http://partsregistry.org/Part:BBa_K200010 K200010]) are produced. This duplicity of restriction enzymes ensures that even when one enzyme becomes mutated and dysfunctional, the other restriction enzyme still works well by itself. Therefore, by using two restriction enzymes, we can be more certain that our DNA has been digested. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td></tr>
</table>Tywang