http://2014.igem.org/wiki/index.php?title=Team:Uppsala/Project_Adhesion&feed=atom&action=historyTeam:Uppsala/Project Adhesion - Revision history2024-03-28T14:39:08ZRevision history for this page on the wikiMediaWiki 1.16.5http://2014.igem.org/wiki/index.php?title=Team:Uppsala/Project_Adhesion&diff=363943&oldid=prevAmaron at 22:48, 17 October 20142014-10-17T22:48:44Z<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 22:48, 17 October 2014</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 70:</td>
<td colspan="2" class="diff-lineno">Line 70:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>If the bacteriocin would survive degradation in stomach like environments and later on find <i>Y. enterocolitica</i>, the bacteriocin, while attaching to the membrane, would fix the probiotic to its target. Even though this connection would be much weaker than a system with collagen, it is safe and still an advantage to the Bactissile.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>If the bacteriocin would survive degradation in stomach like environments and later on find <i>Y. enterocolitica</i>, the bacteriocin, while attaching to the membrane, would fix the probiotic to its target. Even though this connection would be much weaker than a system with collagen, it is safe and still an advantage to the Bactissile.</p></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><h2>System Design</h2></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><a id="ref_point3"></a></ins><h2>System Design</h2></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>The Anchor</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>The Anchor</h3></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>Since we want our system to be usable in a probiotic bacteria we looked for an anchor motif that works both in our well characterised prototype bacteria <i>(E. coli)</i> and in a well known probiotic bacteria like <i>Lactobacillus</i>. We also wanted the anchor motif to be usable with a wide range of protein types since this would be a great addition to the iGEM registry, since no construct for cell surface display could be found by us at the start of our project. In the end we found the gene coding for PgsA from <i>Bacillus subtilis</i> that had been used successfully in both <i>E.coli</i> and a <i>Lactobacillus</i> species. To prevent interaction with the fused domain we added a long (20aa) rigid linker [BBa_J176131] to the C-terminal of the anchor. </p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>Since we want our system to be usable in a probiotic bacteria we looked for an anchor motif that works both in our well characterised prototype bacteria <i>(E. coli)</i> and in a well known probiotic bacteria like <i>Lactobacillus</i>. We also wanted the anchor motif to be usable with a wide range of protein types since this would be a great addition to the iGEM registry, since no construct for cell surface display could be found by us at the start of our project. In the end we found the gene coding for PgsA from <i>Bacillus subtilis</i> that had been used successfully in both <i>E.coli</i> and a <i>Lactobacillus</i> species. To prevent interaction with the fused domain we added a long (20aa) rigid linker [BBa_J176131] to the C-terminal of the anchor. </p></div></td></tr>
</table>Amaronhttp://2014.igem.org/wiki/index.php?title=Team:Uppsala/Project_Adhesion&diff=346166&oldid=prevAmaron at 19:42, 17 October 20142014-10-17T19:42:36Z<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 19:42, 17 October 2014</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 99:</td>
<td colspan="2" class="diff-lineno">Line 99:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><div class="prev"></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><div class="prev"></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><a href="https://2014.igem.org/Team:Uppsala/Project_Killing"><img class="prev_pic" src="https://static.igem.org/mediawiki/2014/<del class="diffchange diffchange-inline">8</del>/<del class="diffchange diffchange-inline">81</del>/Uppsala-igem2014-<del class="diffchange diffchange-inline">Prev</del>.jpg"></a></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><a href="https://2014.igem.org/Team:Uppsala/Project_Killing"><img class="prev_pic" src="https://static.igem.org/mediawiki/2014/<ins class="diffchange diffchange-inline">f</ins>/<ins class="diffchange diffchange-inline">ff</ins>/Uppsala-igem2014-<ins class="diffchange diffchange-inline">Next_Previously-Recovered</ins>.jpg"></a></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: #eee; color:black; font-size: smaller;"><div></div></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></div></div></td></tr>
</table>Amaronhttp://2014.igem.org/wiki/index.php?title=Team:Uppsala/Project_Adhesion&diff=342476&oldid=prevAmaron at 18:51, 17 October 20142014-10-17T18:51:39Z<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 18:51, 17 October 2014</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 60:</td>
<td colspan="2" class="diff-lineno">Line 60:</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;"></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>We studied the ways of infection of <i>Y. enterocolitica</i> and could determine that its main strategies of entering through the gut wall is by using its diverse membrane proteins YadA, invasin and Ail. YadA and invasin work together to bind to our cell membranes. While YadA binds to collagen, invasin structurally resembles fibronectin, that is present in the extracellular matrix, which is recognized by the fibronectin binding membrane protein integrin alpha-5-beta-1 in our cell membranes.<sup><a href="#<del class="diffchange diffchange-inline">reference2</del>">[1]</a></sup> <i>Y. enterocolitica</i> uses our own cell-to-cell binding mechanism to find a target cell and trick it to endocytose by binding to the integrin and to the collagen outside and in between the cells.<sup><a href="#<del class="diffchange diffchange-inline">reference1</del>">[2]</a></sup></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>We studied the ways of infection of <i>Y. enterocolitica</i> and could determine that its main strategies of entering through the gut wall is by using its diverse membrane proteins YadA, invasin and Ail. YadA and invasin work together to bind to our cell membranes. While YadA binds to collagen, invasin structurally resembles fibronectin, that is present in the extracellular matrix, which is recognized by the fibronectin binding membrane protein integrin alpha-5-beta-1 in our cell membranes.<sup><a href="#<ins class="diffchange diffchange-inline">reference1</ins>">[1]</a></sup> <i>Y. enterocolitica</i> uses our own cell-to-cell binding mechanism to find a target cell and trick it to endocytose by binding to the integrin and to the collagen outside and in between the cells.<sup><a href="#<ins class="diffchange diffchange-inline">reference2</ins>">[2]</a></sup></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>At first glance we thought of manipulating our probiotics to also use the same bioorganic systems. That could be done by expressing collagen-like proteins and fusing them with an anchor protein that could transport the whole construct to the outer membrane and keep the collagen attached to the probiotic surface.<sup><a href="#reference3">[3]</a></sup></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>At first glance we thought of manipulating our probiotics to also use the same bioorganic systems. That could be done by expressing collagen-like proteins and fusing them with an anchor protein that could transport the whole construct to the outer membrane and keep the collagen attached to the probiotic surface.<sup><a href="#reference3">[3]</a></sup></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>Amaronhttp://2014.igem.org/wiki/index.php?title=Team:Uppsala/Project_Adhesion&diff=342309&oldid=prevSnowflake at 18:49, 17 October 20142014-10-17T18:49:23Z<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 18:49, 17 October 2014</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 53:</td>
<td colspan="2" class="diff-lineno">Line 53:</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><a id="ref_point"></a><h2>Background</h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><a id="ref_point"></a><h2>Background</h2></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><h3>Purpose of making an adhesion system between <del class="diffchange diffchange-inline">Yersinia </del>and <del class="diffchange diffchange-inline">probiotic bacteria</del></h3></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><h3>Purpose of making an adhesion system between <ins class="diffchange diffchange-inline"><i>Y.enterocolitica</i> </ins>and <ins class="diffchange diffchange-inline">the Bactissile</ins></h3></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p> While creating a biological machine that can efficiently kill specific pathogens without disturbing other cells in its environment, an adhesion system could play a vital role. Getting our Bactissile to attach to its target, in our case <i>Yersinia enterocolitica</i> , could give important advantages in target specificity and delivering medicine in a way that ensures it reaches <i>Y. enterocolitica</i> in a concentration as high as possible.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p> While creating a biological machine that can efficiently kill specific pathogens without disturbing other cells in its environment, an adhesion system could play a vital role. Getting our Bactissile to attach to its target, in our case <i>Yersinia enterocolitica</i> , could give important advantages in target specificity and delivering medicine in a way that ensures it reaches <i>Y. enterocolitica</i> in a concentration as high as possible.</p></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: #eee; color:black; font-size: smaller;"><div><h3>Technical Approach</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Technical Approach</h3></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>The idea was to manipulate our Bactissiles bonding to any of Y. enterocoliticas surface structures and thus preventing the pathogen from ever attaching to the gut wall. This could be done with a DNA construct coding for a membrane protein that either resembles the bonding that <i>Y. enterocolitica</i> makes to our cells or one that itself binds to <i>Y. enterocolitica</i>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>The idea was to manipulate our Bactissiles bonding to any of <ins class="diffchange diffchange-inline"><i></ins>Y. enterocoliticas<ins class="diffchange diffchange-inline"></i> </ins>surface structures and thus preventing the pathogen from ever attaching to the gut wall. This could be done with a DNA construct coding for a membrane protein that either resembles the bonding that <i>Y. enterocolitica</i> makes to our cells or one that itself binds to <i>Y. enterocolitica</i>.</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;"></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>We studied the ways of infection of <i><del class="diffchange diffchange-inline">Yersinia </del>enterocolitica</i> and could determine that its main strategies of entering through the gut wall is by using its diverse membrane proteins YadA, invasin and Ail. YadA and invasin work together to bind to our cell membranes. While YadA binds to collagen, invasin structurally resembles fibronectin, that is present in the extracellular matrix, which is recognized by the fibronectin binding membrane protein integrin alpha-5-beta-1 in our cell membranes.<sup><a href="#reference2">[1]</a></sup> <i>Y. enterocolitica</i> uses our own cell-to-cell binding mechanism to find a target cell and trick it to endocytose by binding to the integrin and to the collagen outside and in between the cells.<sup><a href="#reference1">[2]</a></sup></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>We studied the ways of infection of <i><ins class="diffchange diffchange-inline">Y. </ins>enterocolitica</i> and could determine that its main strategies of entering through the gut wall is by using its diverse membrane proteins YadA, invasin and Ail. YadA and invasin work together to bind to our cell membranes. While YadA binds to collagen, invasin structurally resembles fibronectin, that is present in the extracellular matrix, which is recognized by the fibronectin binding membrane protein integrin alpha-5-beta-1 in our cell membranes.<sup><a href="#reference2">[1]</a></sup> <i>Y. enterocolitica</i> uses our own cell-to-cell binding mechanism to find a target cell and trick it to endocytose by binding to the integrin and to the collagen outside and in between the cells.<sup><a href="#reference1">[2]</a></sup></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>At first glance we thought of manipulating our probiotics to also use the same bioorganic systems. That could be done by expressing collagen-like proteins and fusing them with an anchor protein that could transport the whole construct to the outer membrane and keep the collagen attached to the probiotic surface.<sup><a href="#reference3">[3]</a></sup></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>At first glance we thought of manipulating our probiotics to also use the same bioorganic systems. That could be done by expressing collagen-like proteins and fusing them with an anchor protein that could transport the whole construct to the outer membrane and keep the collagen attached to the probiotic surface.<sup><a href="#reference3">[3]</a></sup></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>By using collagen, and especially putting it on the surface of a bacterial outer membrane that is meant to enter the human body, is an extremely risky procedure. <i>“As antibodies would be produced to target the collagen and anchor protein complex this could lead to an autoimmune reaction as they would also react on the collagen naturally present in our body”</i> says Lars Hellman, a professor in immunology at Uppsala University. More about problems with <del class="diffchange diffchange-inline">Yersinia</del>-adhesion and the development of autoimmune disease can be found in this <a href="https://2014.igem.org/Team:Uppsala/Safety">text</a>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>By using collagen, and especially putting it on the surface of a bacterial outer membrane that is meant to enter the human body, is an extremely risky procedure. <i>“As antibodies would be produced to target the collagen and anchor protein complex this could lead to an autoimmune reaction as they would also react on the collagen naturally present in our body”</i> says Lars Hellman, a professor in immunology at Uppsala University. More about problems with <ins class="diffchange diffchange-inline"><i>Y. enterocolitica</i></ins>-adhesion and the development of autoimmune disease can be found in this <a href="https://2014.igem.org/Team:Uppsala/Safety">text</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: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
</table>Snowflakehttp://2014.igem.org/wiki/index.php?title=Team:Uppsala/Project_Adhesion&diff=322454&oldid=prevGunta at 13:54, 17 October 20142014-10-17T13:54:35Z<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 13:54, 17 October 2014</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 116:</td>
<td colspan="2" class="diff-lineno">Line 116:</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><ul class="reference"></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><ul class="reference"></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><li>[1] (2012) “Bacterial Cell Surface Structures of Yersinia”, N. Bialas</li></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><li<ins class="diffchange diffchange-inline">><a id="reference1"</ins>>[1]<ins class="diffchange diffchange-inline"></a> </ins>(2012) “Bacterial Cell Surface Structures of Yersinia”, N. Bialas</li></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><li>[2] (2011) “Unique Cell Adhesion and Invasion Properties of Yersinia enterocolitica” O:3, the Most Frequent Cause of Human Yersiniosis” by F. Piscano et.al.</li></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><li<ins class="diffchange diffchange-inline">><a id="reference2"</ins>>[2]<ins class="diffchange diffchange-inline"></a> </ins>(2011) “Unique Cell Adhesion and Invasion Properties of Yersinia enterocolitica” O:3, the Most Frequent Cause of Human Yersiniosis” by F. Piscano et.al.</li></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><li>[3] (2001) “YadA, the multifaceted Yersinia adhesin” by J. El Tahir, M. Skurnik.</li></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><li<ins class="diffchange diffchange-inline">><a id="reference3"</ins>>[3]<ins class="diffchange diffchange-inline"></a> </ins>(2001) “YadA, the multifaceted Yersinia adhesin” by J. El Tahir, M. Skurnik.</li></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><li>[4] (2012) “Novel Colicin FY of Yersinia frederiksenii Inhibits Pathogenic Yersinia Strains via YiuR-Mediated Reception, TonB Import, and Cell Membrane Pore Formation“ by J. Bosak et al. </li></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><li<ins class="diffchange diffchange-inline">><a id="reference4"</ins>>[4]<ins class="diffchange diffchange-inline"></a> </ins>(2012) “Novel Colicin FY of Yersinia frederiksenii Inhibits Pathogenic Yersinia Strains via YiuR-Mediated Reception, TonB Import, and Cell Membrane Pore Formation“ by J. Bosak et al. </li></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></ul></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></ul></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>
</table>Guntahttp://2014.igem.org/wiki/index.php?title=Team:Uppsala/Project_Adhesion&diff=283171&oldid=prevTjocko at 18:27, 16 October 20142014-10-16T18:27:12Z<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 18:27, 16 October 2014</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 90:</td>
<td colspan="2" class="diff-lineno">Line 90:</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><table id=partsT style="width:100%"></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><table id=partsT style="width:100%"></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><tr><th></th><th>BioBrick code</th><th>Type</th><th>Construct</th><th>Description</th><th>Designers</th></tr></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><tr><th><ins class="diffchange diffchange-inline">Fav.</ins></th><th>BioBrick code</th><th>Type</th><th>Construct</th><th>Description</th><th>Designers</th></tr></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><tr><td></td><td><a href="http://parts.igem.org/Part:BBa_K1381024">BBa_K1381024</a></td><td>Tag</td><td>B0034-pgsA</td><td></td><td>Adhesion Group</td></tr></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><tr><td></td><td><a href="http://parts.igem.org/Part:BBa_K1381024">BBa_K1381024</a></td><td>Tag</td><td>B0034-pgsA<ins class="diffchange diffchange-inline">-(EAAAR)x4</ins></td><td><ins class="diffchange diffchange-inline">The anchor tag pgsA with the RBS B0034 and a linker</ins></td><td>Adhesion Group</td></tr></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><tr><td></td><td><a href="http://parts.igem.org/Part:BBa_K1381025">BBa_K1381025</a></td><td>Reporter</td><td>B0034-BFP (<del class="diffchange diffchange-inline">fusion standard</del>)</td><td></td><td>Adhesion Group</td></tr></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><tr><td></td><td><a href="http://parts.igem.org/Part:BBa_K1381025">BBa_K1381025</a></td><td>Reporter</td><td>B0034-BFP (<ins class="diffchange diffchange-inline">RFC25</ins>)</td><td><ins class="diffchange diffchange-inline">A mutagenised version of B0034-BFP, compatible with RFC25</ins></td><td>Adhesion Group</td></tr></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></table></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></table></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>
</table>Tjockohttp://2014.igem.org/wiki/index.php?title=Team:Uppsala/Project_Adhesion&diff=280017&oldid=prevAmaron at 16:46, 16 October 20142014-10-16T16:46:53Z<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 16:46, 16 October 2014</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 18:</td>
<td colspan="2" class="diff-lineno">Line 18:</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><h2 class="overview">System design</h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2 class="overview">System design</h2></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></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><p class="box_text">We made two constructs, using a synthesized gene coding for pgsA. In the first one we fused pgsA with colicin Fy and added a promoter aswell as a RBS called B0034. In the other, BFP was put instead of colicin Fy to test the expression of pgsA.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="box_text">We made two constructs, using a synthesized gene coding for pgsA. In the first one we fused pgsA with colicin Fy and added a promoter aswell as a RBS called B0034. In the other, BFP was put instead of colicin Fy to test the expression of pgsA.</p></div></td></tr>
<tr><td colspan="2" class="diff-lineno">Line 70:</td>
<td colspan="2" class="diff-lineno">Line 72:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2>System Design</h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2>System Design</h2></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>The Anchor</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>The Anchor</h3></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>Since we want our system to be usable in a probiotic bacteria we looked for an anchor motif that works both in our well characterised prototype bacteria <i>(E. coli)</i> and in a well known probiotic bacteria like <i>Lactobacillus</i>. We also wanted the anchor motif to be usable with a wide range of protein types since this would be a great addition to the iGEM registry, since no construct for cell surface display could be found by us at the start of our project. In the end we found the gene coding for PgsA from <i>Bacillus subtilis</i> that had been used successfully in both <i>E.coli</i> and a <i>Lactobacillus</i> species. To prevent interaction with the fused domain we added a long (20aa) rigid linker [BBa_J176131] to the C-terminal of the anchor. </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 style="color: red; font-weight: bold; text-decoration: none;"><h3>The fusion</h3></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;"><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 style="color: red; font-weight: bold; text-decoration: none;">To make the fusion protein of anchor and colicin Fy (PgsA-CFY) we chose to synthesize the genes in RFC25 standard (Freiburg). This adds two additional restriction enzyme sites (NgoMIV and AgeI) to the four standard ones, thus enabling simple assembly of the two proteins so that they are translated as one in the same reading frame.</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;"></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 style="color: red; font-weight: bold; text-decoration: none;"><h3>Back up plan</h3></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;"><p>We used normal assembly (RFC10) with re-ligation of XbaI and SpeI sites that leaves a TACTAG-scar where TAG can stop translation and therefore disable production of a fusion protein. A solution would then be to perform PCR-mutagenesis and remove the TACTAG-scar via point mutation.</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;"></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 style="color: red; font-weight: bold; text-decoration: none;"><h3>Characterising the anchor</h3></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;"><p>To test that the anchor attaches to the membrane, we tried to fuse a reporter, the blue fluorescence protein BFP, to the anchor. We chose to mutagenise B0034-BFP [BBa_K592024] to make it compatible with RFC25. It was designed to get an ATG followed by an NgoMIV site upstream of the BFP CDS. The original ATG from the BFP CDS was removed. In the end we would still have to mutagenise BFP, because we never got the RFC25 assembly to work.</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;"></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 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"><a id="ref_point2"></a><h2>Results</h2></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"><p>Mutagenesis of BFP into RFC25 was successful, verified by sequencing. All other constructs were not successful. Mainly because we had trouble with the RFC25-assemblies. The mutagenesis to remove TACTAG also failed because the reverse primer annealed to a region upstream of the desired site causing a frameshift verified by sequencing. The repeat-like sequence of the 60bp linker was probably one of the reasons for this.</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"></p></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><a id="ref_point4"></a><h2>Parts</h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><a id="ref_point4"></a><h2>Parts</h2></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>
</table>Amaronhttp://2014.igem.org/wiki/index.php?title=Team:Uppsala/Project_Adhesion&diff=270941&oldid=prevStephyHe at 11:55, 16 October 20142014-10-16T11:55:04Z<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 11:55, 16 October 2014</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 5:</td>
<td colspan="2" class="diff-lineno">Line 5:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><div class="background"></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><div class="background"></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><a href="#ref_point" style="display:block; width:100%; height: 100%; text-decoration:none !important;"><h2 class="overview">Background</h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><a href="#ref_point" style="display:block; width:100%; height: 100%; text-decoration:none !important;"><h2 class="overview">Background</h2></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p class="box_text">To maximize the specificity in targeting Yersinia enterocolitica, we wanted to design a construct consisting of a membrane protein complex that would bind to the surface of the pathogen. This could be done by fusing an anchor protein with the colicin Fy that aimed at the membrane of Y. enterocolitica. This complex would be expressed on the surface of our probiotic, making it adhere to its target.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p class="box_text">To maximize the specificity in targeting <ins class="diffchange diffchange-inline"><i></ins>Yersinia enterocolitica<ins class="diffchange diffchange-inline"></i></ins>, we wanted to design a construct consisting of a membrane protein complex that would bind to the surface of the pathogen. This could be done by fusing an anchor protein with the colicin Fy that aimed at the membrane of <ins class="diffchange diffchange-inline"><i></ins>Y. enterocolitica<ins class="diffchange diffchange-inline"></i></ins>. This complex would be expressed on the surface of our probiotic, making it adhere to its target.</p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></a></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></a></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></div></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></div></div></td></tr>
</table>StephyHehttp://2014.igem.org/wiki/index.php?title=Team:Uppsala/Project_Adhesion&diff=270918&oldid=prevStephyHe at 11:54, 16 October 20142014-10-16T11:54:15Z<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 11:54, 16 October 2014</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 5:</td>
<td colspan="2" class="diff-lineno">Line 5:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><div class="background"></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><div class="background"></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><a href="#ref_point" style="display:block; width:100%; height: 100%; text-decoration:none !important;"><h2 class="overview">Background</h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><a href="#ref_point" style="display:block; width:100%; height: 100%; text-decoration:none !important;"><h2 class="overview">Background</h2></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p class="box_text"><del class="diffchange diffchange-inline">In our project we developed a system to fight pathogens </del>in <del class="diffchange diffchange-inline">the gut</del>, <del class="diffchange diffchange-inline">which is not a unique idea. Except </del>we wanted <del class="diffchange diffchange-inline">our systems key attribute </del>to <del class="diffchange diffchange-inline">be specificity. To do so, we needed to find </del>a <del class="diffchange diffchange-inline">unique quorum sensing language </del>that <del class="diffchange diffchange-inline">we </del>could <del class="diffchange diffchange-inline">hijack and utilize</del>. <del class="diffchange diffchange-inline">We found the Yen system, from <i>Yersinia </del>enterocolitica<del class="diffchange diffchange-inline"></i></del>. <del class="diffchange diffchange-inline">A homologous sytem to </del>the <del class="diffchange diffchange-inline">well known Lux system</del>, <del class="diffchange diffchange-inline">from <i>Vibrio fischeri</i></del>.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p class="box_text"><ins class="diffchange diffchange-inline">To maximize the specificity </ins>in <ins class="diffchange diffchange-inline">targeting Yersinia enterocolitica</ins>, we wanted to <ins class="diffchange diffchange-inline">design a construct consisting of </ins>a <ins class="diffchange diffchange-inline">membrane protein complex </ins>that <ins class="diffchange diffchange-inline">would bind to the surface of the pathogen. This </ins>could <ins class="diffchange diffchange-inline">be done by fusing an anchor protein with the colicin Fy that aimed at the membrane of Y</ins>. enterocolitica. <ins class="diffchange diffchange-inline">This complex would be expressed on </ins>the <ins class="diffchange diffchange-inline">surface of our probiotic</ins>, <ins class="diffchange diffchange-inline">making it adhere to its target</ins>.</p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></a></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></a></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></div></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></div></div></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><h2 class="overview">System design</h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2 class="overview">System design</h2></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><p class="box_text"><del class="diffchange diffchange-inline">From </del>the <del class="diffchange diffchange-inline">Yen system </del>we <del class="diffchange diffchange-inline">stole the activator YenR and the recognition region, the yenbox, </del>fused with a <del class="diffchange diffchange-inline">wild type </del>promoter<del class="diffchange diffchange-inline">. YenR was synthesized with the </del>RBS B0034 <del class="diffchange diffchange-inline">and codon optimized for <i>E</del>. <del class="diffchange diffchange-inline">coli</i>. A customized version </del>of the <del class="diffchange diffchange-inline">yenbox-promoter was also created</del>.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p class="box_text"><ins class="diffchange diffchange-inline">We made two constructs, using a synthesized gene coding for pgsA. In </ins>the <ins class="diffchange diffchange-inline">first one </ins>we fused <ins class="diffchange diffchange-inline">pgsA </ins>with <ins class="diffchange diffchange-inline">colicin Fy and added </ins>a promoter <ins class="diffchange diffchange-inline">aswell as a </ins>RBS <ins class="diffchange diffchange-inline">called </ins>B0034. <ins class="diffchange diffchange-inline">In the other, BFP was put instead </ins>of <ins class="diffchange diffchange-inline">colicin Fy to test </ins>the <ins class="diffchange diffchange-inline">expression of pgsA</ins>.</p></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: #eee; color:black; font-size: smaller;"><div></a></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></a></div></td></tr>
<tr><td colspan="2" class="diff-lineno">Line 29:</td>
<td colspan="2" class="diff-lineno">Line 29:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2 class="overview">Result</h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2 class="overview">Result</h2></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><p class="box_text"><del class="diffchange diffchange-inline">We managed to get a five</del>-<del class="diffchange diffchange-inline">fold induction relative to the base level of expression</del>. <del class="diffchange diffchange-inline">The cells that </del>were <del class="diffchange diffchange-inline">used during the measurements were double transformed </del>with the <del class="diffchange diffchange-inline">yenbox</del>-<del class="diffchange diffchange-inline">promoter complex with GFP and a plasmid producing the activator YenR</del>.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p class="box_text"><ins class="diffchange diffchange-inline">Mutagenesis of B0034</ins>-<ins class="diffchange diffchange-inline">BFP into RFC25 was successful, verified by sequencing</ins>. <ins class="diffchange diffchange-inline">All other constructs </ins>were <ins class="diffchange diffchange-inline">not successful. Mainly because we had trouble </ins>with the <ins class="diffchange diffchange-inline">RFC25</ins>-<ins class="diffchange diffchange-inline">assemblies. The removal of TACTAG was also not successful</ins>.</p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></a></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></a></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></div></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></div></div></td></tr>
</table>StephyHehttp://2014.igem.org/wiki/index.php?title=Team:Uppsala/Project_Adhesion&diff=258241&oldid=prevAmaron at 23:30, 15 October 20142014-10-15T23:30:40Z<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 23:30, 15 October 2014</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 84:</td>
<td colspan="2" class="diff-lineno">Line 84:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><div class="prev"></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><div class="prev"></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><a href="https://2014.igem.org/Team:Uppsala/<del class="diffchange diffchange-inline">Project_Targeting</del>"><img class="prev_pic" src="https://static.igem.org/mediawiki/2014/8/81/Uppsala-igem2014-Prev.jpg"></a></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><a href="https://2014.igem.org/Team:Uppsala/<ins class="diffchange diffchange-inline">Project_Killing</ins>"><img class="prev_pic" src="https://static.igem.org/mediawiki/2014/8/81/Uppsala-igem2014-Prev.jpg"></a></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: #eee; color:black; font-size: smaller;"><div></div></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></div></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 class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><div class="next"></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><div class="next"></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 93:</td>
<td colspan="2" class="diff-lineno">Line 94:</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></div></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></div></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">--></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></div></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></div></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>
</table>Amaron