http://2014.igem.org/wiki/index.php?title=Team:WPI-Worcester/Proof-of-Principle&feed=atom&action=historyTeam:WPI-Worcester/Proof-of-Principle - Revision history2024-03-29T02:23:16ZRevision history for this page on the wikiMediaWiki 1.16.5http://2014.igem.org/wiki/index.php?title=Team:WPI-Worcester/Proof-of-Principle&diff=365754&oldid=prevCllajeunesse at 23:07, 17 October 20142014-10-17T23:07:10Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h9>Agglutination Assay</h9></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h9>Agglutination Assay</h9></p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>For our proof of principle agglutination assay, we wanted to test if antigens expressed on the cell surface, in this case YFP antigens, would bind to their corresponding antibody and produce visible agglutination. In order to do this, we combined in a well plate GFP antibodies (which is analogous to YFP antibodies) with <i> E.coli</i> expressing YFP externally through the use of both INP and BclA. As a control, we also mixed GFP antibodies with <i> E.coli</i> internally expressed GFP as well as <i>E.coli</i> externally expressing the CAEV antigen using our biobrick(<a href="http://parts.igem.org/Part:BBa_K1423004">BBa_K1423004)</a>. </p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>For our proof of principle agglutination assay, we wanted to test if antigens expressed on the cell surface, in this case YFP antigens, would bind to their corresponding antibody and produce visible agglutination. In order to do this, we <ins class="diffchange diffchange-inline">based our protocol off of <a href="http://www.plosntds.org/article/info%3Adoi%2F10.1371%2Fjournal.pntd.0001946">this</a> paper. We </ins>combined in a well plate GFP antibodies (which is analogous to YFP antibodies) with <i> E.coli</i> expressing YFP externally through the use of both INP and BclA. As a control, we also mixed GFP antibodies with <i> E.coli</i> internally expressed GFP as well as <i>E.coli</i> externally expressing the CAEV antigen using our biobrick(<a href="http://parts.igem.org/Part:BBa_K1423004">BBa_K1423004)</a>. </p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>If our agglutination experiment is successful, we would expect agglutination in the wells with <i>E.coli</i> externally expressing YFP (using BCLA and INP). We would not expect agglutination in the well with <i> E.coli</i> internally expressing GFP because the GFP is internal, meaning the extracellular antigen cannot bind to it. We would also expect no agglutination in the well containing <i>E.coli</i> externally expressing the CAEV antigen because that is the incorrect antigen, and it is not recognized by the GFP antibodies. </p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>If our agglutination experiment is successful, we would expect agglutination in the wells with <i>E.coli</i> externally expressing YFP (using BCLA and INP). We would not expect agglutination in the well with <i> E.coli</i> internally expressing GFP because the GFP is internal, meaning the extracellular antigen cannot bind to it. We would also expect no agglutination in the well containing <i>E.coli</i> externally expressing the CAEV antigen because that is the incorrect antigen, and it is not recognized by the GFP antibodies. </p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>It is important to first establish how to interpret the results of our agglutination assay. When agglutination occurs, antibodies will bind to their matching antigen pairs, creating a mat on the bottom of the well. This is the case because each antibody has two antigen binding sites, meaning it can bind to more than one antigen and form an interwoven antigen-antibody complex. No agglutination, on the other hand, would manifest as a small dot at the bottom of the well, because no interwoven antigen-antibody complexes were formed and the cells just clumped to the bottom due to gravity. A schematic showing the differences between a positive agglutination and a negative agglutination result can be seen below. </p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>It is important to first establish how to interpret the results of our agglutination assay. When agglutination occurs, antibodies will bind to their matching antigen pairs, creating a mat on the bottom of the well. This is the case because each antibody has two antigen binding sites, meaning it can bind to more than one antigen and form an interwoven antigen-antibody complex. No agglutination, on the other hand, would manifest as a small dot at the bottom of the well, because no interwoven antigen-antibody complexes were formed and the cells just clumped to the bottom due to gravity. A schematic showing the differences between a positive agglutination and a negative agglutination result can be seen below. </p></div></td></tr>
</table>Cllajeunessehttp://2014.igem.org/wiki/index.php?title=Team:WPI-Worcester/Proof-of-Principle&diff=364043&oldid=prevCllajeunesse at 22:49, 17 October 20142014-10-17T22:49:41Z<p></p>
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</table>Cllajeunessehttp://2014.igem.org/wiki/index.php?title=Team:WPI-Worcester/Proof-of-Principle&diff=363583&oldid=prevAdturland at 22:45, 17 October 20142014-10-17T22:45:27Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href="https://2014.igem.org/Team:WPI-Worcester/Team">Bios</a></li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href="https://2014.igem.org/Team:WPI-Worcester/Team">Bios</a></li></div></td></tr>
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</table>Adturlandhttp://2014.igem.org/wiki/index.php?title=Team:WPI-Worcester/Proof-of-Principle&diff=362830&oldid=prevAdturland at 22:37, 17 October 20142014-10-17T22:37:52Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><center><img src="https://static.igem.org/mediawiki/2014/thumb/c/cd/AggLine.png/800px-AggLine.png"/></center></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><center><img src="https://static.igem.org/mediawiki/2014/thumb/c/cd/AggLine.png/800px-AggLine.png"/></center></p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><center><img src="https://static.igem.org/mediawiki/2014/8/88/Wpi_2014_agglutination_result_example.png"/></center></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><center><img src="https://static.igem.org/mediawiki/2014/8/88/Wpi_2014_agglutination_result_example.png"/></center></p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p><center><h3><b>Figure 5</b>: Agglutination Schematic- An unmatching antibody on top and a matching antibody on the bottom.</h3></center></p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p><center><h3><b>Figure 5</b>: Agglutination Schematic- An unmatching antibody on top and a matching antibody <ins class="diffchange diffchange-inline">below that; with the final results </ins>on the bottom<ins class="diffchange diffchange-inline">. The final results are a top view of the containing wells</ins>. </h3></center></p></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>The actual results of our assay can be seen below in <b>Figure 6</b>. It can be seen that the wells with cellular surface expression of GFP (the bottom two rows) were positive (a mat) as expected while the controls (the top two rows) were not agglutinated and had a dot of cells in the bottom of the well. This proves that it is possible to see a visual confirmation of agglutination. <p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>The actual results of our assay can be seen below in <b>Figure 6</b>. It can be seen that the wells with cellular surface expression of GFP (the bottom two rows) were positive (a mat) as expected while the controls (the top two rows) were not agglutinated and had a dot of cells in the bottom of the well. This proves that it is possible to see a visual confirmation of agglutination. <p></div></td></tr>
</table>Adturlandhttp://2014.igem.org/wiki/index.php?title=Team:WPI-Worcester/Proof-of-Principle&diff=362703&oldid=prevAdturland at 22:36, 17 October 20142014-10-17T22:36:22Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p><center><h3><b>Figure 5</b>: Agglutination Schematic<del class="diffchange diffchange-inline">: </del> An unmatching antibody on top and a matching antibody on the bottom.</h3></center></p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p><center><h3><b>Figure 5</b>: Agglutination Schematic<ins class="diffchange diffchange-inline">- </ins> An unmatching antibody on top and a matching antibody on the bottom.</h3></center></p></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>The actual results of our assay can be seen below in <b>Figure 6</b>. It can be seen that the wells with cellular surface expression of GFP (the bottom two rows) were positive (a mat) as expected while the controls (the top two rows) were not agglutinated and had a dot of cells in the bottom of the well. This proves that it is possible to see a visual confirmation of agglutination. <p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>The actual results of our assay can be seen below in <b>Figure 6</b>. It can be seen that the wells with cellular surface expression of GFP (the bottom two rows) were positive (a mat) as expected while the controls (the top two rows) were not agglutinated and had a dot of cells in the bottom of the well. This proves that it is possible to see a visual confirmation of agglutination. <p></div></td></tr>
</table>Adturlandhttp://2014.igem.org/wiki/index.php?title=Team:WPI-Worcester/Proof-of-Principle&diff=362674&oldid=prevAdturland at 22:35, 17 October 20142014-10-17T22:35:55Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><center><img src="https://static.igem.org/mediawiki/2014/8/88/Wpi_2014_agglutination_result_example.png"/></center></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><center><img src="https://static.igem.org/mediawiki/2014/8/88/Wpi_2014_agglutination_result_example.png"/></center></p></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"><p><center><h3><b>Figure 5</b>: Agglutination Schematic: An unmatching antibody on top and a matching antibody on the bottom.</h3></center></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><p>The actual results of our assay can be seen below in <b>Figure <del class="diffchange diffchange-inline">5</del></b>. It can be seen that the wells with cellular surface expression of GFP (the bottom two rows) were positive (a mat) as expected while the controls (the top two rows) were not agglutinated and had a dot of cells in the bottom of the well. This proves that it is possible to see a visual confirmation of agglutination. <p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>The actual results of our assay can be seen below in <b>Figure <ins class="diffchange diffchange-inline">6</ins></b>. It can be seen that the wells with cellular surface expression of GFP (the bottom two rows) were positive (a mat) as expected while the controls (the top two rows) were not agglutinated and had a dot of cells in the bottom of the well. This proves that it is possible to see a visual confirmation of agglutination. <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><p><center><img src="https://static.igem.org/mediawiki/2014/7/75/WPI_AgglutinationResults.png"/></center></p><p><center><h3><b>Figure <del class="diffchange diffchange-inline">5</del></b>: Agglutination Assay Results</h3></center></p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p><center><img src="https://static.igem.org/mediawiki/2014/7/75/WPI_AgglutinationResults.png"/></center></p><p><center><h3><b>Figure <ins class="diffchange diffchange-inline">6</ins></b>: Agglutination Assay Results</h3></center></p></div></td></tr>
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</table>Adturlandhttp://2014.igem.org/wiki/index.php?title=Team:WPI-Worcester/Proof-of-Principle&diff=362257&oldid=prevAdturland at 22:31, 17 October 20142014-10-17T22:31:18Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>For our proof of principle agglutination assay, we wanted to test if antigens expressed on the cell surface, in this case YFP antigens, would bind to their corresponding antibody and produce visible agglutination. In order to do this, we combined in a well plate GFP antibodies (which is analogous to YFP antibodies) with <i> E.coli</i> expressing YFP externally through the use of both INP and BclA. As a control, we also mixed GFP antibodies with <i> E.coli</i> internally expressed GFP as well as <i>E.coli</i> externally expressing the CAEV antigen using our biobrick(<a href="http://parts.igem.org/Part:BBa_K1423004">BBa_K1423004)</a>. </p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>For our proof of principle agglutination assay, we wanted to test if antigens expressed on the cell surface, in this case YFP antigens, would bind to their corresponding antibody and produce visible agglutination. In order to do this, we combined in a well plate GFP antibodies (which is analogous to YFP antibodies) with <i> E.coli</i> expressing YFP externally through the use of both INP and BclA. As a control, we also mixed GFP antibodies with <i> E.coli</i> internally expressed GFP as well as <i>E.coli</i> externally expressing the CAEV antigen using our biobrick(<a href="http://parts.igem.org/Part:BBa_K1423004">BBa_K1423004)</a>. </p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>If our agglutination experiment is successful, we would expect agglutination in the wells with <i>E.coli</i> externally expressing YFP (using BCLA and INP). We would not expect agglutination in the well with <i> E.coli</i> internally expressing GFP because the GFP is internal, meaning the extracellular antigen cannot bind to it. We would also expect no agglutination in the well containing <i>E.coli</i> externally expressing the CAEV antigen because that is the incorrect antigen, and it is not recognized by the GFP antibodies. </p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>If our agglutination experiment is successful, we would expect agglutination in the wells with <i>E.coli</i> externally expressing YFP (using BCLA and INP). We would not expect agglutination in the well with <i> E.coli</i> internally expressing GFP because the GFP is internal, meaning the extracellular antigen cannot bind to it. We would also expect no agglutination in the well containing <i>E.coli</i> externally expressing the CAEV antigen because that is the incorrect antigen, and it is not recognized by the GFP antibodies. </p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>It is important to first establish how to interpret the results of our agglutination assay. When agglutination occurs, antibodies will bind to their matching antigen pairs, creating a mat on the bottom of the well. This is the case because each antibody has two antigen binding sites, meaning it can bind to more than one antigen and form an interwoven antigen-antibody complex. No agglutination, on the other hand, would manifest as a small dot at the bottom of the well, because no interwoven antigen-antibody complexes were formed and the cells just clumped to the bottom due to gravity. A schematic showing the <del class="diffchange diffchange-inline">difference </del>between a positive agglutination <del class="diffchange diffchange-inline">well </del>and a negative agglutination <del class="diffchange diffchange-inline">well </del>result can be seen below. </p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>It is important to first establish how to interpret the results of our agglutination assay. When agglutination occurs, antibodies will bind to their matching antigen pairs, creating a mat on the bottom of the well. This is the case because each antibody has two antigen binding sites, meaning it can bind to more than one antigen and form an interwoven antigen-antibody complex. No agglutination, on the other hand, would manifest as a small dot at the bottom of the well, because no interwoven antigen-antibody complexes were formed and the cells just clumped to the bottom due to gravity. A schematic showing the <ins class="diffchange diffchange-inline">differences </ins>between a positive agglutination and a negative agglutination result can be seen below. <ins class="diffchange diffchange-inline"></p></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><p><center><img src="https://static.igem.org/mediawiki/2014/thumb/c/cd/AggLine.png/800px-AggLine.png"/></center></ins></p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><center><img src="https://static.igem.org/mediawiki/2014/8/88/Wpi_2014_agglutination_result_example.png"/></center></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><center><img src="https://static.igem.org/mediawiki/2014/8/88/Wpi_2014_agglutination_result_example.png"/></center></p></div></td></tr>
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</table>Adturlandhttp://2014.igem.org/wiki/index.php?title=Team:WPI-Worcester/Proof-of-Principle&diff=361453&oldid=prevSmhenry at 22:22, 17 October 20142014-10-17T22:22:27Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h9>Agglutination Assay</h9></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h9>Agglutination Assay</h9></p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>For our proof of principle agglutination assay, we wanted to test if antigens expressed on the cell surface, in this case YFP antigens, would bind to their corresponding antibody and produce visible agglutination. In order to do this, we combined in a well plate GFP antibodies (which is analogous to YFP antibodies) with <i> E.coli</i> expressing YFP externally through the use of both INP and BclA. As a control, we also mixed GFP antibodies with <i> E.coli</i> internally expressed GFP as well as <i>E.coli</i> externally expressing the CAEV antigen using our biobrick(<a href="http://parts.igem.org/Part:BBa_K1423004">BBa_K1423004)</a>. </p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>For our proof of principle agglutination assay, we wanted to test if antigens expressed on the cell surface, in this case YFP antigens, would bind to their corresponding antibody and produce visible agglutination. In order to do this, we combined in a well plate GFP antibodies (which is analogous to YFP antibodies) with <i> E.coli</i> expressing YFP externally through the use of both INP and BclA. As a control, we also mixed GFP antibodies with <i> E.coli</i> internally expressed GFP as well as <i>E.coli</i> externally expressing the CAEV antigen using our biobrick(<a href="http://parts.igem.org/Part:BBa_K1423004">BBa_K1423004)</a>. </p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>If our agglutination experiment is successful, we would expect agglutination in the wells with <i>E.coli</i> externally expressing YFP (using BCLA and INP). We would not expect agglutination in the well with <i> E.coli</i> internally expressing GFP because the GFP is internal, meaning the extracellular antigen cannot bind to it. We would also expect no agglutination in the well containing <i>E.coli</i> externally expressing the CAEV antigen because that is the incorrect antigen, and it <del class="diffchange diffchange-inline">will </del>not <del class="diffchange diffchange-inline">be </del>recognized by the GFP antibodies. </p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>If our agglutination experiment is successful, we would expect agglutination in the wells with <i>E.coli</i> externally expressing YFP (using BCLA and INP). We would not expect agglutination in the well with <i> E.coli</i> internally expressing GFP because the GFP is internal, meaning the extracellular antigen cannot bind to it. We would also expect no agglutination in the well containing <i>E.coli</i> externally expressing the CAEV antigen because that is the incorrect antigen, and it <ins class="diffchange diffchange-inline">is </ins>not recognized by the GFP antibodies. </p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>It is important to first establish how to interpret the results of our agglutination assay. When agglutination occurs, antibodies will bind to their matching antigen pairs, creating a mat on the bottom of the well. This is the case because each antibody has two antigen binding sites, meaning it can bind to more than one antigen and form an interwoven antigen-antibody complex. No agglutination, on the other hand, would manifest as a small dot at the bottom of the well, because no interwoven antigen-antibody complexes were formed and the cells just clumped to the bottom due to gravity. A schematic showing the difference between a positive agglutination well and a negative agglutination well result can be seen below. </p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>It is important to first establish how to interpret the results of our agglutination assay. When agglutination occurs, antibodies will bind to their matching antigen pairs, creating a mat on the bottom of the well. This is the case because each antibody has two antigen binding sites, meaning it can bind to more than one antigen and form an interwoven antigen-antibody complex. No agglutination, on the other hand, would manifest as a small dot at the bottom of the well, because no interwoven antigen-antibody complexes were formed and the cells just clumped to the bottom due to gravity. A schematic showing the difference between a positive agglutination well and a negative agglutination well result can be seen below. </p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><center><img src="https://static.igem.org/mediawiki/2014/8/88/Wpi_2014_agglutination_result_example.png"/></center></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><center><img src="https://static.igem.org/mediawiki/2014/8/88/Wpi_2014_agglutination_result_example.png"/></center></p></div></td></tr>
</table>Smhenryhttp://2014.igem.org/wiki/index.php?title=Team:WPI-Worcester/Proof-of-Principle&diff=361297&oldid=prevSmhenry at 22:20, 17 October 20142014-10-17T22:20:47Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><h9>Western Blot</h9></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><h9>Western Blot</h9></p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>We attempted another proof of principle experiment, in order to support the fact that BCLA causes cellular surface expression of proteins. To do this, we performed a fractionation experiment followed by a Western Blot. We were doing the fractionation to isolate the membrane bound proteins from the cytoplasmic proteins. We then performed a Western Blot using a GFP antibody. Our results suggest that their might be some localization of YFP to the cell surface membrane using the BclA-YFP construct. However, the results were not conclusive. We believe that the <b>Figure <del class="diffchange diffchange-inline">1</del></b> below shows the localization of the YFP to the membrane. This is the case because in the BclA-YFP well in the membrane bound protein lane there was an extremely high expression on the blot. This shows that the YFP is being localized to the cell surface on the biochemical level. When performing the Western Blot, it appeared that the wells were loaded evenly but a loading control was not used which is part of the reason we concede these results to be mildly inconclusive. We still feel the results of this Western Blot adds extra support to the efficiency of BclA cell surface protein localization when combined with our microscopy results.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>We attempted another proof of principle experiment, in order to support the fact that BCLA causes cellular surface expression of proteins. To do this, we performed a fractionation experiment followed by a Western Blot. We were doing the fractionation to isolate the membrane bound proteins from the cytoplasmic proteins. We then performed a Western Blot using a GFP antibody. Our results suggest that their might be some localization of YFP to the cell surface membrane using the BclA-YFP construct. However, the results were not conclusive. We believe that the <b>Figure <ins class="diffchange diffchange-inline">4</ins></b> below shows the localization of the YFP to the membrane. This is the case because in the BclA-YFP well in the membrane bound protein lane there was an extremely high expression on the blot. This shows that the YFP is being localized to the cell surface on the biochemical level. When performing the Western Blot, it appeared that the wells were loaded evenly but a loading control was not used which is part of the reason we concede these results to be mildly inconclusive. We still feel the results of this Western Blot adds extra support to the efficiency of BclA cell surface protein localization when combined with our microscopy results.</p></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><center><img src="https://static.igem.org/mediawiki/2014/e/ea/WPI_Biochemical_analysis.png"/></center></p><p></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><center><img src="https://static.igem.org/mediawiki/2014/e/ea/WPI_Biochemical_analysis.png"/></center></p><p></p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p><p><center><h3><b>Figure <del class="diffchange diffchange-inline">1</del></b>: Cell Fractionation and Western Blot Results</h3></center></p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p><p><center><h3><b>Figure <ins class="diffchange diffchange-inline">4</ins></b>: Cell Fractionation and Western Blot Results</h3></center></p></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h9>Agglutination Assay</h9></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h9>Agglutination Assay</h9></p></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><center><img src="https://static.igem.org/mediawiki/2014/8/88/Wpi_2014_agglutination_result_example.png"/></center></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><center><img src="https://static.igem.org/mediawiki/2014/8/88/Wpi_2014_agglutination_result_example.png"/></center></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><p>The actual results of our assay can be seen below in <b>Figure <del class="diffchange diffchange-inline">1</del></b>. It can be seen that the wells with cellular surface expression of GFP (the bottom two rows) were positive (a mat) as expected while the controls (the top two rows) were not agglutinated and had a dot of cells in the bottom of the well. This proves that it is possible to see a visual confirmation of agglutination. <p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>The actual results of our assay can be seen below in <b>Figure <ins class="diffchange diffchange-inline">5</ins></b>. It can be seen that the wells with cellular surface expression of GFP (the bottom two rows) were positive (a mat) as expected while the controls (the top two rows) were not agglutinated and had a dot of cells in the bottom of the well. This proves that it is possible to see a visual confirmation of agglutination. <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><p><center><img src="https://static.igem.org/mediawiki/2014/7/75/WPI_AgglutinationResults.png"/></center></<del class="diffchange diffchange-inline">p></del>p><p><center><h3><b>Figure <del class="diffchange diffchange-inline">1</del></b>: Agglutination Assay Results</h3></center></p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p><center><img src="https://static.igem.org/mediawiki/2014/7/75/WPI_AgglutinationResults.png"/></center></p><p><center><h3><b>Figure <ins class="diffchange diffchange-inline">5</ins></b>: Agglutination Assay Results</h3></center></p></div></td></tr>
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</table>Smhenryhttp://2014.igem.org/wiki/index.php?title=Team:WPI-Worcester/Proof-of-Principle&diff=361179&oldid=prevSmhenry at 22:19, 17 October 20142014-10-17T22:19:34Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><p><center><h3><b>Figure 1</b>: Cell Fractionation and Western Blot Results</h3></center></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><p><center><h3><b>Figure 1</b>: Cell Fractionation and Western Blot Results</h3></center></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><del class="diffchange diffchange-inline"><p></del><h9>Agglutination Assay</h9></p><p><del class="diffchange diffchange-inline">We were able </del>to <del class="diffchange diffchange-inline">successfully achieve </del>the <del class="diffchange diffchange-inline">results </del>we <del class="diffchange diffchange-inline">wanted </del>with our agglutination <del class="diffchange diffchange-inline">assay</del>. <del class="diffchange diffchange-inline">When antibodies are </del>in the <del class="diffchange diffchange-inline">presence </del>of their matching antigen pairs, <del class="diffchange diffchange-inline">they will bind to each other</del>. <del class="diffchange diffchange-inline"> Each </del>antibody can bind to more than one antigen <del class="diffchange diffchange-inline">because they have two antigen binding sites. This means when there is a large number of </del>antigen-antibody <del class="diffchange diffchange-inline">binding a </del>complex <del class="diffchange diffchange-inline">will form</del>. <del class="diffchange diffchange-inline">This network manifests </del>on <del class="diffchange diffchange-inline">a large scale </del>as a <del class="diffchange diffchange-inline">mat of bacteria on </del>the bottom of the <del class="diffchange diffchange-inline">containing vessel</del>, <del class="diffchange diffchange-inline">as opposed </del>to a <del class="diffchange diffchange-inline">solid dot formed by unagglutinated antibodies</del>. <del class="diffchange diffchange-inline">We based our assay off of </del><<del class="diffchange diffchange-inline">a href</del>="<del class="diffchange diffchange-inline">http</del>://<del class="diffchange diffchange-inline">www</del>.<del class="diffchange diffchange-inline">plosntds</del>.org/<del class="diffchange diffchange-inline">article</del>/<del class="diffchange diffchange-inline">info%3Adoi%2F10</del>.<del class="diffchange diffchange-inline">1371%2Fjournal.pntd.0001946</del>"><del class="diffchange diffchange-inline">this paper</del></<del class="diffchange diffchange-inline">a</del>><del class="diffchange diffchange-inline">.</del></p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><h9>Agglutination Assay</h9></p></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p><ins class="diffchange diffchange-inline">For our proof of principle agglutination assay, we wanted </ins>to <ins class="diffchange diffchange-inline">test if antigens expressed on </ins>the <ins class="diffchange diffchange-inline">cell surface, in this case YFP antigens, would bind to their corresponding antibody and produce visible agglutination. In order to do this, </ins>we <ins class="diffchange diffchange-inline">combined in a well plate GFP antibodies (which is analogous to YFP antibodies) </ins>with <ins class="diffchange diffchange-inline"><i> E.coli</i> expressing YFP externally through the use of both INP and BclA. As a control, we also mixed GFP antibodies with <i> E.coli</i> internally expressed GFP as well as <i>E.coli</i> externally expressing the CAEV antigen using our biobrick(<a href="http://parts.igem.org/Part:BBa_K1423004">BBa_K1423004)</a>. </p></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><p>If </ins>our agglutination <ins class="diffchange diffchange-inline">experiment is successful, we would expect agglutination in the wells with <i>E</ins>.<ins class="diffchange diffchange-inline">coli</i> externally expressing YFP (using BCLA and INP). We would not expect agglutination </ins>in the <ins class="diffchange diffchange-inline">well with <i> E.coli</i> internally expressing GFP because the GFP is internal, meaning the extracellular antigen cannot bind to it. We would also expect no agglutination in the well containing <i>E.coli</i> externally expressing the CAEV antigen because that is the incorrect antigen, and it will not be recognized by the GFP antibodies. </p></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><p>It is important to first establish how to interpret the results </ins>of <ins class="diffchange diffchange-inline">our agglutination assay. When agglutination occurs, antibodies will bind to </ins>their matching antigen pairs, <ins class="diffchange diffchange-inline">creating a mat on the bottom of the well</ins>. <ins class="diffchange diffchange-inline">This is the case because each </ins>antibody <ins class="diffchange diffchange-inline">has two antigen binding sites, meaning it </ins>can bind to more than one antigen <ins class="diffchange diffchange-inline">and form an interwoven </ins>antigen-antibody complex. <ins class="diffchange diffchange-inline">No agglutination, </ins>on <ins class="diffchange diffchange-inline">the other hand, would manifest </ins>as a <ins class="diffchange diffchange-inline">small dot at </ins>the bottom of the <ins class="diffchange diffchange-inline">well</ins>, <ins class="diffchange diffchange-inline">because no interwoven antigen-antibody complexes were formed and the cells just clumped </ins>to <ins class="diffchange diffchange-inline">the bottom due to gravity. A schematic showing the difference between </ins>a <ins class="diffchange diffchange-inline">positive agglutination well and a negative agglutination well result can be seen below</ins>. <<ins class="diffchange diffchange-inline">/p></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><p><center><img src</ins>="<ins class="diffchange diffchange-inline">https</ins>://<ins class="diffchange diffchange-inline">static</ins>.<ins class="diffchange diffchange-inline">igem</ins>.org/<ins class="diffchange diffchange-inline">mediawiki</ins>/<ins class="diffchange diffchange-inline">2014/8/88/Wpi_2014_agglutination_result_example</ins>.<ins class="diffchange diffchange-inline">png</ins>"<ins class="diffchange diffchange-inline">/</ins>></<ins class="diffchange diffchange-inline">center</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: #ffa; color:black; font-size: smaller;"><div><p><del class="diffchange diffchange-inline">For our proof </del>of <del class="diffchange diffchange-inline">principle agglutination, we used surface-bound YFP (Yellow Fluorescent Protein) and a corresponding GFP (Green Fluorescent Protein)</del>/<del class="diffchange diffchange-inline">YFP antibody (the part of the protein </del>that the <del class="diffchange diffchange-inline">antibody binds to is not affected by the part that causes the color change.) We used both of the </del>surface expression <del class="diffchange diffchange-inline">proteins we had at our disposal: BclA </del>(the <del class="diffchange diffchange-inline">one we made</del>) <del class="diffchange diffchange-inline">and INP </del>(a <del class="diffchange diffchange-inline">pre-existing biobrick</del>)<del class="diffchange diffchange-inline">. As controls, we used internally expressed YFP </del>as <del class="diffchange diffchange-inline">well as a protein that was externally expressed but did not match with </del>the <del class="diffchange diffchange-inline">antibody </del>(<del class="diffchange diffchange-inline">we used </del>the <del class="diffchange diffchange-inline">CAEV protein we made</del>).</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p><ins class="diffchange diffchange-inline">The actual results </ins>of <ins class="diffchange diffchange-inline">our assay can be seen below in <b>Figure 1<</ins>/<ins class="diffchange diffchange-inline">b>. It can be seen </ins>that the <ins class="diffchange diffchange-inline">wells with cellular </ins>surface expression <ins class="diffchange diffchange-inline">of GFP </ins>(the <ins class="diffchange diffchange-inline">bottom two rows</ins>) <ins class="diffchange diffchange-inline">were positive </ins>(a <ins class="diffchange diffchange-inline">mat</ins>) as <ins class="diffchange diffchange-inline">expected while </ins>the <ins class="diffchange diffchange-inline">controls </ins>(the <ins class="diffchange diffchange-inline">top two rows</ins>) <ins class="diffchange diffchange-inline">were not agglutinated and had a dot of cells in the bottom of the well</ins>. <ins class="diffchange diffchange-inline">This proves that it is possible to see a visual confirmation of agglutination. <p></ins></div></td></tr>
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<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><p><center><img src="https://static.igem.org/mediawiki/2014/7/75/WPI_AgglutinationResults.png"/></center></p>p><p><center><h3><b>Figure 1</b>: Agglutination Assay Results</h3></center></ins></p></div></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;"><p><center><img src="https://static.igem.org/mediawiki/2014/7/75/WPI_AgglutinationResults.png"/></center></p></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;"><p>This photo clearly shows that the wells containing the bacteria with the matching antigen on the surface formed agglutinated mats while the control wells did not agglutinate and formed dots at the bottom of the wells.</del></div></td><td colspan="2"> </td></tr>
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</table>Smhenry