http://2009.igem.org/wiki/index.php?title=Special:Contributions/Acleone&feed=atom&limit=50&target=Acleone&year=&month=2009.igem.org - User contributions [en]2024-03-29T11:30:29ZFrom 2009.igem.orgMediaWiki 1.16.5http://2009.igem.org/Team:Washington/Project/CDSTeam:Washington/Project/CDS2009-10-22T01:26:32Z<p>Acleone: /* Custom Display Vector with wild-type monomeric streptavidin inserted: */</p>
<hr />
<div>__NOTOC__<br />
{{Template:Team:Washington/Templates/Header}}<br />
<br />
<div><br />
<div style="float:right;">'''[[Team:Washington/Project/FoldIt|Fold-It &gt;]]'''</div><br />
<div>'''[[Team:Washington/Future|&lt; Future Directions]]'''</div><br />
</div><br />
<br />
===Custom Display System===<br />
====Problem====<br />
The underlying design of the streptavidin display constructs in the registry has the displayed protein bound close to the cell membrane. This constraint could be preventing streptavidin on the surface of the cell from forming tetramers lowering its effectiveness at binding biotin. Furthermore these existing display systems prevent the addition of another protein into them, preventing the user from displaying other proteins. <br />
<br />
====Idea====<br />
The first goal of making a new display vector was to incorporate a GS linker between the displayed protein and the OmpA protein anchoring in in the cell wall. However when reviewing the construct we decided to add some other useful features as well. Maintaining the Lpp tag (to direct the protein to the periplasm) and the OmpA trans-membrane regions (for anchoring the construct) of the original 2006 parts we added: <br />
#GS Linker (Gly4Ser)4 - allowing for more space between the protein and the cell wall<br />
#TEV - Tobacco Etch Virus (TEV) protease site allowing for cleavage of displayed proteins<br />
#NheI restriction site - allowing for the insert of '''any BioBrick protein''' into the display construct<br />
<br><br><br />
[[Image:PDS_design.png |600px |center]]<br />
<br><br><br />
<br />
====Current Status====<br />
<br />
===== Custom Display Vector: =====<br />
* 1 trans-membrane OmpA: [http://partsregistry.org/Part:BBa_K215200 BBa_K215200]<br />
* 5 trans-membrane OmpA: [http://partsregistry.org/Part:BBa_K215201 BBa_K215201]<br />
<br />
We submitted the Custom Display Vector to the registry as [http://partsregistry.org/Part:BBa_K215200 BBa_K215200] and [http://partsregistry.org/Part:BBa_K215201 BBa_K215201] (for 1 and 5 trans-membrane OmpA). We are currently inserting GFP into the display system to verify that the OmpA display system is working, but we will not have any data by the jamboree.<br />
<br />
===== Custom Display Vector with wild-type monomeric streptavidin inserted: =====<br />
* 1 trans-membrane OmpA: [http://partsregistry.org/Part:BBa_K215210 BBa_K215210] (equivalent to 2006 Harvard iGEM: [http://partsregistry.org/Part:BBa_J36848 BBa_J36848] with an extra linker between OmpA and the streptavidin)<br />
* 5 trans-membrane OmpA: [http://partsregistry.org/Part:BBa_K215211 BBa_K215211] (equivalent to 2006 Harvard iGEM: [http://partsregistry.org/Part:BBa_J36850 BBa_J36850] with the extra linker)<br />
<br />
We inserted wild-type monomeric streptavidin to see if the extra linker would make the streptavidin functional. These parts would be equivalent to the 2006 Harvard iGEM parts, [http://partsregistry.org/Part:BBa_J36848 BBa_J36848] and [http://partsregistry.org/Part:BBa_J36850 BBa_J36850], except with the extra linker and TEV protease site. These parts were submitted to the registry as [http://partsregistry.org/Part:BBa_K215210 BBa_K215210] and [http://partsregistry.org/Part:BBa_K215211 BBa_K215211]. We ran the same flow cytometry assays that we used for the Harvard parts - the streptavidin is non-functional (see [[Team:Washington/Project/Display|Display]] and [[Team:Washington/Notebook|Notebook]] - this is the same cytometry assay that we used for characterizing the 2006 Harvard parts):<br />
<br />
<table align="center"><br />
<tr align="center"><br />
<td>[[Image:New_generic_51.png|375px]]<br>1tmr OmpA, [http://partsregistry.org/Part:BBa_K215210 BBa_K215210]</td><br />
<td>[[Image:New_generic_58.png|375px]]<br>5tmr OmpA, [http://partsregistry.org/Part:BBa_K215211 BBa_K215211]</td><br />
</tr><br />
<tr align="center"><br />
<td colspan="2">''Cytometry showing no difference in bound biotin-conjugated fluorophore for 1mM IPTG induced vs uninduced cells at different fluorophore concentrations. Conclusion: Wild-type monomeric streptavidin shows no measureable activity when expressed as a fusion protein with 1 or 5 trans-membrane OmpA.''</td><br />
</tr><br />
</table><br />
<br />
----<br />
<br />
{{Template:Team:Washington/Templates/Footer}}</div>Acleonehttp://2009.igem.org/Team:Washington/Project/CDSTeam:Washington/Project/CDS2009-10-22T01:25:41Z<p>Acleone: /* Custom Display Vector with wild-type monomeric streptavidin inserted: */</p>
<hr />
<div>__NOTOC__<br />
{{Template:Team:Washington/Templates/Header}}<br />
<br />
<div><br />
<div style="float:right;">'''[[Team:Washington/Project/FoldIt|Fold-It &gt;]]'''</div><br />
<div>'''[[Team:Washington/Future|&lt; Future Directions]]'''</div><br />
</div><br />
<br />
===Custom Display System===<br />
====Problem====<br />
The underlying design of the streptavidin display constructs in the registry has the displayed protein bound close to the cell membrane. This constraint could be preventing streptavidin on the surface of the cell from forming tetramers lowering its effectiveness at binding biotin. Furthermore these existing display systems prevent the addition of another protein into them, preventing the user from displaying other proteins. <br />
<br />
====Idea====<br />
The first goal of making a new display vector was to incorporate a GS linker between the displayed protein and the OmpA protein anchoring in in the cell wall. However when reviewing the construct we decided to add some other useful features as well. Maintaining the Lpp tag (to direct the protein to the periplasm) and the OmpA trans-membrane regions (for anchoring the construct) of the original 2006 parts we added: <br />
#GS Linker (Gly4Ser)4 - allowing for more space between the protein and the cell wall<br />
#TEV - Tobacco Etch Virus (TEV) protease site allowing for cleavage of displayed proteins<br />
#NheI restriction site - allowing for the insert of '''any BioBrick protein''' into the display construct<br />
<br><br><br />
[[Image:PDS_design.png |600px |center]]<br />
<br><br><br />
<br />
====Current Status====<br />
<br />
===== Custom Display Vector: =====<br />
* 1 trans-membrane OmpA: [http://partsregistry.org/Part:BBa_K215200 BBa_K215200]<br />
* 5 trans-membrane OmpA: [http://partsregistry.org/Part:BBa_K215201 BBa_K215201]<br />
<br />
We submitted the Custom Display Vector to the registry as [http://partsregistry.org/Part:BBa_K215200 BBa_K215200] and [http://partsregistry.org/Part:BBa_K215201 BBa_K215201] (for 1 and 5 trans-membrane OmpA). We are currently inserting GFP into the display system to verify that the OmpA display system is working, but we will not have any data by the jamboree.<br />
<br />
===== Custom Display Vector with wild-type monomeric streptavidin inserted: =====<br />
* 1 trans-membrane OmpA: [http://partsregistry.org/Part:BBa_K215210 BBa_K215210] (equivalent to 2006 Harvard iGEM: [http://partsregistry.org/Part:BBa_J36848 BBa_J36848] with an extra linker between OmpA and the streptavidin)<br />
* 5 trans-membrane OmpA: [http://partsregistry.org/Part:BBa_K215211 BBa_K215211] (equivalent to 2006 Harvard iGEM: [http://partsregistry.org/Part:BBa_J36850 BBa_J36850] with the extra linker)<br />
<br />
We inserted wild-type monomeric streptavidin to see if the extra linker would make the streptavidin functional. These parts would be equivalent to the 2006 Harvard iGEM parts, [http://partsregistry.org/Part:BBa_J36848 BBa_J36848] and [http://partsregistry.org/Part:BBa_J36850 BBa_J36850], except with the extra linker and TEV protease site. These parts were submitted to the registry as [http://partsregistry.org/Part:BBa_K215210 BBa_K215210] and [http://partsregistry.org/Part:BBa_K215211 BBa_K215211]. We ran the same flow cytometry assays that we used for the Harvard parts - the streptavidin is non-functional (see [[Team:Washington/Project/Display|Display]] and [[Team:Washington/Notebook|Notebook]] - this is the same cytometry assay that we used for characterizing the 2006 Harvard parts):<br />
<br />
<table align="center"><br />
<tr align="center"><br />
<td>[[Image:New_generic_51.png|350px]]<br>1tmr OmpA, [http://partsregistry.org/Part:BBa_K215210 BBa_K215210]</td><br />
<td>[[Image:New_generic_58.png|350px]]<br>5tmr OmpA, [http://partsregistry.org/Part:BBa_K215211 BBa_K215211]</td><br />
</tr><br />
<tr align="center"><br />
<td colspan="2">''Cytometry showing no difference in bound biotin-conjugated fluorophore for 1mM IPTG induced vs uninduced cells at different fluorophore concentrations. Conclusion: Wild-type monomeric streptavidin shows no measureable activity when expressed as a fusion protein with 1 or 5 trans-membrane OmpA.''</td><br />
</tr><br />
</table><br />
<br />
----<br />
<br />
{{Template:Team:Washington/Templates/Footer}}</div>Acleonehttp://2009.igem.org/Team:Washington/Project/CDSTeam:Washington/Project/CDS2009-10-22T01:23:02Z<p>Acleone: /* Custom Display Vector with wild-type monomeric streptavidin inserted: */</p>
<hr />
<div>__NOTOC__<br />
{{Template:Team:Washington/Templates/Header}}<br />
<br />
<div><br />
<div style="float:right;">'''[[Team:Washington/Project/FoldIt|Fold-It &gt;]]'''</div><br />
<div>'''[[Team:Washington/Future|&lt; Future Directions]]'''</div><br />
</div><br />
<br />
===Custom Display System===<br />
====Problem====<br />
The underlying design of the streptavidin display constructs in the registry has the displayed protein bound close to the cell membrane. This constraint could be preventing streptavidin on the surface of the cell from forming tetramers lowering its effectiveness at binding biotin. Furthermore these existing display systems prevent the addition of another protein into them, preventing the user from displaying other proteins. <br />
<br />
====Idea====<br />
The first goal of making a new display vector was to incorporate a GS linker between the displayed protein and the OmpA protein anchoring in in the cell wall. However when reviewing the construct we decided to add some other useful features as well. Maintaining the Lpp tag (to direct the protein to the periplasm) and the OmpA trans-membrane regions (for anchoring the construct) of the original 2006 parts we added: <br />
#GS Linker (Gly4Ser)4 - allowing for more space between the protein and the cell wall<br />
#TEV - Tobacco Etch Virus (TEV) protease site allowing for cleavage of displayed proteins<br />
#NheI restriction site - allowing for the insert of '''any BioBrick protein''' into the display construct<br />
<br><br><br />
[[Image:PDS_design.png |600px |center]]<br />
<br><br><br />
<br />
====Current Status====<br />
<br />
===== Custom Display Vector: =====<br />
* 1 trans-membrane OmpA: [http://partsregistry.org/Part:BBa_K215200 BBa_K215200]<br />
* 5 trans-membrane OmpA: [http://partsregistry.org/Part:BBa_K215201 BBa_K215201]<br />
<br />
We submitted the Custom Display Vector to the registry as [http://partsregistry.org/Part:BBa_K215200 BBa_K215200] and [http://partsregistry.org/Part:BBa_K215201 BBa_K215201] (for 1 and 5 trans-membrane OmpA). We are currently inserting GFP into the display system to verify that the OmpA display system is working, but we will not have any data by the jamboree.<br />
<br />
===== Custom Display Vector with wild-type monomeric streptavidin inserted: =====<br />
* 1 trans-membrane OmpA: [http://partsregistry.org/Part:BBa_K215210 BBa_K215210] (equivalent to 2006 Harvard iGEM: [http://partsregistry.org/Part:BBa_J36848 BBa_J36848] with an extra linker between OmpA and the streptavidin)<br />
* 5 trans-membrane OmpA: [http://partsregistry.org/Part:BBa_K215211 BBa_K215211] (equivalent to 2006 Harvard iGEM: [http://partsregistry.org/Part:BBa_J36850 BBa_J36850] with the extra linker)<br />
<br />
We inserted wild-type monomeric streptavidin to see if the extra linker would make the streptavidin functional. These parts would be equivalent to the 2006 Harvard iGEM parts, [http://partsregistry.org/Part:BBa_J36848 BBa_J36848] and [http://partsregistry.org/Part:BBa_J36850 BBa_J36850], except with the extra linker and TEV protease site. These parts were submitted to the registry as [http://partsregistry.org/Part:BBa_K215210 BBa_K215210] and [http://partsregistry.org/Part:BBa_K215211 BBa_K215211]. We ran the same flow cytometry assays that we used for the Harvard parts - the streptavidin is non-functional (see [[Team:Washington/Project/Display|Display]] and [[Team:Washington/Notebook|Notebook]] - this is the same cytometry assay that we used for characterizing the 2006 Harvard parts):<br />
<br />
<table align="center"><tr align="center"><br />
<td colspan="2">'''Cytometry showing no difference in bound biotin-conjugated fluorophore for 1mM IPTG induced vs uninduced cells at different fluorophore concentrations'''</td></tr><br />
<tr align="center"><br />
<td>[[Image:New_generic_51.png|350px]]<br>1tmr OmpA, [http://partsregistry.org/Part:BBa_K215210 BBa_K215210]</td><br />
<td>[[Image:New_generic_58.png|350px]]<br>5tmr OmpA, [http://partsregistry.org/Part:BBa_K215211 BBa_K215211]</td><br />
</tr><br />
</table><br />
<br />
----<br />
<br />
{{Template:Team:Washington/Templates/Footer}}</div>Acleonehttp://2009.igem.org/File:New_generic_51.pngFile:New generic 51.png2009-10-22T01:22:45Z<p>Acleone: Cytometry data showing no difference in bound fluorescence for IPTG induced/uninduced samples for the new display construct with wild-type monomeric streptavidin and 1tmr OmpA.</p>
<hr />
<div>Cytometry data showing no difference in bound fluorescence for IPTG induced/uninduced samples for the new display construct with wild-type monomeric streptavidin and 1tmr OmpA.</div>Acleonehttp://2009.igem.org/File:New_generic_58.pngFile:New generic 58.png2009-10-22T01:22:31Z<p>Acleone: Cytometry data showing no difference in bound fluorescence for IPTG induced/uninduced samples for the new display construct with wild-type monomeric streptavidin and 5tmr OmpA.</p>
<hr />
<div>Cytometry data showing no difference in bound fluorescence for IPTG induced/uninduced samples for the new display construct with wild-type monomeric streptavidin and 5tmr OmpA.</div>Acleonehttp://2009.igem.org/Team:Washington/Project/CDSTeam:Washington/Project/CDS2009-10-22T01:20:53Z<p>Acleone: /* Custom Display Vector with wild-type monomeric streptavidin inserted: */</p>
<hr />
<div>__NOTOC__<br />
{{Template:Team:Washington/Templates/Header}}<br />
<br />
<div><br />
<div style="float:right;">'''[[Team:Washington/Project/FoldIt|Fold-It &gt;]]'''</div><br />
<div>'''[[Team:Washington/Future|&lt; Future Directions]]'''</div><br />
</div><br />
<br />
===Custom Display System===<br />
====Problem====<br />
The underlying design of the streptavidin display constructs in the registry has the displayed protein bound close to the cell membrane. This constraint could be preventing streptavidin on the surface of the cell from forming tetramers lowering its effectiveness at binding biotin. Furthermore these existing display systems prevent the addition of another protein into them, preventing the user from displaying other proteins. <br />
<br />
====Idea====<br />
The first goal of making a new display vector was to incorporate a GS linker between the displayed protein and the OmpA protein anchoring in in the cell wall. However when reviewing the construct we decided to add some other useful features as well. Maintaining the Lpp tag (to direct the protein to the periplasm) and the OmpA trans-membrane regions (for anchoring the construct) of the original 2006 parts we added: <br />
#GS Linker (Gly4Ser)4 - allowing for more space between the protein and the cell wall<br />
#TEV - Tobacco Etch Virus (TEV) protease site allowing for cleavage of displayed proteins<br />
#NheI restriction site - allowing for the insert of '''any BioBrick protein''' into the display construct<br />
<br><br><br />
[[Image:PDS_design.png |600px |center]]<br />
<br><br><br />
<br />
====Current Status====<br />
<br />
===== Custom Display Vector: =====<br />
* 1 trans-membrane OmpA: [http://partsregistry.org/Part:BBa_K215200 BBa_K215200]<br />
* 5 trans-membrane OmpA: [http://partsregistry.org/Part:BBa_K215201 BBa_K215201]<br />
<br />
We submitted the Custom Display Vector to the registry as [http://partsregistry.org/Part:BBa_K215200 BBa_K215200] and [http://partsregistry.org/Part:BBa_K215201 BBa_K215201] (for 1 and 5 trans-membrane OmpA). We are currently inserting GFP into the display system to verify that the OmpA display system is working, but we will not have any data by the jamboree.<br />
<br />
===== Custom Display Vector with wild-type monomeric streptavidin inserted: =====<br />
* 1 trans-membrane OmpA: [http://partsregistry.org/Part:BBa_K215210 BBa_K215210] (equivalent to 2006 Harvard iGEM: [http://partsregistry.org/Part:BBa_J36848 BBa_J36848] with an extra linker between OmpA and the streptavidin)<br />
* 5 trans-membrane OmpA: [http://partsregistry.org/Part:BBa_K215211 BBa_K215211] (equivalent to 2006 Harvard iGEM: [http://partsregistry.org/Part:BBa_J36850 BBa_J36850] with the extra linker)<br />
<br />
We inserted wild-type monomeric streptavidin to see if the extra linker would make the streptavidin functional. These parts would be equivalent to the 2006 Harvard iGEM parts, [http://partsregistry.org/Part:BBa_J36848 BBa_J36848] and [http://partsregistry.org/Part:BBa_J36850 BBa_J36850], except with the extra linker and TEV protease site. These parts were submitted to the registry as [http://partsregistry.org/Part:BBa_K215210 BBa_K215210] and [http://partsregistry.org/Part:BBa_K215211 BBa_K215211]. We ran the same flow cytometry assays that we used for the Harvard parts - the streptavidin is non-functional (see [[Team:Washington/Project/Display|Display]] and [[Team:Washington/Notebook|Notebook]] - this is the same cytometry assay that we used for characterizing the 2006 Harvard parts):<br />
<br />
<table align="center"><tr align="center"><br />
<td colspan="2">Cytometry showing no difference in bound biotin-conjugated fluorophore for 1mM IPTG induced vs uninduced cells at different fluorophore concentrations)</td></tr><br />
<tr align="center"><br />
<td>[Image:New_generic_51.png|350px]<br>1tmr OmpA, [http://partsregistry.org/Part:BBa_K215210 BBa_K215210]</td><br />
<td>[Image:New_generic_58.png|350px]<br>5tmr OmpA, [http://partsregistry.org/Part:BBa_K215211 BBa_K215211]</td><br />
</tr><br />
</table><br />
<br />
----<br />
<br />
{{Template:Team:Washington/Templates/Footer}}</div>Acleonehttp://2009.igem.org/Team:Washington/Project/CDSTeam:Washington/Project/CDS2009-10-22T01:19:13Z<p>Acleone: /* Custom Display Vector with wild-type monomeric streptavidin inserted: */</p>
<hr />
<div>__NOTOC__<br />
{{Template:Team:Washington/Templates/Header}}<br />
<br />
<div><br />
<div style="float:right;">'''[[Team:Washington/Project/FoldIt|Fold-It &gt;]]'''</div><br />
<div>'''[[Team:Washington/Future|&lt; Future Directions]]'''</div><br />
</div><br />
<br />
===Custom Display System===<br />
====Problem====<br />
The underlying design of the streptavidin display constructs in the registry has the displayed protein bound close to the cell membrane. This constraint could be preventing streptavidin on the surface of the cell from forming tetramers lowering its effectiveness at binding biotin. Furthermore these existing display systems prevent the addition of another protein into them, preventing the user from displaying other proteins. <br />
<br />
====Idea====<br />
The first goal of making a new display vector was to incorporate a GS linker between the displayed protein and the OmpA protein anchoring in in the cell wall. However when reviewing the construct we decided to add some other useful features as well. Maintaining the Lpp tag (to direct the protein to the periplasm) and the OmpA trans-membrane regions (for anchoring the construct) of the original 2006 parts we added: <br />
#GS Linker (Gly4Ser)4 - allowing for more space between the protein and the cell wall<br />
#TEV - Tobacco Etch Virus (TEV) protease site allowing for cleavage of displayed proteins<br />
#NheI restriction site - allowing for the insert of '''any BioBrick protein''' into the display construct<br />
<br><br><br />
[[Image:PDS_design.png |600px |center]]<br />
<br><br><br />
<br />
====Current Status====<br />
<br />
===== Custom Display Vector: =====<br />
* 1 trans-membrane OmpA: [http://partsregistry.org/Part:BBa_K215200 BBa_K215200]<br />
* 5 trans-membrane OmpA: [http://partsregistry.org/Part:BBa_K215201 BBa_K215201]<br />
<br />
We submitted the Custom Display Vector to the registry as [http://partsregistry.org/Part:BBa_K215200 BBa_K215200] and [http://partsregistry.org/Part:BBa_K215201 BBa_K215201] (for 1 and 5 trans-membrane OmpA). We are currently inserting GFP into the display system to verify that the OmpA display system is working, but we will not have any data by the jamboree.<br />
<br />
===== Custom Display Vector with wild-type monomeric streptavidin inserted: =====<br />
* 1 trans-membrane OmpA: [http://partsregistry.org/Part:BBa_K215210 BBa_K215210] (equivalent to 2006 Harvard iGEM: [http://partsregistry.org/Part:BBa_J36848 BBa_J36848] with an extra linker between OmpA and the streptavidin)<br />
* 5 trans-membrane OmpA: [http://partsregistry.org/Part:BBa_K215211 BBa_K215211] (equivalent to 2006 Harvard iGEM: [http://partsregistry.org/Part:BBa_J36850 BBa_J36850] with the extra linker)<br />
<br />
We inserted wild-type monomeric streptavidin to see if the extra linker would make the streptavidin functional. These parts would be equivalent to the 2006 Harvard iGEM parts, [http://partsregistry.org/Part:BBa_J36848 BBa_J36848] and [http://partsregistry.org/Part:BBa_J36850 BBa_J36850], except with the extra linker and TEV protease site. These parts were submitted to the registry as [http://partsregistry.org/Part:BBa_K215210 BBa_K215210] and [http://partsregistry.org/Part:BBa_K215211 BBa_K215211]. We ran the same flow cytometry assays that we used for the Harvard parts - the streptavidin is non-functional (see [[Team:Washington/Project/Display|Display]] and [[Team:Washington/Notebook|Notebook]] - this is the same cytometry assay that we used for characterizing the 2006 Harvard parts):<br />
<br />
<table align="center"><tr align="center"><br />
<td colspan="2">Cytometry showing no difference in bound biotin-conjugated fluorophore for 1mM IPTG induced vs uninduced cells at different fluorophore concentrations)</td></tr><br />
<tr align="center"><br />
<td>[[Image:New_generic_51.png|350px]]<br>1tmr OmpA, [http://partsregistry.org/Part:BBa_K215210 BBa_K215210]</td><br />
<td>[[Image:New_generic_58.png|350px]]<br>5tmr OmpA, [http://partsregistry.org/Part:BBa_K215211 BBa_K215211]</td><br />
</tr><br />
</table><br />
<br />
----<br />
<br />
{{Template:Team:Washington/Templates/Footer}}</div>Acleonehttp://2009.igem.org/Team:Washington/Project/CDSTeam:Washington/Project/CDS2009-10-22T01:17:07Z<p>Acleone: /* Custom Display Vector with wild-type monomeric streptavidin inserted: */</p>
<hr />
<div>__NOTOC__<br />
{{Template:Team:Washington/Templates/Header}}<br />
<br />
<div><br />
<div style="float:right;">'''[[Team:Washington/Project/FoldIt|Fold-It &gt;]]'''</div><br />
<div>'''[[Team:Washington/Future|&lt; Future Directions]]'''</div><br />
</div><br />
<br />
===Custom Display System===<br />
====Problem====<br />
The underlying design of the streptavidin display constructs in the registry has the displayed protein bound close to the cell membrane. This constraint could be preventing streptavidin on the surface of the cell from forming tetramers lowering its effectiveness at binding biotin. Furthermore these existing display systems prevent the addition of another protein into them, preventing the user from displaying other proteins. <br />
<br />
====Idea====<br />
The first goal of making a new display vector was to incorporate a GS linker between the displayed protein and the OmpA protein anchoring in in the cell wall. However when reviewing the construct we decided to add some other useful features as well. Maintaining the Lpp tag (to direct the protein to the periplasm) and the OmpA trans-membrane regions (for anchoring the construct) of the original 2006 parts we added: <br />
#GS Linker (Gly4Ser)4 - allowing for more space between the protein and the cell wall<br />
#TEV - Tobacco Etch Virus (TEV) protease site allowing for cleavage of displayed proteins<br />
#NheI restriction site - allowing for the insert of '''any BioBrick protein''' into the display construct<br />
<br><br><br />
[[Image:PDS_design.png |600px |center]]<br />
<br><br><br />
<br />
====Current Status====<br />
<br />
===== Custom Display Vector: =====<br />
* 1 trans-membrane OmpA: [http://partsregistry.org/Part:BBa_K215200 BBa_K215200]<br />
* 5 trans-membrane OmpA: [http://partsregistry.org/Part:BBa_K215201 BBa_K215201]<br />
<br />
We submitted the Custom Display Vector to the registry as [http://partsregistry.org/Part:BBa_K215200 BBa_K215200] and [http://partsregistry.org/Part:BBa_K215201 BBa_K215201] (for 1 and 5 trans-membrane OmpA). We are currently inserting GFP into the display system to verify that the OmpA display system is working, but we will not have any data by the jamboree.<br />
<br />
===== Custom Display Vector with wild-type monomeric streptavidin inserted: =====<br />
* 1 trans-membrane OmpA: [http://partsregistry.org/Part:BBa_K215210 BBa_K215210] (equivalent to 2006 Harvard iGEM: [http://partsregistry.org/Part:BBa_J36848 BBa_J36848] with an extra linker between OmpA and the streptavidin)<br />
* 5 trans-membrane OmpA: [http://partsregistry.org/Part:BBa_K215211 BBa_K215211] (equivalent to 2006 Harvard iGEM: [http://partsregistry.org/Part:BBa_J36850 BBa_J36850] with the extra linker)<br />
<br />
We inserted wild-type monomeric streptavidin to see if the extra linker would make the streptavidin functional. These parts would be equivalent to the 2006 Harvard iGEM parts, [http://partsregistry.org/Part:BBa_J36848 BBa_J36848] and [http://partsregistry.org/Part:BBa_J36850 BBa_J36850], except with the extra linker and TEV protease site. These parts were submitted to the registry as [http://partsregistry.org/Part:BBa_K215210 BBa_K215210] and [http://partsregistry.org/Part:BBa_K215211 BBa_K215211]. We ran the same flow cytometry assays that we used for the Harvard parts - the streptavidin is non-functional (see [[Team:Washington/Project/Display|Display]] and [[Team:Washington/Notebook|Notebook]] - this is the same cytometry assay that we used for characterizing the 2006 Harvard parts):<br />
<br />
{| align="center"<br />
| colspan="2" Cytometry showing no difference in bound biotin-conjugated fluorophore for 1mM IPTG induced vs uninduced cells at different fluorophore concentrations) |<br />
|-<br />
| align="center" [[Image:New_generic_51.png|350px]]<br>1tmr OmpA, [http://partsregistry.org/Part:BBa_K215210 BBa_K215210] || [[Image:New_generic_58.png|350px]]<br>5tmr OmpA, [http://partsregistry.org/Part:BBa_K215211 BBa_K215211] |<br />
|}<br />
<br />
----<br />
<br />
{{Template:Team:Washington/Templates/Footer}}</div>Acleonehttp://2009.igem.org/Team:Washington/Project/CDSTeam:Washington/Project/CDS2009-10-21T02:10:39Z<p>Acleone: </p>
<hr />
<div>__NOTOC__<br />
{{Template:Team:Washington/Templates/Header}}<br />
<br />
<div><br />
<div style="float:right;">'''[[Team:Washington/Project/FoldIt|Fold-It &gt;]]'''</div><br />
<div>'''[[Team:Washington/Future|&lt; Future Directions]]'''</div><br />
</div><br />
<br />
===Custom Display System===<br />
====Problem====<br />
The underlying design of the streptavidin display constructs in the registry has the displayed protein bound close to the cell membrane. This constraint could be preventing streptavidin on the surface of the cell from forming tetramers lowering its effectiveness at binding biotin. Furthermore these existing display systems prevent the addition of another protein into them, preventing the user from displaying other proteins. <br />
<br />
====Idea====<br />
The first goal of making a new display vector was to incorporate a GS linker between the displayed protein and the OmpA protein anchoring in in the cell wall. However when reviewing the construct we decided to add some other useful features as well. Maintaining the Lpp tag (to direct the protein to the periplasm) and the OmpA trans-membrane regions (for anchoring the construct) of the original 2006 parts we added: <br />
#GS Linker (Gly4Ser)4 - allowing for more space between the protein and the cell wall<br />
#TEV - Tobacco Etch Virus (TEV) protease site allowing for cleavage of displayed proteins<br />
#NheI restriction site - allowing for the insert of '''any BioBrick protein''' into the display construct<br />
<br><br><br />
[[Image:PDS_design.png |600px |center]]<br />
<br><br><br />
<br />
====Current Status====<br />
<br />
===== Custom Display Vector: =====<br />
* 1 trans-membrane OmpA: [http://partsregistry.org/Part:BBa_K215200 BBa_K215200]<br />
* 5 trans-membrane OmpA: [http://partsregistry.org/Part:BBa_K215201 BBa_K215201]<br />
<br />
We submitted the Custom Display Vector to the registry as [http://partsregistry.org/Part:BBa_K215200 BBa_K215200] and [http://partsregistry.org/Part:BBa_K215201 BBa_K215201] (for 1 and 5 trans-membrane OmpA). We are currently inserting GFP into the display system to verify that the OmpA display system is working, but we will not have any data by the jamboree.<br />
<br />
===== Custom Display Vector with wild-type monomeric streptavidin inserted: =====<br />
* 1 trans-membrane OmpA: [http://partsregistry.org/Part:BBa_K215210 BBa_K215210] (equivalent to 2006 Harvard iGEM: [http://partsregistry.org/Part:BBa_J36848 BBa_J36848] with an extra linker between OmpA and the streptavidin)<br />
* 5 trans-membrane OmpA: [http://partsregistry.org/Part:BBa_K215211 BBa_K215211] (equivalent to 2006 Harvard iGEM: [http://partsregistry.org/Part:BBa_J36850 BBa_J36850] with the extra linker)<br />
<br />
We inserted wild-type monomeric streptavidin to see if the extra linker would make the streptavidin functional. These parts would be equivalent to the 2006 Harvard iGEM parts, [http://partsregistry.org/Part:BBa_J36848 BBa_J36848] and [http://partsregistry.org/Part:BBa_J36850 BBa_J36850], except with the extra linker and TEV protease site. These parts were submitted to the registry as [http://partsregistry.org/Part:BBa_K215210 BBa_K215210] and [http://partsregistry.org/Part:BBa_K215211 BBa_K215211]. We ran the same flow cytometry assays that we used for the Harvard parts - the streptavidin is non-functional:<br />
<br />
----<br />
<br />
{{Template:Team:Washington/Templates/Footer}}</div>Acleonehttp://2009.igem.org/Team:Washington/Project/CDSTeam:Washington/Project/CDS2009-10-20T21:14:55Z<p>Acleone: /* Current Status */</p>
<hr />
<div>__NOTOC__<br />
{{Template:Team:Washington/Templates/Header}}<br />
<br />
<div><br />
<div style="float:right;">'''[[Team:Washington/Project/FoldIt|Fold-It &gt;]]'''</div><br />
<div>'''[[Team:Washington/Future|&lt; Future Directions]]'''</div><br />
</div><br />
<br />
===Custom Display System===<br />
====Problem====<br />
The underlying design of the streptavidin display constructs in the registry has the displayed protein bound close to the cell membrane. This constraint could be preventing streptavidin on the surface of the cell from forming tetramers lowering its effectiveness at binding biotin. Furthermore these existing display systems prevent the addition of another protein into them, preventing the user from displaying other proteins. <br />
<br />
====Idea====<br />
The first goal of making a new display vector was to incorporate a GS linker between the displayed protein and the OmpA protein anchoring in in the cell wall. However when reviewing the construct we decided to add some other useful features as well. Maintaining the Lpp tag (to direct the protein to the periplasm) and the OmpA trans-membrane regions (for anchoring the construct) of the original 2006 parts we added: <br />
#GS Linker (Gly4Ser)4 - allowing for more space between the protein and the cell wall<br />
#TEV - Tobacco Etch Virus (TEV) protease site allowing for cleavage of displayed proteins<br />
#NheI restriction site - allowing for the insert of '''any BioBrick protein''' into the display construct<br />
<br><br><br />
[[Image:PDS_design.png |600px |center]]<br />
<br><br><br />
<br />
====Current Status====<br />
<br />
Custom Display Vector:<br />
* 1 trans-membrane OmpA: [http://partsregistry.org/Part:BBa_K215200 BBa_K215200]<br />
* 5 trans-membrane OmpA: [http://partsregistry.org/Part:BBa_K215201 BBa_K215201]<br />
<br />
We submitted the Custom Display Vector to the registry as [http://partsregistry.org/Part:BBa_K215200 BBa_K215200] and [http://partsregistry.org/Part:BBa_K215201 BBa_K215201] (for 1 and 5 trans-membrane OmpA). We are currently inserting GFP into the display system to verify that the OmpA display system is working, but we will not have any data by the jamboree.<br />
<br />
Custom Display Vector with wild-type monomeric streptavidin inserted:<br />
* 1 trans-membrane OmpA: [http://partsregistry.org/Part:BBa_K215210 BBa_K215210] (equivalent to 2006 Harvard iGEM: [http://partsregistry.org/Part:BBa_J36848 BBa_J36848] with an extra linker between OmpA and the streptavidin)<br />
* 5 trans-membrane OmpA: [http://partsregistry.org/Part:BBa_K215211 BBa_K215211] (equivalent to 2006 Harvard iGEM: [http://partsregistry.org/Part:BBa_J36850 BBa_J36850] with the extra linker)<br />
<br />
We inserted wild-type monomeric streptavidin to see if the extra linker would make the streptavidin functional. These parts would be equivalent to the 2006 Harvard iGEM parts, [http://partsregistry.org/Part:BBa_J36848 BBa_J36848] and [http://partsregistry.org/Part:BBa_J36850 BBa_J36850], except with the extra linker and TEV protease site. These parts were submitted to the registry as [http://partsregistry.org/Part:BBa_K215210 BBa_K215210] and [http://partsregistry.org/Part:BBa_K215211 BBa_K215211]. We ran the same flow cytometry assays that we used for the Harvard parts - the streptavidin is non-functional:<br />
<br />
----<br />
<br />
{{Template:Team:Washington/Templates/Footer}}</div>Acleonehttp://2009.igem.org/Team:Washington/Project/CDSTeam:Washington/Project/CDS2009-10-20T21:14:15Z<p>Acleone: /* Current Status */</p>
<hr />
<div>__NOTOC__<br />
{{Template:Team:Washington/Templates/Header}}<br />
<br />
<div><br />
<div style="float:right;">'''[[Team:Washington/Project/FoldIt|Fold-It &gt;]]'''</div><br />
<div>'''[[Team:Washington/Future|&lt; Future Directions]]'''</div><br />
</div><br />
<br />
===Custom Display System===<br />
====Problem====<br />
The underlying design of the streptavidin display constructs in the registry has the displayed protein bound close to the cell membrane. This constraint could be preventing streptavidin on the surface of the cell from forming tetramers lowering its effectiveness at binding biotin. Furthermore these existing display systems prevent the addition of another protein into them, preventing the user from displaying other proteins. <br />
<br />
====Idea====<br />
The first goal of making a new display vector was to incorporate a GS linker between the displayed protein and the OmpA protein anchoring in in the cell wall. However when reviewing the construct we decided to add some other useful features as well. Maintaining the Lpp tag (to direct the protein to the periplasm) and the OmpA trans-membrane regions (for anchoring the construct) of the original 2006 parts we added: <br />
#GS Linker (Gly4Ser)4 - allowing for more space between the protein and the cell wall<br />
#TEV - Tobacco Etch Virus (TEV) protease site allowing for cleavage of displayed proteins<br />
#NheI restriction site - allowing for the insert of '''any BioBrick protein''' into the display construct<br />
<br><br><br />
[[Image:PDS_design.png |600px |center]]<br />
<br><br><br />
<br />
====Current Status====<br />
<br />
Custom Display Vector:<br />
* [http://partsregistry.org/Part:BBa_K215200 BBa_K215200]<br />
* [http://partsregistry.org/Part:BBa_K215201 BBa_K215201]<br />
<br />
We submitted the Custom Display Vector to the registry as [http://partsregistry.org/Part:BBa_K215200 BBa_K215200] and [http://partsregistry.org/Part:BBa_K215201 BBa_K215201] (for 1 and 5 trans-membrane OmpA). We are currently inserting GFP into the display system to verify that the OmpA display system is working, but we will not have any data by the jamboree.<br />
<br />
Custom Display Vector with wild-type monomeric streptavidin inserted:<br />
* 1 trans-membrane OmpA: [http://partsregistry.org/Part:BBa_K215210 BBa_K215210] (equivalent to 2006 Harvard iGEM: [http://partsregistry.org/Part:BBa_J36848 BBa_J36848] with an extra linker between OmpA and the streptavidin)<br />
* 5 trans-membrane OmpA: [http://partsregistry.org/Part:BBa_K215211 BBa_K215211] (equivalent to 2006 Harvard iGEM: [http://partsregistry.org/Part:BBa_J36850 BBa_J36850] with the extra linker)<br />
<br />
We inserted wild-type monomeric streptavidin to see if the extra linker would make the streptavidin functional. These parts would be equivalent to the 2006 Harvard iGEM parts, [http://partsregistry.org/Part:BBa_J36848 BBa_J36848] and [http://partsregistry.org/Part:BBa_J36850 BBa_J36850], except with the extra linker and TEV protease site. These parts were submitted to the registry as [http://partsregistry.org/Part:BBa_K215210 BBa_K215210] and [http://partsregistry.org/Part:BBa_K215211 BBa_K215211]. We ran the same flow cytometry assays that we used for the Harvard parts - the streptavidin is non-functional:<br />
<br />
----<br />
<br />
{{Template:Team:Washington/Templates/Footer}}</div>Acleonehttp://2009.igem.org/Team:Washington/Project/CDSTeam:Washington/Project/CDS2009-10-20T20:51:09Z<p>Acleone: /* Current Status */</p>
<hr />
<div>__NOTOC__<br />
{{Template:Team:Washington/Templates/Header}}<br />
<br />
<div><br />
<div style="float:right;">'''[[Team:Washington/Project/FoldIt|Fold-It &gt;]]'''</div><br />
<div>'''[[Team:Washington/Future|&lt; Future Directions]]'''</div><br />
</div><br />
<br />
===Custom Display System===<br />
====Problem====<br />
The underlying design of the streptavidin display constructs in the registry has the displayed protein bound close to the cell membrane. This constraint could be preventing streptavidin on the surface of the cell from forming tetramers lowering its effectiveness at binding biotin. Furthermore these existing display systems prevent the addition of another protein into them, preventing the user from displaying other proteins. <br />
<br />
====Idea====<br />
The first goal of making a new display vector was to incorporate a GS linker between the displayed protein and the OmpA protein anchoring in in the cell wall. However when reviewing the construct we decided to add some other useful features as well. Maintaining the Lpp tag (to direct the protein to the periplasm) and the OmpA trans-membrane regions (for anchoring the construct) of the original 2006 parts we added: <br />
#GS Linker (Gly4Ser)4 - allowing for more space between the protein and the cell wall<br />
#TEV - Tobacco Etch Virus (TEV) protease site allowing for cleavage of displayed proteins<br />
#NheI restriction site - allowing for the insert of '''any BioBrick protein''' into the display construct<br />
<br><br><br />
[[Image:PDS_design.png |600px |center]]<br />
<br><br><br />
<br />
====Current Status====<br />
We have built the Custom Display Vector and have inserted streptavidin into it, however we ran out of time and were not able to characterize this part. It was submitted to the registry with streptavidin ([http://partsregistry.org/Part:BBa_K215210 BBa_K215210] and [http://partsregistry.org/Part:BBa_K215211 BBa_K215211]) and without a displayed protein ([http://partsregistry.org/Part:BBa_K215200 BBa_K215200] and [http://partsregistry.org/Part:BBa_K215201 BBa_K215201]).<br />
----<br />
<br />
{{Template:Team:Washington/Templates/Footer}}</div>Acleonehttp://2009.igem.org/Team:Washington/Project/CDSTeam:Washington/Project/CDS2009-10-20T20:50:43Z<p>Acleone: </p>
<hr />
<div>__NOTOC__<br />
{{Template:Team:Washington/Templates/Header}}<br />
<br />
<div><br />
<div style="float:right;">'''[[Team:Washington/Project/FoldIt|Fold-It &gt;]]'''</div><br />
<div>'''[[Team:Washington/Future|&lt; Future Directions]]'''</div><br />
</div><br />
<br />
===Custom Display System===<br />
====Problem====<br />
The underlying design of the streptavidin display constructs in the registry has the displayed protein bound close to the cell membrane. This constraint could be preventing streptavidin on the surface of the cell from forming tetramers lowering its effectiveness at binding biotin. Furthermore these existing display systems prevent the addition of another protein into them, preventing the user from displaying other proteins. <br />
<br />
====Idea====<br />
The first goal of making a new display vector was to incorporate a GS linker between the displayed protein and the OmpA protein anchoring in in the cell wall. However when reviewing the construct we decided to add some other useful features as well. Maintaining the Lpp tag (to direct the protein to the periplasm) and the OmpA trans-membrane regions (for anchoring the construct) of the original 2006 parts we added: <br />
#GS Linker (Gly4Ser)4 - allowing for more space between the protein and the cell wall<br />
#TEV - Tobacco Etch Virus (TEV) protease site allowing for cleavage of displayed proteins<br />
#NheI restriction site - allowing for the insert of '''any BioBrick protein''' into the display construct<br />
<br><br><br />
[[Image:PDS_design.png |600px |center]]<br />
<br><br><br />
<br />
====Current Status====<br />
We have built the Custom Display Vector and have inserted streptavidin into it, however we ran out of time and were not able to characterize this part. It was submitted to the registry with streptavidin ([http://partsregistry.org/Part:BBa_K215210 BBa_K215210] and [http://partsregistry.org/Part:BBa_K215211 BBa_K215211]) and without a displayed protein ([http://partsregistry.org/Part:BBa_K215200 BBa_K215200] and [http://partsregistry.org/Part:BBa_K215200 BBa_K215200]).<br />
----<br />
<br />
{{Template:Team:Washington/Templates/Footer}}</div>Acleonehttp://2009.igem.org/Team:Washington/Project/FoldItTeam:Washington/Project/FoldIt2009-10-20T20:20:35Z<p>Acleone: </p>
<hr />
<div>__NOTOC__<br />
{{Template:Team:Washington/Templates/Header}}<br />
<div><br />
<div>'''[[Team:Washington/Future|&lt; Future Directions]]'''</div><br />
</div><br />
<html><script type="text/javascript"><br />
$(function() {<br />
$("#uw_foldit_div a").attr("href", "http://www.fold.it/");<br />
});<br />
</script></html><br />
<br />
<div id="uw_foldit_div" style="text-align:center;font-size:200%;"><br />
'''[http://www.fold.it/ Fold-It]'''<br />
<br />
[[image:FoldIt_Link.png]]<br />
</div><br />
<br />
====Problem====<br />
Streptavidin in its native form exists as a homotetramer, where adjacent subunits interact allowing for a strong interaction with biotin. This interaction is strong (Kd = 1.5E-15 M at pH 5.0) and can withstand most strong denaturing agents<sup>1</sup>. However when in its monomeric form, streptavidin does not maintain this strong interaction and its usefulness as a strong binder diminishes. For our system we needed a protein that could: be easily displayed on the surface of the cell, specifically bind a ligand, and release this ligand in the presence of biotin. The ability to display a protein on the cell surface is trivial, however there is difficulty in trying to get a protein to be functional on the surface of the cell. In the case of streptavidin the ability of the protein to form tetramers on the cell surface seems to be hindered, due to the poor ability of cells displaying streptavidin to bind biotinylated fluorophore (observed above). From this issue the idea of using a monomeric protein to bind biotin arose. <br />
<br />
====The Idea====<br />
There are engineered forms of streptavidin that have mutations preventing the formation of tetrameric structures. However as mentioned before, as a monomer streptavidin has a weaker affinity to biotin than would be desired. Instead of screening proteins from the literature for ability to bind biotin our group approached the Baker lab at our university. After mentioning our problem, it was recommended that we design a biotin binding protein using the [http://boinc.bakerlab.org/rosetta/ Rosetta software] they developed. Rosetta in conjunction with [http://www.folt.it Fold-It] (also developed at the University of Washington) would allow use to design and optimize proteins for binding biotin. <br />
<br />
====The Trench Work====<br />
The first step in designing our protein was looking at the native biotin-streptavidin interaction and taking measurements between key amino acids and the biotin molecule. From here we entered the constraints into Rosetta where it matched our measurements into proteins from a protein scaffold library. This produced a large set of scaffolds with different ways each one could be used to bind biotin. These scaffolds must be screened manually, and the scaffolds that look the most promising can be placed into Fold-It. Once in Fold-It, the public has access to your protein design and can tweak and tune the protein to optimize its interaction with biotin. This allows anyone (with or without prior protein knowledge) to optimize your protein scaffold. <br />
<br />
As can be seen below Fold-It uses an easily learned user interface and uses a score board to show the players who is the best folder. <br />
-----<br />
<br><br />
<html><br />
<br />
<table><tr><td><br />
<object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/P-UR3G7TBb4&hl=en&fs=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/P-UR3G7TBb4&hl=en&fs=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object><br />
</td><td><br />
Here we see a video of Fold-It, showing one of our biotin puzzles "Hold Me Tightly". The protein is represented as a cartoon model, showing off its secondary structure as well as key amino acid groups. Steric clashes of the amino acid side chains show up as red balls and can also be observed in the video. These steric clashs can be removed with the Shake Function. The Shake function in Fold-It performs coarse sampling of the amino acid conformations, looking for a global-minima. <br />
</td></tr></table><br />
<br />
<table><tr><td><br />
<object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/_Ugmw69_94g&hl=en&fs=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/_Ugmw69_94g&hl=en&fs=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object><br />
</td><td><br />
The function we see here is the Mutate function. This allows the user to sample many amino acids at a particular site, or the whole protein. Mutate looks for global-minima while sampling amino acids. As is seen here Alanine is mutated to Asparagine. The blue and white striped band indicates that a hydrogen bond has been formed, which is a favorable interaction between two polar residues.<br />
</td></tr></table><br />
<br />
<table><tr><td><br />
<object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/3V2OpBGruzQ&hl=en&fs=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/3V2OpBGruzQ&hl=en&fs=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object><br />
</td><td><br />
Another nice feature of Fold-It is the ability to select a sphere of amino acids around your ligand, and optimize these amino acids based off of a fine sampling of conformations. Here we see the amino acids surrounding the ligand being selected and having the Wiggle function performed on them. The Wiggle function in Fold-It allows the user to fine tune the protein structure. Finding a local-minima for the amino acid conformations.<br />
</td></tr></table><br />
<br />
</html><br />
<br><br />
-----<br />
<br />
This accessible format has allowed over 100,000 users to help design proteins. Currently we have published protein puzzles on Fold-It and are screening though the top scoring designs. An undergrad in our group will be active throughout the next year testing the designs and looking for biotin binding proteins.<br />
<br />
<br />
<br />
<div id="uw_foldit_div" style="text-align:center;font-size:200%;"><br />
'''[http://www.fold.it/ Try Fold-It!]'''<br />
</div><br />
<br />
=== References ===<br />
#[http://www.ncbi.nlm.nih.gov/pubmed/18287646?ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum Haugland RP, "Coupling of antibodies with biotin".]<br />
<br />
<br />
{{Template:Team:Washington/Templates/Footer}}</div>Acleonehttp://2009.igem.org/File:Uw_js.txtFile:Uw js.txt2009-10-20T20:16:01Z<p>Acleone: uploaded a new version of "Image:Uw js.txt"</p>
<hr />
<div></div>Acleonehttp://2009.igem.org/Team:Washington/Project/FoldItTeam:Washington/Project/FoldIt2009-10-19T05:06:54Z<p>Acleone: </p>
<hr />
<div>__NOTOC__<br />
{{Template:Team:Washington/Templates/Header}}<br />
<div><br />
<div>'''[[Team:Washington/Project/CDS|&lt; Custom Display System]]'''</div><br />
</div><br />
<html><script type="text/javascript"><br />
$(function() {<br />
$("#uw_foldit_div a").attr("href", "http://www.fold.it/");<br />
});<br />
</script></html><br />
<br />
<div id="uw_foldit_div" style="text-align:center;font-size:200%;"><br />
'''[http://www.fold.it/ Fold-It]'''<br />
<br />
[[image:FoldIt_Link.png]]<br />
</div><br />
<br />
====Problem====<br />
Streptavidin in its native form exists as a homotetramer, where adjacent subunits interact allowing for a strong interaction with biotin. This interaction is strong (Kd = 1.5E-15 M at pH 5.0) and can withstand most strong denaturing agents<sup>1</sup>. However when in its monomeric form, streptavidin does not maintain this strong interaction and its usefulness as a strong binder diminishes. For our system we needed a protein that could: be easily displayed on the surface of the cell, specifically bind a ligand, and release this ligand in the presence of biotin. The ability to display a protein on the cell surface is trivial, however there is difficulty in trying to get a protein to be functional on the surface of the cell. In the case of streptavidin the ability of the protein to form tetramers on the cell surface seems to be hindered, due to the poor ability of cells displaying streptavidin to bind biotinylated fluorophore (observed above). From this issue the idea of using a monomeric protein to bind biotin arose. <br />
<br />
====The Idea====<br />
There are engineered forms of streptavidin that have mutations preventing the formation of tetrameric structures. However as mentioned before, as a monomer streptavidin has a weaker affinity to biotin than would be desired. Instead of screening proteins from the literature for ability to bind biotin our group approached the Baker lab at our university. After mentioning our problem, it was recommended that we design a biotin binding protein using the [http://boinc.bakerlab.org/rosetta/ Rosetta software] they developed. Rosetta in conjunction with [http://www.folt.it Fold-It] (also developed at the University of Washington) would allow use to design and optimize proteins for binding biotin. <br />
<br />
====The Trench Work====<br />
The first step in designing our protein was looking at the native biotin-streptavidin interaction and taking measurements between key amino acids and the biotin molecule. From here we entered the constraints into Rosetta where it matched our measurements into proteins from a protein scaffold library. This produced a large set of scaffolds with different ways each one could be used to bind biotin. These scaffolds must be screened manually, and the scaffolds that look the most promising can be placed into Fold-It. Once in Fold-It, the public has access to your protein design and can tweak and tune the protein to optimize its interaction with biotin. This allows anyone (with or without prior protein knowledge) to optimize your protein scaffold. <br />
<br />
As can be seen below Fold-It uses an easily learned user interface and uses a score board to show the players who is the best folder. <br />
-----<br />
<br><br />
<html><br />
<br />
<table><tr><td><br />
<object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/P-UR3G7TBb4&hl=en&fs=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/P-UR3G7TBb4&hl=en&fs=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object><br />
</td><td><br />
Here we see a video of Fold-It, showing one of our biotin puzzles "Hold Me Tightly". The protein is represented as a cartoon model, showing off its secondary structure as well as key amino acid groups. Steric clashes of the amino acid side chains show up as red balls and can also be observed in the video. These steric clashs can be removed with the Shake Function. The Shake function in Fold-It performs coarse sampling of the amino acid conformations, looking for a global-minima. <br />
</td></tr></table><br />
<br />
<table><tr><td><br />
<object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/_Ugmw69_94g&hl=en&fs=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/_Ugmw69_94g&hl=en&fs=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object><br />
</td><td><br />
The function we see here is the Mutate function. This allows the user to sample many amino acids at a particular site, or the whole protein. Mutate looks for global-minima while sampling amino acids. As is seen here Alanine is mutated to Asparagine. The blue and white striped band indicates that a hydrogen bond has been formed, which is a favorable interaction between two polar residues.<br />
</td></tr></table><br />
<br />
<table><tr><td><br />
<object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/3V2OpBGruzQ&hl=en&fs=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/3V2OpBGruzQ&hl=en&fs=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object><br />
</td><td><br />
Another nice feature of Fold-It is the ability to select a sphere of amino acids around your ligand, and optimize these amino acids based off of a fine sampling of conformations. Here we see the amino acids surrounding the ligand being selected and having the Wiggle function performed on them. The Wiggle function in Fold-It allows the user to fine tune the protein structure. Finding a local-minima for the amino acid conformations.<br />
</td></tr></table><br />
<br />
</html><br />
<br><br />
-----<br />
<br />
This accessible format has allowed over 100,000 users to help design proteins. Currently we have published protein puzzles on Fold-It and are screening though the top scoring designs. An undergrad in our group will be active throughout the next year testing the designs and looking for biotin binding proteins.<br />
<br />
=== References ===<br />
#[http://www.ncbi.nlm.nih.gov/pubmed/18287646?ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum Haugland RP, "Coupling of antibodies with biotin".]<br />
<br />
<br />
{{Template:Team:Washington/Templates/Footer}}</div>Acleonehttp://2009.igem.org/Team:Washington/Project/CDSTeam:Washington/Project/CDS2009-10-19T05:03:31Z<p>Acleone: </p>
<hr />
<div>__NOTOC__<br />
{{Template:Team:Washington/Templates/Header}}<br />
<br />
<div><br />
<div style="float:right;">'''[[Team:Washington/Project/FoldIt|Fold-It &gt;]]'''</div><br />
<div>'''[[Team:Washington/Future|&lt; Future Directions]]'''</div><br />
</div><br />
<br />
===Custom Display System===<br />
====Problem====<br />
The underlying design of the streptavidin display constructs in the registry has the displayed protein bound close to the cell membrane. This constraint could be preventing streptavidin on the surface of the cell from forming tetramers lowering its effectiveness at binding biotin. Furthermore these existing display systems prevent the addition of another protein into them, preventing the user from displaying other proteins. <br />
<br />
====Idea====<br />
The first goal of making a new display vector was to incorporate a GS linker between the displayed protein and the ompA protein anchoring in in the cell wall. However when reviewing the construct we decided to add some other useful features as well. Maintaining the Lpp tag (to direct the protein to the periplasm) and the OmpA trans-membrane regions (for anchoring the construct) of the original 2006 parts we added: <br />
#GS Linker (Gly4Ser)4 - allowing for more space between the protein and the cell wall<br />
#TEV (Tyrosine-Glutamate-Valine) - protease site allowing for cleavage of displayed proteins<br />
#NheI restriction site - allowing for the insert of '''any BioBrick protein''' into the display construct<br />
<br><br><br />
[[Image:PDS_design.png |600px |center]]<br />
<br><br><br />
<br />
====Current Status====<br />
We have built the Custom Display Vector and have inserted streptavidin into it, however we ran out of time and were not able to characterize this part. It was subbmited to the registry with streptavidin (BBa_K215210 and BBa_K215211) and without a displayed protein(BBa_K215200 and BBa_K215200).<br />
----<br />
<br />
{{Template:Team:Washington/Templates/Footer}}</div>Acleonehttp://2009.igem.org/Team:Washington/Project/CDSTeam:Washington/Project/CDS2009-10-19T05:03:16Z<p>Acleone: </p>
<hr />
<div>__NOTOC__<br />
{{Template:Team:Washington/Templates/Header}}<br />
<br />
<div><br />
<div style="float:right;">'''[[Team:Washington/Project/FoldIt|Fold-It &gt;]'''</div><br />
<div>'''[[Team:Washington/Future|&lt; Future Directions]]'''</div><br />
</div><br />
<br />
===Custom Display System===<br />
====Problem====<br />
The underlying design of the streptavidin display constructs in the registry has the displayed protein bound close to the cell membrane. This constraint could be preventing streptavidin on the surface of the cell from forming tetramers lowering its effectiveness at binding biotin. Furthermore these existing display systems prevent the addition of another protein into them, preventing the user from displaying other proteins. <br />
<br />
====Idea====<br />
The first goal of making a new display vector was to incorporate a GS linker between the displayed protein and the ompA protein anchoring in in the cell wall. However when reviewing the construct we decided to add some other useful features as well. Maintaining the Lpp tag (to direct the protein to the periplasm) and the OmpA trans-membrane regions (for anchoring the construct) of the original 2006 parts we added: <br />
#GS Linker (Gly4Ser)4 - allowing for more space between the protein and the cell wall<br />
#TEV (Tyrosine-Glutamate-Valine) - protease site allowing for cleavage of displayed proteins<br />
#NheI restriction site - allowing for the insert of '''any BioBrick protein''' into the display construct<br />
<br><br><br />
[[Image:PDS_design.png |600px |center]]<br />
<br><br><br />
<br />
====Current Status====<br />
We have built the Custom Display Vector and have inserted streptavidin into it, however we ran out of time and were not able to characterize this part. It was subbmited to the registry with streptavidin (BBa_K215210 and BBa_K215211) and without a displayed protein(BBa_K215200 and BBa_K215200).<br />
----<br />
<br />
{{Template:Team:Washington/Templates/Footer}}</div>Acleonehttp://2009.igem.org/Team:Washington/Project/DisplayTeam:Washington/Project/Display2009-10-19T04:55:27Z<p>Acleone: </p>
<hr />
<div>__NOTOC__<br />
{{Template:Team:Washington/Templates/Header}}<br />
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<html><style><br />
#uw_himg {<br />
display:none;<br />
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<div style="float:left;">'''[https://2009.igem.org/Team:Washington/Project/Secretion &lt; Secretion]'''</div><br />
<div style="float:right;">'''[https://2009.igem.org/Team:Washington/Project/Release Release &gt;]'''</div><br />
</div><br />
<br />
[[Image:Main_graphic3_display_banner.png|center]]<br />
<br />
='''Background'''=<br />
Our system hinged on finding a protein which could bind other proteins to the outside of the cell, but whose interaction with these proteins was weak enough to be disrupted by another small molecule. streptavidin presented itself as a logical choice. Several protein tags (such as the Nano-Tag <sup>1</sup>) have been developed such that they bind streptavidin at the biotin binding site, but can be released when biotin is added to the system and binds to streptavidin. The ability for peptides to bind streptavidin and be released upon the addition of biotin is a technology currently used on a daily basis world-wide by labs purifying proteins. The major difference between the traditional system and our system is that streptavidin is attached to beads which are used in a column format in the traditional protein purification method. Our system will have streptavidin attached to the surface of the cell. Combining the display system with our target and secretion vector our vision is complete. A single cell can then produce, secrete, bind, and release any protein of interest.<br />
{| cellpadding="1" cellspacing="8"<br />
|-<br />
|[[Image:Strept-biotin.png | 175px]]<br />
|The binding of the Nano-Tag by streptavidin has an affinity of 4nM and has been used to purify multiple proteins. The ability of biotin to compete off the Nano-Tag in biding assays indicates that the binding occurs at the same site of streptavidin. Of great importance for incorporating streptavidin into our display system is that streptavidin is a common protein used in bio-chemical assays. Its ability to bind biotin is one of the strongest non-covalent interactions known. This allows for the utilization of biotin and streptavidin to act as molecular connectors in protein systems. Furthermore the availability of biotinylated and streptavidin linked fluorophores allows for an easy assay of their presence, using fluorescence microscopy<sup>2</sup>.<br />
|-<br />
|}<br />
<br />
{| cellpadding="1" cellspacing="8"<br />
|-<br />
|Next we needed a way to anchor the streptavidin to the outer membrane. The obvious choice was to use the LPP signal peptide and Outer Membrane Protein A (ompA) because this system has been extensively characterized as an expression system to display proteins on the cell surface. This LPP-ompA system has been used to display a diverse group of proteins, including Green Fluorescent Protein (GFP) <sup>3</sup>, Organophosphorus Hydrolase (OPH) <sup>4</sup>, Cyclodextrin Glucanotransferase (CGTase) <sup>5</sup>, Methyl Parathion Hydrolase (MPH) <sup>6</sup>, Enhanced Green Fluorescent Protein (EGFP) <sup>6</sup>. Thus, an appropriate display construct for our system would be Lpp-OmpA display construct fused to streptavidin, which would be anchored in the outer membrane and display streptavidin on the surface of the cell - which would bind a nanotag on our target protein in an interaction that could be disrupted upon the addition of biotin. <br />
|[[Image:Display image.jpg|400px|right]]<br />
|-<br />
|}<br><br />
==== Harvard 2006 Surface Display System: Legacy Parts ====<br />
<br />
<br />
When we checked the Parts Registry to see if someone had already built this or sometime similar that we could use as a starting part for our Idealized Protein Purification Display System, we found this Lpp-OmpA-streptavidin construct, submitted by Harvard iGEM team as part of their project in 2006 <sup>7</sup>. In fact, Harvard had submitted four biobrick parts which were all variations on the same theme. All four are fusion proteins which have a LPP signal peptide, either one or five trans-membrane ompA, and either monomeric or dimeric streptavidin. All the variations and the resulting biobricks are shown below.<br />
<br />
<br />
<br />
{|cellpadding="3" cellspacing="1" border="2" width="75%" align="center"<br />
! Bio Brick<br />
! OmpA trans-membrane domains<br />
! Type of Streptavidin<br />
|- align="center"<br />
|J36848<br />
|1<br />
|monomeric<br />
|- align="center"<br />
|J36849<br />
|1<br />
|dimeric<br />
|- align="center"<br />
|J36850<br />
|5<br />
|monomeric<br />
|- align="center"<br />
|J36851<br />
|5<br />
|dimeric<br />
|-<br />
|}<br />
<br><br />
<br />
==== Harvard 2006 Surface Display System: Documented Data ====<br />
<br />
Data regarding the activity of the Harvard iGEM 2006 Lpp-OmpA-Streptavidin construct has been published in IET Synthetic Biology in 2007 <sup>7</sup>. The author showed, by Western blot against the His tag fused to this construct, that their protein was being expressed within the cell. In order to demonstrate binding of their cell-surface-anchored streptavidin to biotin, the author incubated cells expressing Lpp-OmpA-streptavidin with an aptamer that linked biotin to a fluorophore using an oligonucleotide linker (see figure below). Any cells that express streptavidin on the surface of the cell should fluoresce in their assay - concordantly, when cells were incubated with this biotin-oligo-fluorescein aptamer, the author saw fluorescence on cells expressing Lpp-OmpA-Streptavidin, but not on Lpp-OmpA negative control. However, cells that expressed streptavidin on the surface also bound to the negative aptamer control (oligo-fluorescein) that lacked biotin, indicating that the oligo aptamer itself (and not biotin) may have been responsible for the association of the fluorophore to the cell surface. Thus, the Harvard 2006 iGEM team was never able to definitively show specific affinity of their Lpp-OmpA-Streptavidin construct.<br />
<br />
{|<br />
|-<br />
|[[Image:HarvardIETSYNTH.jpg|400px|center]]<br />
|''<br />
L01W: Lpp-OmpA-Streptavidin monomer, no His Tag.<br><br />
L01H: Lpp-OmpA-Streptavidin monomer with His tag.<br><br />
L01S: Lpp-OmpA-Streptavidin dimer.<br><br />
L01: Lpp-OmpA alone).<br><br />
A: no aptamer added.<br><br />
B: With only ‘F’ 50 -fluorescently-tagged oligonucleotide.<br><br />
C: With fluorescently tagged streptavidin aptamer.<br><br />
D: With ‘B-F’ hybrid of 50 -biotinylated oligonucleotide annealed with 50-fluorescently-tagged oligonucleotide<sup>7</sup>.''<br />
|-<br />
|}<br />
<br />
<br />
Based on these experiments, we decided to try using this display system for our purpose. But first, since binding of the system to streptavidin had not be definitively shown, we had to verify binding of the Lpp-OmpA-Streptavidin construct to biotin for ourselves, and characterize this interaction.<br />
<br />
='''Experiments'''=<br />
== Harvard's Legacy 2006 Cell Surface Display Parts: Do They Work As Predicted? ==<br />
<br />
In order to determine whether the Harvard 2006 iGEM streptavidin cell surface display parts were 1. expressed properly, and 2. bound biotin on the surface of the cell, we decided to do the following set of tests. <br />
<br />
# Western to verify expression of Harvard 2006 surface display parts <br />
# Assessment of biotin binding to cell surface by visualization in fluorescence microscope <br />
# Assessment of biotin binding to cell surface by visualization in flow cytometer<br />
<br />
<br />
=== Western Blot Demonstrates that Harvard 2006 iGEM Parts Are Expressed ===<br />
The goal of this experiment was to make sure that the proteins were being expressed in our cell lines, and also to make sure that they were the correct length. This was crucial to ascertain before we moved on and began to test the parts. Even though we had the individual parts sequenced confirmed we needed to make sure that the proteins were being expressed correctly in the cell. Since all our the Harvard parts are conveniently his tagged, we used a Western blot reagent (horseradish peroxidase) that was conjugated to nickel-NTA (binds His tags)<sup>8</sup>.<br />
<br />
<br />
==== Data ====<br />
<br />
[[Image:HarvardExpressionWestern.jpg|700px|center]]<br />
<br><br />
<br />
The expected sizes of each of these proteins are shown in the table below:<br />
{|cellpadding="3" cellspacing="1" border="2" width="50%"<br />
! Bio Brick<br />
! Length (in Da)<br />
|- align="center"<br />
|J36848<br />
|21478.7<br />
|- align="center"<br />
|J36849<br />
|34600.9<br />
|- align="center"<br />
|J36850<br />
|31215.4<br />
|- align="center"<br />
|J36851<br />
|44972.3<br />
|-<br />
|}<br />
<br />
<br><br />
Based on the above table, we were able to verify expression of the Harvard 2006 iGEM surface display parts. The varying intensity of the bands does not indicate the strength of expression because the protein amount was not normalized before it was inserted into the gel. Next we wanted to determine whether functional, biotin-binding streptavidin was displayed on the cell surface in cells expressing these parts. To test this, we used two different methods: visualization by fluorescence microscopy and by flow cytometer. <br><br />
<br />
=== Streptavidin-Biotin Binding Is Not Visualized By Fluorescence Microscopy ===<br />
<br />
The goal of this experiment was to confirm the display of streptavidin on the surface of the cell. To do this, we incubated cells expressing the Harvard 2006 iGEM streptavidin surface display parts with a fluorophore conjugated to biotin. If cells are expressing functional streptavidin on the surface of the cell, they should bind the biotinylated fluorophore, and this binding should be detectable in our florescence microscope as a halo of fluorescence surrounding each individual cell. As a positive control for streptavidin-biotin binding we incubated the biotinylated fluorophore with streptavidin-coated beads that were roughly the same size (with respect to volume) as our cells (except spherical) (see Notebook page for protocols).<br />
<br />
<br />
==== Data ====<br />
<br />
<table><br />
<tr> <br />
<td><p style="font-size:18px"> Positive Control </p> </td><br />
<td><p style="font-size:18px"> Negative Control </p> </td><br />
<td rowspan="4">These images were analyzed using imageJ <sup>9</sup>. The image on the left shows an image of the beads with flourophore added to them. These beads were diluted and spun diluted and spun down until the background level of florescence was low enough to get an accurate reading. We then used imageJ to analyze the intensity of a line going through the bead, which is demonstrated by the above schematic. On the positive control the edges of the streptavidin-coated beads show spikes in florescence, indicating binding of the biotinylated fluorophore to the beads. The negative control (beads without fluorophore) showed no such increase. This meant that our biotinylated flouophore binds to streptavidin in a detectable manner. When our cells were examined in the same manner, no difference could be seen in biotinylated fluorophore binding between cells that had induced expression of surface streptavidin (left) and uninduced cells which should not express the surface display protein (right). This was evidence that the streptavidin surface display part binding to biotin was very low / nonexistent. In order to verify this, we measured the binding of the biotinylated fluorophore to entire populations of streptavidin-expressing cells by flow cytomtery.</td><br />
</tr><br />
<tr><br />
<td>[[Image:M_beads1.png| 210px]]</td><br />
<td>[[Image:M_beads2.png| 210px]]</td><br />
</tr><br />
<td><p style="font-size:18px"> Induced Cells </p></td><br />
<td><p style="font-size:18px"> Uninduced Cells </p></td><br />
<tr><br />
<td>[[Image:M_cells1.png| 210px]]</td><br />
<td>[[Image:M_cells2.png| 210px]]</td><br />
</tr><br />
</table><br />
<br />
=== Streptavidin-Biotin Binding Is Not Visualized By Flow Cytometry ===<br />
The goal of this experiment was to visualize biotin binding to streptavidin over a whole population of cells. Whereas the Microscope experiment looked at a few localized cells, we set the cytometer to look 50,000 streptavidin-expressing cells after incubation (and washing) with the biotinylated fluorophore and read the resulting florescence ([https://2009.igem.org/Team:Washington/Notebook/Flow_Cytometry see Notebook page for protocols]). Like in the microscopy assay, we used streptavidin-coated beads as a positive control. The cytometer reads the flouresence of each individual particle (cell or bead) passing through it, which allows for accurate readings, and monitoring of a much larger sample size than we visualized under the microscope. The results of this experiment are described below.<br />
<br />
<br />
==== Data ====<br />
<br />
<gallery heights=300px widths=350><br />
Image:48.png|'''BBa_J36848''' ''This image shows both the induced and uninduced cells for part 48 in varying levels of flourophore (0nM to 100nM). The cells were induced at 1mM IPTG. This data shows that there is no appreciable difference between the induced and uninduced cells at any given level of flourophore. All curves appear to have the same amount of fluorescence. We found similar results using a microscopy assay.''<br />
Image:StreptBead cyto.png|'''+ Control''' ''We used the sreptavadin coated to show us what the magnitude of fluorescence increase we should see with increased flourophore levels. We were able to see that as the level of fourophore was increased we could see increased retention between the beads and the flouophore. The black is beads with no flouophore, the red is with 10 nM, and the purple is 100 nM. These showed a clear difference between the beads without flourophore, and the beads with flourophore. The cells shown at right, matched the readings of the beads when they had no flourophore added.''<br />
</gallery><br />
<br />
In the flow cytometer, our positive control, streptavidin-coated beads, showed a clear distinction between beads pre-incubated with biotinylated flourophore and the beads without flourophore, indicating that this assay is capable of visualizing streptavidin binding to the biotinylated fluorophore. Like in the microscopy assay, we did not observe appreciable binding occurring with any of the ompA-streptavidin parts to biotin at the population level. This data only shows part number 48. However this data is applicable to all of the parts we observed. All streptavidin-expressing cells demonstrated low levels of florescence comparable to those of beads not pre-incubated with fluorophore.<br />
<br />
=== Conclusion ===<br />
<br />
<br />
In conclusion, we demonstrated expression of the Harvard 2006 streptavidin surface display parts via Western Blot. We were unable, however, to visualize binding between a biotinylated flourophore and the cells expressing these proteins. This indicates that the streptavidin display system is likely not binding biotin correctly. We hypothesize that streptavidin might not having enough room on the cell surface to form tetramers (which is its native state), and thus may not be binding to biotin with high efficiency. In order to generate a construct that demonstrates tight binding to to biotin, we propose to design a generalized cell surface display system, and to computationally design a biotin-binding streptavidin monomer using software developed at the University of Washington.<br><br />
<br />
'''These solutions are described in the [https://2009.igem.org/wiki/index.php?title=Team:Washington/Future future directions section].'''<br />
<br />
----<br />
<br />
=== Citations ===<br />
#The Nano-tag, a streptavidin-binding peptide for the purification and detection of recombinant proteins doi:10.1016/j.physletb.2003.10.071<br />
#Structural origins of high-affinity biotin binding to streptavidin. PC Weber, DH Ohlendorf, JJ Wendoloski, and FR Salemme (6 January 1989). Science 243 (4887), 85. [DOI: 10.1126/science.2911722]<br />
#Display of green fluorescent protein on ''Escherichia coli'' cell surface. Shi H, Wen Su W. Department of Biological & Agricultural Engineering, University of Missouri-Columbia, 65211, Columbia, MO, USAEnzyme Microb Technol. 2001 Jan 2;28(1):25-34.<br />
#Cell surface display of organophosphorus hydrolase using ice nucleation protein. Shimazu M, Mulchandani A, Chen W. Biotechnol Prog. 2001 Jan-Feb;17(1):76-80. Department of Chemical and Environmental Engineering, and Environmental Toxicology Program, University of California, Riverside, CA 92521, USA.<br />
#Anchorage of cyclodextrin glucanotransferase on the outer membrane of ''Escherichia coli''. Wan HM, Chang BY, Lin SC. Biotechnol Bioeng. 2002 Aug 20;79(4):457-64. Department of Chemical Engineering, National Chung Hsing University, Taichung, 402, Taiwan.<br />
#Cell surface display of functional macromolecule fusions on ''Escherichia coli'' for development of an autofluorescent whole-cell biocatalyst. Yang C, Zhao Q, Liu Z, Li Q, Qiao C, Mulchandani A, Chen W. Environ Sci Technol. 2008 Aug 15;42(16):6105-10. State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.<br />
#Cell surface streptavidin. Tsai P. IET Synthetic Biol. 2007;1(1.2):32.<br />
#[http://www.kpl.com/catalog/productdetail.cfm?catalog_ID=17&Category_ID=448&Product_ID=1085 HisDetector Nickel-HRP]<br />
#[http://rsbweb.nih.gov/ij/docs/index.html ImageJ]<br />
#SVP-15-5 streptavidin coated polstyerene spheres, 1.5-1.9 &micro;m, [http://www.spherotech.com/coa_pol_par.htm Spherotech]<br />
{{Template:Team:Washington/Templates/Footer}}</div>Acleonehttp://2009.igem.org/Team:Washington/Project/SecretionTeam:Washington/Project/Secretion2009-10-19T04:55:15Z<p>Acleone: </p>
<hr />
<div>__NOTOC__<br />
{{Template:Team:Washington/Templates/Header}}<br />
<br />
<html><style><br />
#uw_himg {<br />
display:none;<br />
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<div><br />
<div style="float:left;">'''[https://2009.igem.org/Team:Washington/Project/Target &lt; Target]'''</div><br />
<div style="float:right;">'''[https://2009.igem.org/Team:Washington/Project/Display Display &gt;]'''</div><br />
</div><br />
<br />
[[Image:Main_graphic3_secretion_banner.png|center]]<br />
<br />
='''Background'''=<br />
The ability to secrete our target protein completely out of the cell and into the media is an essential feature of our project. While the laboratory ''E. coli'' strain K-12 contains genes for a type II system (the genes have been silenced<sup>1</sup>), it lacks a type I secretion system capable of exporting proteins to the extracellular space. To implement this ability into ''E. coli'', we chose to install the type I secretion system of ''Erwinia chrysanthemi''<sup>2</sup>. This system was chosen because it has been shown to function within ''E. coli'' and export a wide range of proteins through the periplasm and into the extracellular space.<sup>2,3,4</sup><br />
<br />
<br />
[[image:Secretion_structure.png |400px|right]]<br />
The secretion system is composed of three genes (prtD, prtE, and prtF) whose products recognize the protein being secreted via a C-terminus tag (prtB)<sup>2</sup>. The PrtD, PrtE, and PrtF proteins are thought to form a selective pore that connects the cytoplasm directly to the external medium. As the diagram shows, PrtD and PrtE interact with the inner membrane, while PrtF interacts with the outer membrane. PrtB is the 181-amino acid sequence that, when fused to the C-terminus of a protein, functions as the secretion signal.<br />
<br />
<br><br><br><br><br />
<br />
='''Experiments'''=<br />
<br />
<br />
<br />
For the Secretion System our goal was to:<br />
* Construct the Secretion Plasmid<br />
* Characterize the Secretion System<br />
<br />
==Constructing the Secretion System==<br />
<br />
To create the secretion system, we first synthesized a coding sequence that would produce the 3 gene constructs described above<sup>2</sup>. To do this, we synthesized biobrick compatible versions of each gene and their native ribosome binding sites from oligos as described in our [https://2009.igem.org/Team:Washington/Notebook/gene_synthesis| gene synthesis protocol].<br />
[[image:Secretion_plas.PNG |300px | right]]<br />
<br><br />
After synthesis of each individual gene w/RBS biobrick, they were then pieced together using biobrick standard assembly to form a single construct. Three different versions of the secretion construct were then created with the placement of one of three different promoters in front of the gene construct, a high ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 BBa_J23100]), a medium ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J23114 BBa_J23114]), or a low ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J23113 BBa_J23113]) strength promoter . All three designs were placed in pSB3T5. For our experiments, only the high strength promoter was used.<br />
<br />
<br><br />
<br><br><br />
<br />
==Characterization of Secretion System (Secreting the Target Protein)==<br />
<br />
To test the functionality of our secretion system, we decided to see how well the system could export the Target protein into the media. To do this, we cloned GFP (BBa_E0040) into our Target vector, and transformed the vector into cells (strain BL21 lacq) containing the Secretion plasmid. The cells containing our TargetGFP vector and Secretion plasmid were then grown in a large culture (50 ml) and expression of the Target protein was induced via IPTG. After a period of growth, the culture was then spun down to separate the cells from the media. The amount of fluorescence found in the supernatant was then used to quantify the amount of Target protein being secreted (a detailed protocol of the experiment can be found [[Team:Washington/Notebook/50mL_purification|here]]).<br />
<br />
==Results==<br />
In our first round of experiments with the cells containing both the TargetGFP construct and Secretion plasmid, two controls were also tested: cells containing only the TargetGFP construct and cells containing the TargetOpdA construct and the Secretion plasmid.<br />
<br />
The plot below shows the results of our first secretion test.<br />
<br />
[[Image:Secretion_data_plot1.png|center|600px]]<br />
<br />
As shown above, the cells containing both the TargetGFP and Secretion plasmid released a much higher amount of the target protein into the media when compared to the two controls. This result was quite exciting as it appeared that our secretion system was working great. <br />
<br />
However, after verifying these results, we realized that one of the controls was not entirely fair since the culture containing only the TargetGFP vector had just one plasmid and thus was only grown with one antibiotic (ampicillin). To make a more comparable control, the cells containing only the TargetGFP construct were also transformed with a promoter-less Secretion plasmid, and the experiment was repeated. <br />
<br />
The plot below shows the results of the second secretion test. <br />
<br />
[[Image:Secretion_data_plot2.png|center|600px]]<br />
<br />
As shown above, the addition of the promoter-less secretion plasmid into the TargetGFP control also caused the culture to release an elevated amount of target protein into the media. From this result, it appeared that the Secretion system was not responsible for the elevated amount of TargetGFP in the media, but that it was actually an artifact caused by the extra plasmid that the Secretion system was on (pSB3T5). We hypothesize that this artifact could be the result of a couple different reasons: the extra plasmid (and thus extra antibiotic resistance needed) causes an elevated amount of cell stress that causes premature cell lysis, or the cell membrane was rendered more permeable since the extra plasmid encoded tetracycline resistance via a membrane pump protein.<br />
<br />
A couple different variations of the above experiments were then tried to show secretion:<br />
*We tried varying the amount of IPTG used for induction (1-500 uM) to make sure we weren't overloading the cells<br />
*We changed the cell strain from BL21(lacq) to DH5a, the cell strain used in reference 2<br />
*We used PSB3T5 with out an insert, to eliminate the potential leaky expression from a promoter less gene<br />
*We varied the temperature of expression to see if the proteins would be more stable<br />
<br />
Unfortunately we obtained the same results for all of these additional experiments, in which there was no increase of protein concentration in the media when the secretion system was present (data not shown).<br />
<br />
<br />
====Conclusion====<br />
<br />
Based on these results, we have yet to show that the Secretion system is functioning. However, this system has been shown to work properly in the literature<sup>2</sup>, and there are many parameters that we have yet to reproduce and optimize. Proteins that have been secreted by this Type I system and other similar ones include GFP<sup>2,3</sup>, lipase<sup>3</sup>, Trichoderma harzianum endochitinase<sup>2</sup>, trout growth hormone<sup>2</sup>, ompC<sup>2</sup>, and lacZ<sup>2</sup>. This causes us to believe that our part, which has been sequenced verified, should work given a little more tweaking, to see our ideas see [[Team:Washington/Future|Future Directions]]<br />
<br><br><br />
'''Continue to [https://2009.igem.org/Team:Washington/Project/Display Display]'''<br />
<br />
='''Citations'''=<br />
<br />
#Olivera. Expression of the endogenous type II secretion pathway in Escherichia coli leads to chitinase secretion. <br />
#Palacios et al. [http://www.ncbi.nlm.nih.gov/pubmed/11157948 Subset of Hybrid Eukaryotic Proteins Is Exported by the Type I Secretion System of Erwinia chrysanthemi, Secrevtion of GFP in E. Coli]<br />
#Chung et al. [http://www.ncbi.nlm.nih.gov/pubmed/19178697 Export of recombinant proteins in Escherichia coli using ABC transporter, Secretion of GFP in E. Coli]<br />
#Holland et al. [http://www.ncbi.nlm.nih.gov/pubmed/16092522 Type 1 protein secretion in bacteria, the ABC-transporter dependent pathway]<br />
<br />
{{Template:Team:Washington/Templates/Footer}}</div>Acleonehttp://2009.igem.org/Team:Washington/Project/TargetTeam:Washington/Project/Target2009-10-19T04:55:02Z<p>Acleone: </p>
<hr />
<div>__NOTOC__<br />
{{Template:Team:Washington/Templates/Header}}<br />
<br />
<html><style><br />
#uw_himg {<br />
display:none;<br />
}<br />
</style></html><br />
<div><br />
<div style="float:left;">'''[https://2009.igem.org/Team:Washington/Project &lt; Project Description]'''</div><br />
<div style="float:right;">'''[https://2009.igem.org/Team:Washington/Project/Secretion Secretion &gt;]'''</div><br />
</div><br />
<br />
[[Image:Main_graphic3_target_banner.png|center]]<br />
<br />
='''Background'''=<br />
<br />
When a favorite protein (Afp) is cloned into the target vector ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K215002 BBa_K215002]) two tags are fused onto the N and C terminal of Afp. These tags are depicted below: <br> <br />
<br />
[[Image:Targ_map.PNG | 700px | center]]<br />
<br />
The first key feature of the target vector is the NheI restriction site, where afp get's inserted. NheI is compatible with XbaI and SpeI, meaning that a biobrick digested at the X and S sites can be ligated into the target vector at the NheI site (for detailed protocol see: [https://2009.igem.org/Team:Washington/Notebook/NheI| NheI Insertion Protocol]). <br><br />
<br />
At the N-terminus of the Target is the display (aka Nano<sup>1</sup>) tag, which is a 15 amino acid sequence that binds to streptavidin. Since streptavidin is being displayed on the surface of the cell this allows our protein to stick to the outside of the cell, but can still be released by the addition of biotin. For more details see: [https://2009.igem.org/Team:Washington/Project/Display| Surface Display System ]. <br><br />
<br />
At the C-terminus of the Target is a secretion tag<sup>2</sup> (prtB) that is recognized by a Type I secretion system, which secretes proteins from the cytosol, through the periplasim, and into the media. For more details go to: [https://2009.igem.org/Team:Washington/Project/Secretion| Secretion System ]. <br> <br />
<br />
Flanking each side of the NheI site are 6 consecutive hisitidines (6x-His) and TEV protease sites <sup>3</sup>. The histidines allow for traditional immobilized metal affinity chromatography ([https://2009.igem.org/Team:Washington/Notebook/IMAC_protocol IMAC]) protein purification. The TEV sites allows for the N and C terminal tags to be cleaved off of Afp, and due to the strategic placement of the 6x-His tags these tags can then be seperated from Afp by simply running the cleaved solution over a column in which the tags stick but Afp flows right through. <br><br />
<br />
='''Experiments'''=<br />
<br />
<br />
For the target vector our goal was to:<br />
* Construct the target vector<br />
* Insert GFP into the target vector and characterize expression and function<br />
* Biobrick and characterize OpdA, a nerve agent degrading enzyme<br />
* Insert OpdA into the target vector and characterize expression and function<br />
<br />
<br />
<br />
==Target Vector Construction==<br />
<br />
To create the target vector we first synthesized a coding sequence that would produce the protein as described above. To do this we synthesized a gene from oligo's as described in our [https://2009.igem.org/Team:Washington/Notebook/gene_synthesis| Gene Synthesis Protocol].<br />
<br />
==Expression Vector Construction==<br />
<br />
After creating the target construct, we created and BioBricked an expression vector ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K215000 BBa_K215000]) which would express the target protein upon induction with IPTG. We then added the target construct into the expression vector using standard assembly making [http://partsregistry.org/wiki/index.php?title=Part:BBa_K215002 BBa_K215002].<br />
<br />
==GFP Insertion and Characterization==<br />
<br />
Upon completion of constructing the target vector our first experiment was to determine if GFP was still functional as the fusion protein. To do this GFP ([http://partsregistry.org/wiki/index.php/Part:BBa_E0040 BBa_E0040]) was inserted into the target construct use the NheI method as described above. After insertion of E0040 into the target construct, the tagged GFP (target-GFP) was transformed into BL21(lacIq) cells, and subsequently grown in the presence or absence of IPTG. As a control an untagged E0040 was cloned into our expression vector and also grown in the presence or absence of IPTG. This would allow us to determine the effects of the tags on fluorescence. The cells were then washed with PBS, normalized to the same cell density, and fluorescence measured using an excitation of 485 and emission of 525 (cutoff at 515) in a SpectraMax M5e plate reader. The data is show below:<br />
<br />
[[image:GFP_Fluroescense_corrected_for_OD.png |500px | center]]<br />
<br />
<br />
From this data we were able to conclude that our expression vector was functional, as is evident from the large increase in fluorescence with the addition of IPTG. We were also able to conclude that the Target-GFP is functional, but fluorescence was significantly decreased. <br />
<br />
In order to ensure that the Target-GFP had the appropriate 6x-His tags and that fluorescence was a function of protein concentration we purified Target-GFP using a traditional IMAC techniques. The protein concentration was measured from its absorbence a 280nm. A serial dilution of the protein was then made and the resulting fluorescence measured as described earlier. The data is shown below:<br />
<br />
[[Image:Standard_curve_targGFP.png | 500px | center]]<br />
<br />
<br />
As expected the fluorescence intensity is linear with respect to protein concentration.<br />
<br />
==BioBricking and Characterization of OpdA==<br />
<br />
====OpdA Background====<br />
<br />
OpdA ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K215090 BBa_K215090]) is an organophosphate-degrading enzyme from ''Agrobacterium radiobacter''. It is capable of degrading a wide range of organophosphates, most notably pesticides that are poisonous to humans, such as paraoxon. We chose to biobrick and submit this enzyme to the registry for a number of reasons. First and foremost, this enzyme is easy to assay for since it can hydrolyze substrates very quickly (e.g. paraoxon) and form a bright yellow product. This yellow product would make it easy to see that the OpdA was present and functioning in our system. And secondly, OpdA is a very useful enzyme that could have applications in future iGEM and other synthetic biology projects, so its presence in the Standard Registry of Biological Parts is beneficial.<br />
<br />
====OpdA Characterization====<br />
<br />
The Baker lab donated the source plasmid for OpdA (a synthetic gene optimized for ''E. coli'' expression). SOEing PCR was used to remove BioBrick cut sites ([https://2009.igem.org/Team:Washington/Notebook/SOEingPCR SOE PCR Protocol]). Upon removal of the unwanted restriction sites the gene was cloned into pSB1A2 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K215090 BBa_K215090]), our expression vector ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K215091 BBa_K215091]), and the target vector. <br />
<br />
The first experiment carried out was to validate that we could express and purify functional OpdA. To do this we transformed BB# into a BL21(lacIq) cell line and followed a traditional protein production and purification procedure ([https://2009.igem.org/Team:Washington/Notebook/IMAC_protocol IMAC Protocol]). The resulting purified protein was then dialyzed overnight in 1x PBS to remove the elution buffer which we were worried would interfere with the activity assay. The concnetration of the dialysed protein was dteremined by meauring its absorbance at 280nM and using its predicted extinction coefficient (29575 M-1 cm-1,[http://ca.expasy.org/tools/protparam.html ProtParam]). We obtained ~1mL of 10microM protein. To determine the catalytic constants the nerve agent paroxoan was used a a substrate. As shown below, hydrolysis product of paroxoan is p-nitrophenol which has a strong absorbance at 400nM (and turns bright yellow).<br><br />
[[image:ParoxoanRxn2.png | 400px | center]]<br />
<br />
<br />
A serial dilution, ranging from 5 millimolar to 5 micromolar, of paroxoan was made in a reaction buffer (100mM HEPES pH=7, 500mM NaCl, 2mM CoCl2). To this a reaction OpdA was added so that it's final concentration was 1nM (dilutions were made in the reaction buffer). At all substrate concentrations no appreciable hydrolysis was observed without enzyme. The rate of hydrolysis with enzyme is shown below, the left hand plot is the full substrate range, and the right hand plot is a zoom in of the lower substrate concentrations:<br />
<br />
<gallery heights=400px widths=350><br />
image:OpdA_full.png<br />
image:OpdA_zoom.png<br />
</gallery><br />
<br />
From the above plot, it obvious that this enzyme efficiently catalysis paroxoan hydrolysis, but does not exhibit the usual Michaelis-Menten dynamics. It can be seen that at high enough concentrations, the enzyme actually undergoes substrate-inhibition, wherein the extra substrate actually slows the enzyme's velocity. When fit to a cononical substrate inhibition curve we obtain the following kinetic parameters:<br />
<br />
kcat (M-1 s-1): 17.6 <br><br />
Km (mM): 0.011 <br><br />
Ksi (mM): 1.06 <br><br />
<br />
These parameters confirm that this is an extremely efficient enzyme, and our kinetic parameters are comparable to previously published data for this enzyme on this substrate <sup>4,5</sup>. Also, substrate inhibition for this enzyme has been observed previously on similar substrates, so this was not an enitrely surprising results.<br />
<br />
Since the OpdA BioBrick was characterized and worked as expected we decided to continue and insert it into the target vector. Unfortinately when OpdA-Target was expressed and purified as described above no observable paroxoan hydrolysis was observed.<br />
<br />
<div style="text-align:right">'''Continue to [https://2009.igem.org/Team:Washington/Project/Secretion Secretion &gt;]'''</div><br />
<br />
='''Citations'''=<br />
<br />
<br />
1. Lamla and Erdmann. [http://www.ncbi.nlm.nih.gov/pubmed/14680960 The Nano-tag, a streptavidin-binding peptide for the purification and detection of recombinant proteins.]<br />
<br />
2. Palacios et al. [http://www.ncbi.nlm.nih.gov/pubmed/11157948 Subset of Hybrid Eukaryotic Proteins Is Exported by the Type I Secretion System of Erwinia chrysanthemi, Secrevtion of GFP in E. Coli].<br />
<br />
3. [http://www.cardiff.ac.uk/biosi/staffinfo/ehrmann/tools/TEVprot.html Tobacco Etch Virus (TEV) Protease general information]<br />
<br />
4. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC126808/ Irene Horne, et al. Identification of an opd (Organophosphate Degradation) Gene in an Agrobacterium Isolate.]<br />
<br />
5. [http://peds.oxfordjournals.org/cgi/content/abstract/16/2/135 H.Yang, et al. Evolution of an organophosphate-degrading enzyme:a comparison of natural and directed evolution.]<br />
<br />
<br />
<br />
<br />
{{Template:Team:Washington/Templates/Footer}}</div>Acleonehttp://2009.igem.org/Team:Washington/ProjectTeam:Washington/Project2009-10-19T04:53:41Z<p>Acleone: </p>
<hr />
<div>__NOTOC__<br />
{{Template:Team:Washington/Templates/Header}}<br />
<br />
== Traditional Protein Purification vs Our System: Ideal Protein Purification ==<br />
<br />
The ability to quickly and cheaply generate purified proteins is a useful skill for aspiring synthetic biologists and iGEM participants alike, since purified proteins are used in many key laboratory processes. Although some commonly used proteins (like the restriction enzymes EcoRI, XbaI, SpeI, and PstI) can be bought, many applications require synthetic biologists to purify their own proteins, either to study or for use in atypical biological processes. Many members of our team have personal experience purifying proteins and have found it to be a slow and tedious process. For these reasons, the UW 2009 iGEM team has designed an Idealized Protein Purification (IPP) scheme designed to speed up protein purification, in the hopes of making protein purification facile and accessible for synthetic biologists, as well as potentially making previously difficult-to-purify proteins easier to generate.<br />
<br />
==Traditional Protein Production and Purification==<br />
<br />
[[Image:OldPurificationGraphic.png|500px|center]]<br />
<br />
Recombinant protein purification<sup>1</sup> is a method whose basic steps have not changed significantly in at least the last 20 years - thus we believe this process is due for an overhaul. Traditional protein purification steps require expensive and specialized equipment (such as an ultracentrifuge and columns that bind His tags) that may not be accessible to the average synthetic biologist. Traditional protein production and purification typically consists of the following three protein production steps and six purification steps:<br />
<br />
<br />
*TRADITIONAL PROTEIN PRODUCTION<sup>1</sup><br />
# Clone in a favorite protein (referenced here as afp) into an expression plasmid designed to generate lots of protein fused with a His tag. <br />
# Transform this plasmid into an expression strain of bacteria specialized in protein production.<br />
# Culture cells and induce protein expression, allowing cells to grow to high density.<br />
*TRADITIONAL PROTEIN PURIFICATION<sup>1</sup><br />
# Pellet cells, re-suspend in a lysis buffer and incubate to ensure full cell lysis, releasing proteins trapped within the cell into the media. <br />
# Subject cell debris to a long, high-speed spin in an ultra-centrifuge to remove remaining insoluble matter (membrane, insoluble proteins, etc) and leave soluble proteins in the supernatant.<br />
# Filter the supernatant to remove any lingering debris before being added to an affinity column. This ensures that the columns do not get clogged.<br />
# Run the filtered supernatant over an affinity column several times to ensure thorough binding of His-tagged Afp to affinity column.<br />
# Flow wash buffer over column to eliminate proteins that non-specifically bind to the column.<br />
# Collect Afp by running an elution buffer over the column to release Afp.<br />
<br />
<br />
Though not necessarily hard, the traditional purification process is tedious and time consuming, taking us about three and a half hours to complete on purification day. With this in mind, we created an idealized protein purification scheme that is easier, cheaper and faster than the traditional method.<br />
<br />
==Idealized Protein Production and Purification==<br />
<br />
[[Image:NewPurificationGraphic.png|500px|center]]<br />
<br />
To avoid all the laborious purification steps present in traditional protein purification we designed a system (Ideal Protein Purification) in which the cell, not the researcher, performs the purification steps to make recombinant protein product. Ideal Protein Purification reduces purification to two easy steps (cloning remains the same):<br />
<br />
<br />
*IDEALIZED PROTEIN PRODUCTION (IPP)<br />
# Clone afp into the IPP "target vector". This creates a fusion protein in which a "display tag" <sup>2</sup> (called the Nano-Tag) is fused to the to the N-terminus of Afp, and a "secretion tag" to the C-terminus. <br />
# Transform the target vector with the afp insert into cells already containing two additional plasmids. The first is a "Secretion plasmid", which contains all the parts of a type 1 secretion system from ''Erwinia chrysanthemi'' <sup>3</sup> that specifically recognizes proteins that harbor the secretion tag and exports them outside of the cell. The second is a "Display plasmid" which expresses a protein that is directed to the cell surface <sup>4</sup> and can bind to the "display tag", and then subsequently release the display tag when a specific small molecule is introduced to the system<sup>2</sup>. <br />
# Grow up cells in our specialized cell line that contains both Secretion and Display plasmids and induce the expression of afp, just as in the traditional protein purification method. As the protein is produced, it gets secreted to the media via the secretion system, and then binds to the outside of the cells via the display system. Now your protein of interest is non-covalently attached to the outside of the cell.<br />
*IDEALIZED PROTEIN PURIFICATION<br />
# Spin down the cells and re-suspend in an elution buffer containing a small molecule which disrupts the interaction between the display tag and the protein it was binding to on the surface of the cells. This causes Afp to be released from the surface of the cell.<br />
# Collect Afp by pelleting the cells again and keeping the supernatant, which contains your purified protein.<br />
<br />
<br />
This process should take about 10 minutes, quick enough to do between classes! In addition to saving time, our idealized protein purification method has other potential advantages over older methods. By keeping the cells intact, contaminants are not released into the media, which should result in purer protein product. A host of proteins normally toxic to ''E. coli'' in the traditional protein purification system could be grown in the Ideal Protein Purification system since they would be secreted outside of the cell before reaching high intra-cellular concentrations. Alternately, some proteins cannot properly fold in ''E. coli'', but by secreting these proteins into the appropriate media, they might fold correctly. Finally, there are some environmental applications, such as detoxification, where it is desirable to have a bacterium capable of secreting protein into the environment, which our system could be easily modified to do.<br />
<br />
'''Continue to [https://2009.igem.org/Team:Washington/Project/Target Target &gt;]'''<br />
<br />
----<br />
====Citations====<br />
#Protein production and purification, Structural Genomics Consortium et.al. Nature Methods Vol.5 NO.2, February 2008<br />
#TAG: Lamla and Erdmann. [http://www.ncbi.nlm.nih.gov/pubmed/14680960 The Nano-tag, a streptavidin-binding peptide for the purification and detection of recombinant proteins.]<br />
#Palacios et al. [http://www.ncbi.nlm.nih.gov/pubmed/11157948 Subset of Hybrid Eukaryotic Proteins Is Exported by the Type I Secretion System of Erwinia chrysanthemi, Secretion of GFP in E. Coli]<br />
#Cell Surface Streptavidin, P. Tsai. IET Synth. Biol., Vol. 1, No. 1–2, 2007<br />
<br />
{{Template:Team:Washington/Templates/Footer}}</div>Acleonehttp://2009.igem.org/Team:Washington/ProjectTeam:Washington/Project2009-10-19T04:53:22Z<p>Acleone: </p>
<hr />
<div>__NOTOC__<br />
{{Template:Team:Washington/Templates/Header}}<br />
<br />
== Traditional Protein Purification vs Our System: Ideal Protein Purification ==<br />
<br />
The ability to quickly and cheaply generate purified proteins is a useful skill for aspiring synthetic biologists and iGEM participants alike, since purified proteins are used in many key laboratory processes. Although some commonly used proteins (like the restriction enzymes EcoRI, XbaI, SpeI, and PstI) can be bought, many applications require synthetic biologists to purify their own proteins, either to study or for use in atypical biological processes. Many members of our team have personal experience purifying proteins and have found it to be a slow and tedious process. For these reasons, the UW 2009 iGEM team has designed an Idealized Protein Purification (IPP) scheme designed to speed up protein purification, in the hopes of making protein purification facile and accessible for synthetic biologists, as well as potentially making previously difficult-to-purify proteins easier to generate.<br />
<br />
==Traditional Protein Production and Purification==<br />
<br />
[[Image:OldPurificationGraphic.png|500px|center]]<br />
<br />
Recombinant protein purification<sup>1</sup> is a method whose basic steps have not changed significantly in at least the last 20 years - thus we believe this process is due for an overhaul. Traditional protein purification steps require expensive and specialized equipment (such as an ultracentrifuge and columns that bind His tags) that may not be accessible to the average synthetic biologist. Traditional protein production and purification typically consists of the following three protein production steps and six purification steps:<br />
<br />
<br />
*TRADITIONAL PROTEIN PRODUCTION<sup>1</sup><br />
# Clone in a favorite protein (referenced here as afp) into an expression plasmid designed to generate lots of protein fused with a His tag. <br />
# Transform this plasmid into an expression strain of bacteria specialized in protein production.<br />
# Culture cells and induce protein expression, allowing cells to grow to high density.<br />
*TRADITIONAL PROTEIN PURIFICATION<sup>1</sup><br />
# Pellet cells, re-suspend in a lysis buffer and incubate to ensure full cell lysis, releasing proteins trapped within the cell into the media. <br />
# Subject cell debris to a long, high-speed spin in an ultra-centrifuge to remove remaining insoluble matter (membrane, insoluble proteins, etc) and leave soluble proteins in the supernatant.<br />
# Filter the supernatant to remove any lingering debris before being added to an affinity column. This ensures that the columns do not get clogged.<br />
# Run the filtered supernatant over an affinity column several times to ensure thorough binding of His-tagged Afp to affinity column.<br />
# Flow wash buffer over column to eliminate proteins that non-specifically bind to the column.<br />
# Collect Afp by running an elution buffer over the column to release Afp.<br />
<br />
<br />
Though not necessarily hard, the traditional purification process is tedious and time consuming, taking us about three and a half hours to complete on purification day. With this in mind, we created an idealized protein purification scheme that is easier, cheaper and faster than the traditional method.<br />
<br />
==Idealized Protein Production and Purification==<br />
<br />
[[Image:NewPurificationGraphic.png|500px|center]]<br />
<br />
To avoid all the laborious purification steps present in traditional protein purification we designed a system (Ideal Protein Purification) in which the cell, not the researcher, performs the purification steps to make recombinant protein product. Ideal Protein Purification reduces purification to two easy steps (cloning remains the same):<br />
<br />
<br />
*IDEALIZED PROTEIN PRODUCTION (IPP)<br />
# Clone afp into the IPP "target vector". This creates a fusion protein in which a "display tag" <sup>2</sup> (called the Nano-Tag) is fused to the to the N-terminus of Afp, and a "secretion tag" to the C-terminus. <br />
# Transform the target vector with the afp insert into cells already containing two additional plasmids. The first is a "Secretion plasmid", which contains all the parts of a type 1 secretion system from ''Erwinia chrysanthemi'' <sup>3</sup> that specifically recognizes proteins that harbor the secretion tag and exports them outside of the cell. The second is a "Display plasmid" which expresses a protein that is directed to the cell surface <sup>4</sup> and can bind to the "display tag", and then subsequently release the display tag when a specific small molecule is introduced to the system<sup>2</sup>. <br />
# Grow up cells in our specialized cell line that contains both Secretion and Display plasmids and induce the expression of afp, just as in the traditional protein purification method. As the protein is produced, it gets secreted to the media via the secretion system, and then binds to the outside of the cells via the display system. Now your protein of interest is non-covalently attached to the outside of the cell.<br />
*IDEALIZED PROTEIN PURIFICATION<br />
# Spin down the cells and re-suspend in an elution buffer containing a small molecule which disrupts the interaction between the display tag and the protein it was binding to on the surface of the cells. This causes Afp to be released from the surface of the cell.<br />
# Collect Afp by pelleting the cells again and keeping the supernatant, which contains your purified protein.<br />
<br />
<br />
This process should take about 10 minutes, quick enough to do between classes! In addition to saving time, our idealized protein purification method has other potential advantages over older methods. By keeping the cells intact, contaminants are not released into the media, which should result in purer protein product. A host of proteins normally toxic to ''E. coli'' in the traditional protein purification system could be grown in the Ideal Protein Purification system since they would be secreted outside of the cell before reaching high intra-cellular concentrations. Alternately, some proteins cannot properly fold in ''E. coli'', but by secreting these proteins into the appropriate media, they might fold correctly. Finally, there are some environmental applications, such as detoxification, where it is desirable to have a bacterium capable of secreting protein into the environment, which our system could be easily modified to do.<br />
<br />
'''Continue to [https://2009.igem.org/Team:Washington/Project/Target Target]'''<br />
<br />
----<br />
====Citations====<br />
#Protein production and purification, Structural Genomics Consortium et.al. Nature Methods Vol.5 NO.2, February 2008<br />
#TAG: Lamla and Erdmann. [http://www.ncbi.nlm.nih.gov/pubmed/14680960 The Nano-tag, a streptavidin-binding peptide for the purification and detection of recombinant proteins.]<br />
#Palacios et al. [http://www.ncbi.nlm.nih.gov/pubmed/11157948 Subset of Hybrid Eukaryotic Proteins Is Exported by the Type I Secretion System of Erwinia chrysanthemi, Secretion of GFP in E. Coli]<br />
#Cell Surface Streptavidin, P. Tsai. IET Synth. Biol., Vol. 1, No. 1–2, 2007<br />
<br />
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<br />
=== The Idealized Protein Purification System: Improving the lives of molecular biologists ===<br />
<br />
The use of recombinant protein production using ''E. coli''-based protein expression systems has revolutionized the fields of biotechnology and medicine. Although generating purified protein plays a central role in the field of biotechnology, the production of purified protein is a time-consuming and laborious procedure that requires expensive and specialized equipment, and, we believe, is therefore not suited to today's thrifty synthetic biologist. Our project, the Ideal Protein Purification System, aims to create an all-in-one protein expression and purification system using BioBrick standards to greatly simplify protein production for synthetic biologists, reducing the time and cost involved in standard protein purification methods. Our method uses a novel combination of two systems: secretion and display. In our method, your protein of interest is fused to two separate tags: a secretion tag and a display tag. The secretion tag directs protein secretion via our BioBricked Secretion System outside of the cell into the growth media. The display tag binds to the Display System which is located on the cell surface. Collecting your purified target protein is as simple as pelleting cells and re-suspending them in an elution buffer, releasing the protein of interest. Our research exhibits the utility of synthetic biology for developing new techniques that improve upon established practices.<br />
<br />
'''Continue to [https://2009.igem.org/Team:Washington/Project Project Description &gt;]'''<br />
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<br /><br />
<br />
=== The Idealized Protein Purification System: Improving the lives of molecular biologists ===<br />
<br />
The use of recombinant protein production using ''E. coli''-based protein expression systems has revolutionized the fields of biotechnology and medicine. Although generating purified protein plays a central role in the field of biotechnology, the production of purified protein is a time-consuming and laborious procedure that requires expensive and specialized equipment, and, we believe, is therefore not suited to today's thrifty synthetic biologist. Our project, the Ideal Protein Purification System, aims to create an all-in-one protein expression and purification system using BioBrick standards to greatly simplify protein production for synthetic biologists, reducing the time and cost involved in standard protein purification methods. Our method uses a novel combination of two systems: secretion and display. In our method, your protein of interest is fused to two separate tags: a secretion tag and a display tag. The secretion tag directs protein secretion via our BioBricked Secretion System outside of the cell into the growth media. The display tag binds to the Display System which is located on the cell surface. Collecting your purified target protein is as simple as pelleting cells and re-suspending them in an elution buffer, releasing the protein of interest. Our research exhibits the utility of synthetic biology for developing new techniques that improve upon established practices.<br />
<br />
'''Continue to [https://2009.igem.org/Team:Washington/Project Project Description &gt;]'''<br />
<br />
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<br /><br />
<br />
=== The Idealized Protein Purification System: Improving the lives of molecular biologists ===<br />
<br />
The use of recombinant protein production using ''E. coli''-based protein expression systems has revolutionized the fields of biotechnology and medicine. Although generating purified protein plays a central role in the field of biotechnology, the production of purified protein is a time-consuming and laborious procedure that requires expensive and specialized equipment, and, we believe, is therefore not suited to today's thrifty synthetic biologist. Our project, the Ideal Protein Purification System, aims to create an all-in-one protein expression and purification system using BioBrick standards to greatly simplify protein production for synthetic biologists, reducing the time and cost involved in standard protein purification methods. Our method uses a novel combination of two systems: secretion and display. In our method, your protein of interest is fused to two separate tags: a secretion tag and a display tag. The secretion tag directs protein secretion via our BioBricked Secretion System outside of the cell into the growth media. The display tag binds to the Display System which is located on the cell surface. Collecting your purified target protein is as simple as pelleting cells and re-suspending them in an elution buffer, releasing the protein of interest. Our research exhibits the utility of synthetic biology for developing new techniques that improve upon established practices.<br />
<br />
'''Continue to [https://2009.igem.org/Team:Washington/Project Project Description]'''<br />
<br />
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<br />
== Traditional Protein Purification vs Our System: Ideal Protein Purification ==<br />
<br />
The ability to quickly and cheaply generate purified proteins is a useful skill for aspiring synthetic biologists and iGEM participants alike, since purified proteins are used in many key laboratory processes. Although some commonly used proteins (like the restriction enzymes EcoRI, XbaI, SpeI, and PstI) can be bought, many applications require synthetic biologists to purify their own proteins, either to study or for use in atypical biological processes. Many members of our team have personal experience purifying proteins and have found it to be a slow and tedious process. For these reasons, the UW 2009 iGEM team has designed an Idealized Protein Purification (IPP) scheme designed to speed up protein purification, in the hopes of making protein purification facile and accessible for synthetic biologists, as well as potentially making previously difficult-to-purify proteins easier to generate.<br />
<br />
==Traditional Protein Production and Purification==<br />
<br />
[[Image:OldPurificationGraphic.png|500px|center]]<br />
<br />
Recombinant protein purification<sup>1</sup> is a method whose basic steps have not changed significantly in at least the last 20 years - thus we believe this process is due for an overhaul. Traditional protein purification steps require expensive and specialized equipment (such as an ultracentrifuge and columns that bind His tags) that may not be accessible to the average synthetic biologist. Traditional protein production and purification typically consists of the following three protein production steps and six purification steps:<br />
<br />
<br />
*TRADITIONAL PROTEIN PRODUCTION<sup>1</sup><br />
# Clone in a favorite protein (referenced here as afp) into an expression plasmid designed to generate lots of protein fused with a His tag. <br />
# Transform this plasmid into an expression strain of bacteria specialized in protein production.<br />
# Culture cells and induce protein expression, allowing cells to grow to high density.<br />
*TRADITIONAL PROTEIN PURIFICATION<sup>1</sup><br />
# Pellet cells, re-suspend in a lysis buffer and incubate to ensure full cell lysis, releasing proteins trapped within the cell into the media. <br />
# Subject cell debris to a long, high-speed spin in an ultra-centrifuge to remove remaining insoluble matter (membrane, insoluble proteins, etc) and leave soluble proteins in the supernatant.<br />
# Filter the supernatant to remove any lingering debris before being added to an affinity column. This ensures that the columns do not get clogged.<br />
# Run the filtered supernatant over an affinity column several times to ensure thorough binding of His-tagged Afp to affinity column.<br />
# Flow wash buffer over column to eliminate proteins that non-specifically bind to the column.<br />
# Collect Afp by running an elution buffer over the column to release Afp.<br />
<br />
<br />
Though not necessarily hard, the traditional purification process is tedious and time consuming, taking us about three and a half hours to complete on purification day. With this in mind, we created an idealized protein purification scheme that is easier, cheaper and faster than the traditional method.<br />
<br />
==Idealized Protein Production and Purification==<br />
<br />
[[Image:NewPurificationGraphic.png|500px|center]]<br />
<br />
To avoid all the laborious purification steps present in traditional protein purification we designed a system (Ideal Protein Purification) in which the cell, not the researcher, performs the purification steps to make recombinant protein product. Ideal Protein Purification reduces purification to two easy steps (cloning remains the same):<br />
<br />
<br />
*IDEALIZED PROTEIN PRODUCTION (IPP)<br />
# Clone afp into the IPP "target vector". This creates a fusion protein in which a "display tag" <sup>2</sup> (called the Nano-Tag) is fused to the to the N-terminus of Afp, and a "secretion tag" to the C-terminus. <br />
# Transform the target vector with the afp insert into cells already containing two additional plasmids. The first is a "Secretion plasmid", which contains all the parts of a type 1 secretion system from ''Erwinia chrysanthemi'' <sup>3</sup> that specifically recognizes proteins that harbor the secretion tag and exports them outside of the cell. The second is a "Display plasmid" which expresses a protein that is directed to the cell surface <sup>4</sup> and can bind to the "display tag", and then subsequently release the display tag when a specific small molecule is introduced to the system<sup>2</sup>. <br />
# Grow up cells in our specialized cell line that contains both Secretion and Display plasmids and induce the expression of afp, just as in the traditional protein purification method. As the protein is produced, it gets secreted to the media via the secretion system, and then binds to the outside of the cells via the display system. Now your protein of interest is non-covalently attached to the outside of the cell.<br />
*IDEALIZED PROTEIN PURIFICATION<br />
# Spin down the cells and re-suspend in an elution buffer containing a small molecule which disrupts the interaction between the display tag and the protein it was binding to on the surface of the cells. This causes Afp to be released from the surface of the cell.<br />
# Collect Afp by pelleting the cells again and keeping the supernatant, which contains your purified protein.<br />
<br />
<br />
This process should take about 10 minutes, quick enough to do between classes! In addition to saving time, our idealized protein purification method has other potential advantages over older methods. By keeping the cells intact, contaminants are not released into the media, which should result in purer protein product. A host of proteins normally toxic to ''E. coli'' in the traditional protein purification system could be grown in the Ideal Protein Purification system since they would be secreted outside of the cell before reaching high intra-cellular concentrations. Alternately, some proteins cannot properly fold in ''E. coli'', but by secreting these proteins into the appropriate media, they might fold correctly. Finally, there are some environmental applications, such as detoxification, where it is desirable to have a bacterium capable of secreting protein into the environment, which our system could be easily modified to do.<br />
<br />
'''Continue to [https://2009.igem.org/Team:Washington/Project/Target Target]'''<br />
<br />
----<br />
====Citations====<br />
#Protein production and purification, Structural Genomics Consortium et.al. Nature Methods Vol.5 NO.2, February 2008<br />
#TAG: Lamla and Erdmann. [http://www.ncbi.nlm.nih.gov/pubmed/14680960 The Nano-tag, a streptavidin-binding peptide for the purification and detection of recombinant proteins.]<br />
#Palacios et al. [http://www.ncbi.nlm.nih.gov/pubmed/11157948 Subset of Hybrid Eukaryotic Proteins Is Exported by the Type I Secretion System of Erwinia chrysanthemi, Secretion of GFP in E. Coli]<br />
#Cell Surface Streptavidin, P. Tsai. IET Synth. Biol., Vol. 1, No. 1–2, 2007<br />
<br />
{{Template:Team:Washington/Templates/Footer}}</div>Acleonehttp://2009.igem.org/Team:Washington/Project/DisplayTeam:Washington/Project/Display2009-10-19T04:46:03Z<p>Acleone: </p>
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<br />
[[Image:Main_graphic3_display_banner.png|center]]<br />
<br />
='''Background'''=<br />
Our system hinged on finding a protein which could bind other proteins to the outside of the cell, but whose interaction with these proteins was weak enough to be disrupted by another small molecule. streptavidin presented itself as a logical choice. Several protein tags (such as the Nano-Tag <sup>1</sup>) have been developed such that they bind streptavidin at the biotin binding site, but can be released when biotin is added to the system and binds to streptavidin. The ability for peptides to bind streptavidin and be released upon the addition of biotin is a technology currently used on a daily basis world-wide by labs purifying proteins. The major difference between the traditional system and our system is that streptavidin is attached to beads which are used in a column format in the traditional protein purification method. Our system will have streptavidin attached to the surface of the cell. Combining the display system with our target and secretion vector our vision is complete. A single cell can then produce, secrete, bind, and release any protein of interest.<br />
{| cellpadding="1" cellspacing="8"<br />
|-<br />
|[[Image:Strept-biotin.png | 175px]]<br />
|The binding of the Nano-Tag by streptavidin has an affinity of 4nM and has been used to purify multiple proteins. The ability of biotin to compete off the Nano-Tag in biding assays indicates that the binding occurs at the same site of streptavidin. Of great importance for incorporating streptavidin into our display system is that streptavidin is a common protein used in bio-chemical assays. Its ability to bind biotin is one of the strongest non-covalent interactions known. This allows for the utilization of biotin and streptavidin to act as molecular connectors in protein systems. Furthermore the availability of biotinylated and streptavidin linked fluorophores allows for an easy assay of their presence, using fluorescence microscopy<sup>2</sup>.<br />
|-<br />
|}<br />
<br />
{| cellpadding="1" cellspacing="8"<br />
|-<br />
|Next we needed a way to anchor the streptavidin to the outer membrane. The obvious choice was to use the LPP signal peptide and Outer Membrane Protein A (ompA) because this system has been extensively characterized as an expression system to display proteins on the cell surface. This LPP-ompA system has been used to display a diverse group of proteins, including Green Fluorescent Protein (GFP) <sup>3</sup>, Organophosphorus Hydrolase (OPH) <sup>4</sup>, Cyclodextrin Glucanotransferase (CGTase) <sup>5</sup>, Methyl Parathion Hydrolase (MPH) <sup>6</sup>, Enhanced Green Fluorescent Protein (EGFP) <sup>6</sup>. Thus, an appropriate display construct for our system would be Lpp-OmpA display construct fused to streptavidin, which would be anchored in the outer membrane and display streptavidin on the surface of the cell - which would bind a nanotag on our target protein in an interaction that could be disrupted upon the addition of biotin. <br />
|[[Image:Display image.jpg|400px|right]]<br />
|-<br />
|}<br><br />
==== Harvard 2006 Surface Display System: Legacy Parts ====<br />
<br />
<br />
When we checked the Parts Registry to see if someone had already built this or sometime similar that we could use as a starting part for our Idealized Protein Purification Display System, we found this Lpp-OmpA-streptavidin construct, submitted by Harvard iGEM team as part of their project in 2006 <sup>7</sup>. In fact, Harvard had submitted four biobrick parts which were all variations on the same theme. All four are fusion proteins which have a LPP signal peptide, either one or five trans-membrane ompA, and either monomeric or dimeric streptavidin. All the variations and the resulting biobricks are shown below.<br />
<br />
<br />
<br />
{|cellpadding="3" cellspacing="1" border="2" width="75%" align="center"<br />
! Bio Brick<br />
! OmpA trans-membrane domains<br />
! Type of Streptavidin<br />
|- align="center"<br />
|J36848<br />
|1<br />
|monomeric<br />
|- align="center"<br />
|J36849<br />
|1<br />
|dimeric<br />
|- align="center"<br />
|J36850<br />
|5<br />
|monomeric<br />
|- align="center"<br />
|J36851<br />
|5<br />
|dimeric<br />
|-<br />
|}<br />
<br><br />
<br />
==== Harvard 2006 Surface Display System: Documented Data ====<br />
<br />
Data regarding the activity of the Harvard iGEM 2006 Lpp-OmpA-Streptavidin construct has been published in IET Synthetic Biology in 2007 <sup>7</sup>. The author showed, by Western blot against the His tag fused to this construct, that their protein was being expressed within the cell. In order to demonstrate binding of their cell-surface-anchored streptavidin to biotin, the author incubated cells expressing Lpp-OmpA-streptavidin with an aptamer that linked biotin to a fluorophore using an oligonucleotide linker (see figure below). Any cells that express streptavidin on the surface of the cell should fluoresce in their assay - concordantly, when cells were incubated with this biotin-oligo-fluorescein aptamer, the author saw fluorescence on cells expressing Lpp-OmpA-Streptavidin, but not on Lpp-OmpA negative control. However, cells that expressed streptavidin on the surface also bound to the negative aptamer control (oligo-fluorescein) that lacked biotin, indicating that the oligo aptamer itself (and not biotin) may have been responsible for the association of the fluorophore to the cell surface. Thus, the Harvard 2006 iGEM team was never able to definitively show specific affinity of their Lpp-OmpA-Streptavidin construct.<br />
<br />
{|<br />
|-<br />
|[[Image:HarvardIETSYNTH.jpg|400px|center]]<br />
|''<br />
L01W: Lpp-OmpA-Streptavidin monomer, no His Tag.<br><br />
L01H: Lpp-OmpA-Streptavidin monomer with His tag.<br><br />
L01S: Lpp-OmpA-Streptavidin dimer.<br><br />
L01: Lpp-OmpA alone).<br><br />
A: no aptamer added.<br><br />
B: With only ‘F’ 50 -fluorescently-tagged oligonucleotide.<br><br />
C: With fluorescently tagged streptavidin aptamer.<br><br />
D: With ‘B-F’ hybrid of 50 -biotinylated oligonucleotide annealed with 50-fluorescently-tagged oligonucleotide<sup>7</sup>.''<br />
|-<br />
|}<br />
<br />
<br />
Based on these experiments, we decided to try using this display system for our purpose. But first, since binding of the system to streptavidin had not be definitively shown, we had to verify binding of the Lpp-OmpA-Streptavidin construct to biotin for ourselves, and characterize this interaction.<br />
<br />
='''Experiments'''=<br />
== Harvard's Legacy 2006 Cell Surface Display Parts: Do They Work As Predicted? ==<br />
<br />
In order to determine whether the Harvard 2006 iGEM streptavidin cell surface display parts were 1. expressed properly, and 2. bound biotin on the surface of the cell, we decided to do the following set of tests. <br />
<br />
# Western to verify expression of Harvard 2006 surface display parts <br />
# Assessment of biotin binding to cell surface by visualization in fluorescence microscope <br />
# Assessment of biotin binding to cell surface by visualization in flow cytometer<br />
<br />
<br />
=== Western Blot Demonstrates that Harvard 2006 iGEM Parts Are Expressed ===<br />
The goal of this experiment was to make sure that the proteins were being expressed in our cell lines, and also to make sure that they were the correct length. This was crucial to ascertain before we moved on and began to test the parts. Even though we had the individual parts sequenced confirmed we needed to make sure that the proteins were being expressed correctly in the cell. Since all our the Harvard parts are conveniently his tagged, we used a Western blot reagent (horseradish peroxidase) that was conjugated to nickel-NTA (binds His tags)<sup>8</sup>.<br />
<br />
<br />
==== Data ====<br />
<br />
[[Image:HarvardExpressionWestern.jpg|700px|center]]<br />
<br><br />
<br />
The expected sizes of each of these proteins are shown in the table below:<br />
{|cellpadding="3" cellspacing="1" border="2" width="50%"<br />
! Bio Brick<br />
! Length (in Da)<br />
|- align="center"<br />
|J36848<br />
|21478.7<br />
|- align="center"<br />
|J36849<br />
|34600.9<br />
|- align="center"<br />
|J36850<br />
|31215.4<br />
|- align="center"<br />
|J36851<br />
|44972.3<br />
|-<br />
|}<br />
<br />
<br><br />
Based on the above table, we were able to verify expression of the Harvard 2006 iGEM surface display parts. The varying intensity of the bands does not indicate the strength of expression because the protein amount was not normalized before it was inserted into the gel. Next we wanted to determine whether functional, biotin-binding streptavidin was displayed on the cell surface in cells expressing these parts. To test this, we used two different methods: visualization by fluorescence microscopy and by flow cytometer. <br><br />
<br />
=== Streptavidin-Biotin Binding Is Not Visualized By Fluorescence Microscopy ===<br />
<br />
The goal of this experiment was to confirm the display of streptavidin on the surface of the cell. To do this, we incubated cells expressing the Harvard 2006 iGEM streptavidin surface display parts with a fluorophore conjugated to biotin. If cells are expressing functional streptavidin on the surface of the cell, they should bind the biotinylated fluorophore, and this binding should be detectable in our florescence microscope as a halo of fluorescence surrounding each individual cell. As a positive control for streptavidin-biotin binding we incubated the biotinylated fluorophore with streptavidin-coated beads that were roughly the same size (with respect to volume) as our cells (except spherical) (see Notebook page for protocols).<br />
<br />
<br />
==== Data ====<br />
<br />
<table><br />
<tr> <br />
<td><p style="font-size:18px"> Positive Control </p> </td><br />
<td><p style="font-size:18px"> Negative Control </p> </td><br />
<td rowspan="4">These images were analyzed using imageJ <sup>9</sup>. The image on the left shows an image of the beads with flourophore added to them. These beads were diluted and spun diluted and spun down until the background level of florescence was low enough to get an accurate reading. We then used imageJ to analyze the intensity of a line going through the bead, which is demonstrated by the above schematic. On the positive control the edges of the streptavidin-coated beads show spikes in florescence, indicating binding of the biotinylated fluorophore to the beads. The negative control (beads without fluorophore) showed no such increase. This meant that our biotinylated flouophore binds to streptavidin in a detectable manner. When our cells were examined in the same manner, no difference could be seen in biotinylated fluorophore binding between cells that had induced expression of surface streptavidin (left) and uninduced cells which should not express the surface display protein (right). This was evidence that the streptavidin surface display part binding to biotin was very low / nonexistent. In order to verify this, we measured the binding of the biotinylated fluorophore to entire populations of streptavidin-expressing cells by flow cytomtery.</td><br />
</tr><br />
<tr><br />
<td>[[Image:M_beads1.png| 210px]]</td><br />
<td>[[Image:M_beads2.png| 210px]]</td><br />
</tr><br />
<td><p style="font-size:18px"> Induced Cells </p></td><br />
<td><p style="font-size:18px"> Uninduced Cells </p></td><br />
<tr><br />
<td>[[Image:M_cells1.png| 210px]]</td><br />
<td>[[Image:M_cells2.png| 210px]]</td><br />
</tr><br />
</table><br />
<br />
=== Streptavidin-Biotin Binding Is Not Visualized By Flow Cytometry ===<br />
The goal of this experiment was to visualize biotin binding to streptavidin over a whole population of cells. Whereas the Microscope experiment looked at a few localized cells, we set the cytometer to look 50,000 streptavidin-expressing cells after incubation (and washing) with the biotinylated fluorophore and read the resulting florescence ([https://2009.igem.org/Team:Washington/Notebook/Flow_Cytometry see Notebook page for protocols]). Like in the microscopy assay, we used streptavidin-coated beads as a positive control. The cytometer reads the flouresence of each individual particle (cell or bead) passing through it, which allows for accurate readings, and monitoring of a much larger sample size than we visualized under the microscope. The results of this experiment are described below.<br />
<br />
<br />
==== Data ====<br />
<br />
<gallery heights=300px widths=350><br />
Image:48.png|'''BBa_J36848''' ''This image shows both the induced and uninduced cells for part 48 in varying levels of flourophore (0nM to 100nM). The cells were induced at 1mM IPTG. This data shows that there is no appreciable difference between the induced and uninduced cells at any given level of flourophore. All curves appear to have the same amount of fluorescence. We found similar results using a microscopy assay.''<br />
Image:StreptBead cyto.png|'''+ Control''' ''We used the sreptavadin coated to show us what the magnitude of fluorescence increase we should see with increased flourophore levels. We were able to see that as the level of fourophore was increased we could see increased retention between the beads and the flouophore. The black is beads with no flouophore, the red is with 10 nM, and the purple is 100 nM. These showed a clear difference between the beads without flourophore, and the beads with flourophore. The cells shown at right, matched the readings of the beads when they had no flourophore added.''<br />
</gallery><br />
<br />
In the flow cytometer, our positive control, streptavidin-coated beads, showed a clear distinction between beads pre-incubated with biotinylated flourophore and the beads without flourophore, indicating that this assay is capable of visualizing streptavidin binding to the biotinylated fluorophore. Like in the microscopy assay, we did not observe appreciable binding occurring with any of the ompA-streptavidin parts to biotin at the population level. This data only shows part number 48. However this data is applicable to all of the parts we observed. All streptavidin-expressing cells demonstrated low levels of florescence comparable to those of beads not pre-incubated with fluorophore.<br />
<br />
=== Conclusion ===<br />
<br />
<br />
In conclusion, we demonstrated expression of the Harvard 2006 streptavidin surface display parts via Western Blot. We were unable, however, to visualize binding between a biotinylated flourophore and the cells expressing these proteins. This indicates that the streptavidin display system is likely not binding biotin correctly. We hypothesize that streptavidin might not having enough room on the cell surface to form tetramers (which is its native state), and thus may not be binding to biotin with high efficiency. In order to generate a construct that demonstrates tight binding to to biotin, we propose to design a generalized cell surface display system, and to computationally design a biotin-binding streptavidin monomer using software developed at the University of Washington.<br><br />
<br />
'''These solutions are described in the [https://2009.igem.org/wiki/index.php?title=Team:Washington/Future future directions section].'''<br />
<br />
----<br />
<br />
=== Citations ===<br />
#The Nano-tag, a streptavidin-binding peptide for the purification and detection of recombinant proteins doi:10.1016/j.physletb.2003.10.071<br />
#Structural origins of high-affinity biotin binding to streptavidin. PC Weber, DH Ohlendorf, JJ Wendoloski, and FR Salemme (6 January 1989). Science 243 (4887), 85. [DOI: 10.1126/science.2911722]<br />
#Display of green fluorescent protein on ''Escherichia coli'' cell surface. Shi H, Wen Su W. Department of Biological & Agricultural Engineering, University of Missouri-Columbia, 65211, Columbia, MO, USAEnzyme Microb Technol. 2001 Jan 2;28(1):25-34.<br />
#Cell surface display of organophosphorus hydrolase using ice nucleation protein. Shimazu M, Mulchandani A, Chen W. Biotechnol Prog. 2001 Jan-Feb;17(1):76-80. Department of Chemical and Environmental Engineering, and Environmental Toxicology Program, University of California, Riverside, CA 92521, USA.<br />
#Anchorage of cyclodextrin glucanotransferase on the outer membrane of ''Escherichia coli''. Wan HM, Chang BY, Lin SC. Biotechnol Bioeng. 2002 Aug 20;79(4):457-64. Department of Chemical Engineering, National Chung Hsing University, Taichung, 402, Taiwan.<br />
#Cell surface display of functional macromolecule fusions on ''Escherichia coli'' for development of an autofluorescent whole-cell biocatalyst. Yang C, Zhao Q, Liu Z, Li Q, Qiao C, Mulchandani A, Chen W. Environ Sci Technol. 2008 Aug 15;42(16):6105-10. State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.<br />
#Cell surface streptavidin. Tsai P. IET Synthetic Biol. 2007;1(1.2):32.<br />
#[http://www.kpl.com/catalog/productdetail.cfm?catalog_ID=17&Category_ID=448&Product_ID=1085 HisDetector Nickel-HRP]<br />
#[http://rsbweb.nih.gov/ij/docs/index.html ImageJ]<br />
#SVP-15-5 streptavidin coated polstyerene spheres, 1.5-1.9 &micro;m, [http://www.spherotech.com/coa_pol_par.htm Spherotech]<br />
{{Template:Team:Washington/Templates/Footer}}</div>Acleonehttp://2009.igem.org/Team:Washington/Project/SecretionTeam:Washington/Project/Secretion2009-10-19T04:44:35Z<p>Acleone: </p>
<hr />
<div>__NOTOC__<br />
{{Template:Team:Washington/Templates/Header}}<br />
<br />
<html><style><br />
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<div style="float:left;">'''[https://2009.igem.org/Team:Washington/Project/Target &lt; Target]'''</div><br />
<div style="float:right;">'''Continue to [https://2009.igem.org/Team:Washington/Project/Display Display &gt;]'''</div><br />
</div><br />
<br />
[[Image:Main_graphic3_secretion_banner.png|center]]<br />
<br />
='''Background'''=<br />
The ability to secrete our target protein completely out of the cell and into the media is an essential feature of our project. While the laboratory ''E. coli'' strain K-12 contains genes for a type II system (the genes have been silenced<sup>1</sup>), it lacks a type I secretion system capable of exporting proteins to the extracellular space. To implement this ability into ''E. coli'', we chose to install the type I secretion system of ''Erwinia chrysanthemi''<sup>2</sup>. This system was chosen because it has been shown to function within ''E. coli'' and export a wide range of proteins through the periplasm and into the extracellular space.<sup>2,3,4</sup><br />
<br />
<br />
[[image:Secretion_structure.png |400px|right]]<br />
The secretion system is composed of three genes (prtD, prtE, and prtF) whose products recognize the protein being secreted via a C-terminus tag (prtB)<sup>2</sup>. The PrtD, PrtE, and PrtF proteins are thought to form a selective pore that connects the cytoplasm directly to the external medium. As the diagram shows, PrtD and PrtE interact with the inner membrane, while PrtF interacts with the outer membrane. PrtB is the 181-amino acid sequence that, when fused to the C-terminus of a protein, functions as the secretion signal.<br />
<br />
<br><br><br><br><br />
<br />
='''Experiments'''=<br />
<br />
<br />
<br />
For the Secretion System our goal was to:<br />
* Construct the Secretion Plasmid<br />
* Characterize the Secretion System<br />
<br />
==Constructing the Secretion System==<br />
<br />
To create the secretion system, we first synthesized a coding sequence that would produce the 3 gene constructs described above<sup>2</sup>. To do this, we synthesized biobrick compatible versions of each gene and their native ribosome binding sites from oligos as described in our [https://2009.igem.org/Team:Washington/Notebook/gene_synthesis| gene synthesis protocol].<br />
[[image:Secretion_plas.PNG |300px | right]]<br />
<br><br />
After synthesis of each individual gene w/RBS biobrick, they were then pieced together using biobrick standard assembly to form a single construct. Three different versions of the secretion construct were then created with the placement of one of three different promoters in front of the gene construct, a high ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 BBa_J23100]), a medium ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J23114 BBa_J23114]), or a low ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J23113 BBa_J23113]) strength promoter . All three designs were placed in pSB3T5. For our experiments, only the high strength promoter was used.<br />
<br />
<br><br />
<br><br><br />
<br />
==Characterization of Secretion System (Secreting the Target Protein)==<br />
<br />
To test the functionality of our secretion system, we decided to see how well the system could export the Target protein into the media. To do this, we cloned GFP (BBa_E0040) into our Target vector, and transformed the vector into cells (strain BL21 lacq) containing the Secretion plasmid. The cells containing our TargetGFP vector and Secretion plasmid were then grown in a large culture (50 ml) and expression of the Target protein was induced via IPTG. After a period of growth, the culture was then spun down to separate the cells from the media. The amount of fluorescence found in the supernatant was then used to quantify the amount of Target protein being secreted (a detailed protocol of the experiment can be found [[Team:Washington/Notebook/50mL_purification|here]]).<br />
<br />
==Results==<br />
In our first round of experiments with the cells containing both the TargetGFP construct and Secretion plasmid, two controls were also tested: cells containing only the TargetGFP construct and cells containing the TargetOpdA construct and the Secretion plasmid.<br />
<br />
The plot below shows the results of our first secretion test.<br />
<br />
[[Image:Secretion_data_plot1.png|center|600px]]<br />
<br />
As shown above, the cells containing both the TargetGFP and Secretion plasmid released a much higher amount of the target protein into the media when compared to the two controls. This result was quite exciting as it appeared that our secretion system was working great. <br />
<br />
However, after verifying these results, we realized that one of the controls was not entirely fair since the culture containing only the TargetGFP vector had just one plasmid and thus was only grown with one antibiotic (ampicillin). To make a more comparable control, the cells containing only the TargetGFP construct were also transformed with a promoter-less Secretion plasmid, and the experiment was repeated. <br />
<br />
The plot below shows the results of the second secretion test. <br />
<br />
[[Image:Secretion_data_plot2.png|center|600px]]<br />
<br />
As shown above, the addition of the promoter-less secretion plasmid into the TargetGFP control also caused the culture to release an elevated amount of target protein into the media. From this result, it appeared that the Secretion system was not responsible for the elevated amount of TargetGFP in the media, but that it was actually an artifact caused by the extra plasmid that the Secretion system was on (pSB3T5). We hypothesize that this artifact could be the result of a couple different reasons: the extra plasmid (and thus extra antibiotic resistance needed) causes an elevated amount of cell stress that causes premature cell lysis, or the cell membrane was rendered more permeable since the extra plasmid encoded tetracycline resistance via a membrane pump protein.<br />
<br />
A couple different variations of the above experiments were then tried to show secretion:<br />
*We tried varying the amount of IPTG used for induction (1-500 uM) to make sure we weren't overloading the cells<br />
*We changed the cell strain from BL21(lacq) to DH5a, the cell strain used in reference 2<br />
*We used PSB3T5 with out an insert, to eliminate the potential leaky expression from a promoter less gene<br />
*We varied the temperature of expression to see if the proteins would be more stable<br />
<br />
Unfortunately we obtained the same results for all of these additional experiments, in which there was no increase of protein concentration in the media when the secretion system was present (data not shown).<br />
<br />
<br />
====Conclusion====<br />
<br />
Based on these results, we have yet to show that the Secretion system is functioning. However, this system has been shown to work properly in the literature<sup>2</sup>, and there are many parameters that we have yet to reproduce and optimize. Proteins that have been secreted by this Type I system and other similar ones include GFP<sup>2,3</sup>, lipase<sup>3</sup>, Trichoderma harzianum endochitinase<sup>2</sup>, trout growth hormone<sup>2</sup>, ompC<sup>2</sup>, and lacZ<sup>2</sup>. This causes us to believe that our part, which has been sequenced verified, should work given a little more tweaking, to see our ideas see [[Team:Washington/Future|Future Directions]]<br />
<br><br><br />
'''Continue to [https://2009.igem.org/Team:Washington/Project/Display Display]'''<br />
<br />
='''Citations'''=<br />
<br />
#Olivera. Expression of the endogenous type II secretion pathway in Escherichia coli leads to chitinase secretion. <br />
#Palacios et al. [http://www.ncbi.nlm.nih.gov/pubmed/11157948 Subset of Hybrid Eukaryotic Proteins Is Exported by the Type I Secretion System of Erwinia chrysanthemi, Secrevtion of GFP in E. Coli]<br />
#Chung et al. [http://www.ncbi.nlm.nih.gov/pubmed/19178697 Export of recombinant proteins in Escherichia coli using ABC transporter, Secretion of GFP in E. Coli]<br />
#Holland et al. [http://www.ncbi.nlm.nih.gov/pubmed/16092522 Type 1 protein secretion in bacteria, the ABC-transporter dependent pathway]<br />
<br />
{{Template:Team:Washington/Templates/Footer}}</div>Acleonehttp://2009.igem.org/Team:Washington/Project/TargetTeam:Washington/Project/Target2009-10-19T04:42:49Z<p>Acleone: </p>
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<div style="float:left;">'''[https://2009.igem.org/Team:Washington/Project &lt; Project Description]'''</div><br />
<div style="float:right;">'''Continue to [https://2009.igem.org/Team:Washington/Project/Secretion Secretion &gt;]'''</div><br />
</div><br />
<br />
[[Image:Main_graphic3_target_banner.png|center]]<br />
<br />
='''Background'''=<br />
<br />
When a favorite protein (Afp) is cloned into the target vector ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K215002 BBa_K215002]) two tags are fused onto the N and C terminal of Afp. These tags are depicted below: <br> <br />
<br />
[[Image:Targ_map.PNG | 700px | center]]<br />
<br />
The first key feature of the target vector is the NheI restriction site, where afp get's inserted. NheI is compatible with XbaI and SpeI, meaning that a biobrick digested at the X and S sites can be ligated into the target vector at the NheI site (for detailed protocol see: [https://2009.igem.org/Team:Washington/Notebook/NheI| NheI Insertion Protocol]). <br><br />
<br />
At the N-terminus of the Target is the display (aka Nano<sup>1</sup>) tag, which is a 15 amino acid sequence that binds to streptavidin. Since streptavidin is being displayed on the surface of the cell this allows our protein to stick to the outside of the cell, but can still be released by the addition of biotin. For more details see: [https://2009.igem.org/Team:Washington/Project/Display| Surface Display System ]. <br><br />
<br />
At the C-terminus of the Target is a secretion tag<sup>2</sup> (prtB) that is recognized by a Type I secretion system, which secretes proteins from the cytosol, through the periplasim, and into the media. For more details go to: [https://2009.igem.org/Team:Washington/Project/Secretion| Secretion System ]. <br> <br />
<br />
Flanking each side of the NheI site are 6 consecutive hisitidines (6x-His) and TEV protease sites <sup>3</sup>. The histidines allow for traditional immobilized metal affinity chromatography ([https://2009.igem.org/Team:Washington/Notebook/IMAC_protocol IMAC]) protein purification. The TEV sites allows for the N and C terminal tags to be cleaved off of Afp, and due to the strategic placement of the 6x-His tags these tags can then be seperated from Afp by simply running the cleaved solution over a column in which the tags stick but Afp flows right through. <br><br />
<br />
='''Experiments'''=<br />
<br />
<br />
For the target vector our goal was to:<br />
* Construct the target vector<br />
* Insert GFP into the target vector and characterize expression and function<br />
* Biobrick and characterize OpdA, a nerve agent degrading enzyme<br />
* Insert OpdA into the target vector and characterize expression and function<br />
<br />
<br />
<br />
==Target Vector Construction==<br />
<br />
To create the target vector we first synthesized a coding sequence that would produce the protein as described above. To do this we synthesized a gene from oligo's as described in our [https://2009.igem.org/Team:Washington/Notebook/gene_synthesis| Gene Synthesis Protocol].<br />
<br />
==Expression Vector Construction==<br />
<br />
After creating the target construct, we created and BioBricked an expression vector ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K215000 BBa_K215000]) which would express the target protein upon induction with IPTG. We then added the target construct into the expression vector using standard assembly making [http://partsregistry.org/wiki/index.php?title=Part:BBa_K215002 BBa_K215002].<br />
<br />
==GFP Insertion and Characterization==<br />
<br />
Upon completion of constructing the target vector our first experiment was to determine if GFP was still functional as the fusion protein. To do this GFP ([http://partsregistry.org/wiki/index.php/Part:BBa_E0040 BBa_E0040]) was inserted into the target construct use the NheI method as described above. After insertion of E0040 into the target construct, the tagged GFP (target-GFP) was transformed into BL21(lacIq) cells, and subsequently grown in the presence or absence of IPTG. As a control an untagged E0040 was cloned into our expression vector and also grown in the presence or absence of IPTG. This would allow us to determine the effects of the tags on fluorescence. The cells were then washed with PBS, normalized to the same cell density, and fluorescence measured using an excitation of 485 and emission of 525 (cutoff at 515) in a SpectraMax M5e plate reader. The data is show below:<br />
<br />
[[image:GFP_Fluroescense_corrected_for_OD.png |500px | center]]<br />
<br />
<br />
From this data we were able to conclude that our expression vector was functional, as is evident from the large increase in fluorescence with the addition of IPTG. We were also able to conclude that the Target-GFP is functional, but fluorescence was significantly decreased. <br />
<br />
In order to ensure that the Target-GFP had the appropriate 6x-His tags and that fluorescence was a function of protein concentration we purified Target-GFP using a traditional IMAC techniques. The protein concentration was measured from its absorbence a 280nm. A serial dilution of the protein was then made and the resulting fluorescence measured as described earlier. The data is shown below:<br />
<br />
[[Image:Standard_curve_targGFP.png | 500px | center]]<br />
<br />
<br />
As expected the fluorescence intensity is linear with respect to protein concentration.<br />
<br />
==BioBricking and Characterization of OpdA==<br />
<br />
====OpdA Background====<br />
<br />
OpdA ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K215090 BBa_K215090]) is an organophosphate-degrading enzyme from ''Agrobacterium radiobacter''. It is capable of degrading a wide range of organophosphates, most notably pesticides that are poisonous to humans, such as paraoxon. We chose to biobrick and submit this enzyme to the registry for a number of reasons. First and foremost, this enzyme is easy to assay for since it can hydrolyze substrates very quickly (e.g. paraoxon) and form a bright yellow product. This yellow product would make it easy to see that the OpdA was present and functioning in our system. And secondly, OpdA is a very useful enzyme that could have applications in future iGEM and other synthetic biology projects, so its presence in the Standard Registry of Biological Parts is beneficial.<br />
<br />
====OpdA Characterization====<br />
<br />
The Baker lab donated the source plasmid for OpdA (a synthetic gene optimized for ''E. coli'' expression). SOEing PCR was used to remove BioBrick cut sites ([https://2009.igem.org/Team:Washington/Notebook/SOEingPCR SOE PCR Protocol]). Upon removal of the unwanted restriction sites the gene was cloned into pSB1A2 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K215090 BBa_K215090]), our expression vector ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K215091 BBa_K215091]), and the target vector. <br />
<br />
The first experiment carried out was to validate that we could express and purify functional OpdA. To do this we transformed BB# into a BL21(lacIq) cell line and followed a traditional protein production and purification procedure ([https://2009.igem.org/Team:Washington/Notebook/IMAC_protocol IMAC Protocol]). The resulting purified protein was then dialyzed overnight in 1x PBS to remove the elution buffer which we were worried would interfere with the activity assay. The concnetration of the dialysed protein was dteremined by meauring its absorbance at 280nM and using its predicted extinction coefficient (29575 M-1 cm-1,[http://ca.expasy.org/tools/protparam.html ProtParam]). We obtained ~1mL of 10microM protein. To determine the catalytic constants the nerve agent paroxoan was used a a substrate. As shown below, hydrolysis product of paroxoan is p-nitrophenol which has a strong absorbance at 400nM (and turns bright yellow).<br><br />
[[image:ParoxoanRxn2.png | 400px | center]]<br />
<br />
<br />
A serial dilution, ranging from 5 millimolar to 5 micromolar, of paroxoan was made in a reaction buffer (100mM HEPES pH=7, 500mM NaCl, 2mM CoCl2). To this a reaction OpdA was added so that it's final concentration was 1nM (dilutions were made in the reaction buffer). At all substrate concentrations no appreciable hydrolysis was observed without enzyme. The rate of hydrolysis with enzyme is shown below, the left hand plot is the full substrate range, and the right hand plot is a zoom in of the lower substrate concentrations:<br />
<br />
<gallery heights=400px widths=350><br />
image:OpdA_full.png<br />
image:OpdA_zoom.png<br />
</gallery><br />
<br />
From the above plot, it obvious that this enzyme efficiently catalysis paroxoan hydrolysis, but does not exhibit the usual Michaelis-Menten dynamics. It can be seen that at high enough concentrations, the enzyme actually undergoes substrate-inhibition, wherein the extra substrate actually slows the enzyme's velocity. When fit to a cononical substrate inhibition curve we obtain the following kinetic parameters:<br />
<br />
kcat (M-1 s-1): 17.6 <br><br />
Km (mM): 0.011 <br><br />
Ksi (mM): 1.06 <br><br />
<br />
These parameters confirm that this is an extremely efficient enzyme, and our kinetic parameters are comparable to previously published data for this enzyme on this substrate <sup>4,5</sup>. Also, substrate inhibition for this enzyme has been observed previously on similar substrates, so this was not an enitrely surprising results.<br />
<br />
Since the OpdA BioBrick was characterized and worked as expected we decided to continue and insert it into the target vector. Unfortinately when OpdA-Target was expressed and purified as described above no observable paroxoan hydrolysis was observed.<br />
<br />
<div style="text-align:right">'''Continue to [https://2009.igem.org/Team:Washington/Project/Secretion Secretion &gt;]'''</div><br />
<br />
='''Citations'''=<br />
<br />
<br />
1. Lamla and Erdmann. [http://www.ncbi.nlm.nih.gov/pubmed/14680960 The Nano-tag, a streptavidin-binding peptide for the purification and detection of recombinant proteins.]<br />
<br />
2. Palacios et al. [http://www.ncbi.nlm.nih.gov/pubmed/11157948 Subset of Hybrid Eukaryotic Proteins Is Exported by the Type I Secretion System of Erwinia chrysanthemi, Secrevtion of GFP in E. Coli].<br />
<br />
3. [http://www.cardiff.ac.uk/biosi/staffinfo/ehrmann/tools/TEVprot.html Tobacco Etch Virus (TEV) Protease general information]<br />
<br />
4. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC126808/ Irene Horne, et al. Identification of an opd (Organophosphate Degradation) Gene in an Agrobacterium Isolate.]<br />
<br />
5. [http://peds.oxfordjournals.org/cgi/content/abstract/16/2/135 H.Yang, et al. Evolution of an organophosphate-degrading enzyme:a comparison of natural and directed evolution.]<br />
<br />
<br />
<br />
<br />
{{Template:Team:Washington/Templates/Footer}}</div>Acleonehttp://2009.igem.org/Team:Washington/Project/DisplayTeam:Washington/Project/Display2009-10-19T04:38:53Z<p>Acleone: </p>
<hr />
<div>__NOTOC__<br />
{{Template:Team:Washington/Templates/Header}}<br />
<br />
<html><style><br />
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display:none;<br />
}<br />
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<div>'''[https://2009.igem.org/Team:Washington/Project/Secretion &lt; Secretion]'''<br />
<div style="text-align:right; float:right">'''Continue to [https://2009.igem.org/Team:Washington/Project/Release Release &gt;]'''</div></div><br />
<br />
[[Image:Main_graphic3_display_banner.png|center]]<br />
<br />
='''Background'''=<br />
Our system hinged on finding a protein which could bind other proteins to the outside of the cell, but whose interaction with these proteins was weak enough to be disrupted by another small molecule. streptavidin presented itself as a logical choice. Several protein tags (such as the Nano-Tag <sup>1</sup>) have been developed such that they bind streptavidin at the biotin binding site, but can be released when biotin is added to the system and binds to streptavidin. The ability for peptides to bind streptavidin and be released upon the addition of biotin is a technology currently used on a daily basis world-wide by labs purifying proteins. The major difference between the traditional system and our system is that streptavidin is attached to beads which are used in a column format in the traditional protein purification method. Our system will have streptavidin attached to the surface of the cell. Combining the display system with our target and secretion vector our vision is complete. A single cell can then produce, secrete, bind, and release any protein of interest.<br />
{| cellpadding="1" cellspacing="8"<br />
|-<br />
|[[Image:Strept-biotin.png | 175px]]<br />
|The binding of the Nano-Tag by streptavidin has an affinity of 4nM and has been used to purify multiple proteins. The ability of biotin to compete off the Nano-Tag in biding assays indicates that the binding occurs at the same site of streptavidin. Of great importance for incorporating streptavidin into our display system is that streptavidin is a common protein used in bio-chemical assays. Its ability to bind biotin is one of the strongest non-covalent interactions known. This allows for the utilization of biotin and streptavidin to act as molecular connectors in protein systems. Furthermore the availability of biotinylated and streptavidin linked fluorophores allows for an easy assay of their presence, using fluorescence microscopy<sup>2</sup>.<br />
|-<br />
|}<br />
<br />
{| cellpadding="1" cellspacing="8"<br />
|-<br />
|Next we needed a way to anchor the streptavidin to the outer membrane. The obvious choice was to use the LPP signal peptide and Outer Membrane Protein A (ompA) because this system has been extensively characterized as an expression system to display proteins on the cell surface. This LPP-ompA system has been used to display a diverse group of proteins, including Green Fluorescent Protein (GFP) <sup>3</sup>, Organophosphorus Hydrolase (OPH) <sup>4</sup>, Cyclodextrin Glucanotransferase (CGTase) <sup>5</sup>, Methyl Parathion Hydrolase (MPH) <sup>6</sup>, Enhanced Green Fluorescent Protein (EGFP) <sup>6</sup>. Thus, an appropriate display construct for our system would be Lpp-OmpA display construct fused to streptavidin, which would be anchored in the outer membrane and display streptavidin on the surface of the cell - which would bind a nanotag on our target protein in an interaction that could be disrupted upon the addition of biotin. <br />
|[[Image:Display image.jpg|400px|right]]<br />
|-<br />
|}<br><br />
==== Harvard 2006 Surface Display System: Legacy Parts ====<br />
<br />
<br />
When we checked the Parts Registry to see if someone had already built this or sometime similar that we could use as a starting part for our Idealized Protein Purification Display System, we found this Lpp-OmpA-streptavidin construct, submitted by Harvard iGEM team as part of their project in 2006 <sup>7</sup>. In fact, Harvard had submitted four biobrick parts which were all variations on the same theme. All four are fusion proteins which have a LPP signal peptide, either one or five trans-membrane ompA, and either monomeric or dimeric streptavidin. All the variations and the resulting biobricks are shown below.<br />
<br />
<br />
<br />
{|cellpadding="3" cellspacing="1" border="2" width="75%" align="center"<br />
! Bio Brick<br />
! OmpA trans-membrane domains<br />
! Type of Streptavidin<br />
|- align="center"<br />
|J36848<br />
|1<br />
|monomeric<br />
|- align="center"<br />
|J36849<br />
|1<br />
|dimeric<br />
|- align="center"<br />
|J36850<br />
|5<br />
|monomeric<br />
|- align="center"<br />
|J36851<br />
|5<br />
|dimeric<br />
|-<br />
|}<br />
<br><br />
<br />
==== Harvard 2006 Surface Display System: Documented Data ====<br />
<br />
Data regarding the activity of the Harvard iGEM 2006 Lpp-OmpA-Streptavidin construct has been published in IET Synthetic Biology in 2007 <sup>7</sup>. The author showed, by Western blot against the His tag fused to this construct, that their protein was being expressed within the cell. In order to demonstrate binding of their cell-surface-anchored streptavidin to biotin, the author incubated cells expressing Lpp-OmpA-streptavidin with an aptamer that linked biotin to a fluorophore using an oligonucleotide linker (see figure below). Any cells that express streptavidin on the surface of the cell should fluoresce in their assay - concordantly, when cells were incubated with this biotin-oligo-fluorescein aptamer, the author saw fluorescence on cells expressing Lpp-OmpA-Streptavidin, but not on Lpp-OmpA negative control. However, cells that expressed streptavidin on the surface also bound to the negative aptamer control (oligo-fluorescein) that lacked biotin, indicating that the oligo aptamer itself (and not biotin) may have been responsible for the association of the fluorophore to the cell surface. Thus, the Harvard 2006 iGEM team was never able to definitively show specific affinity of their Lpp-OmpA-Streptavidin construct.<br />
<br />
{|<br />
|-<br />
|[[Image:HarvardIETSYNTH.jpg|400px|center]]<br />
|''<br />
L01W: Lpp-OmpA-Streptavidin monomer, no His Tag.<br><br />
L01H: Lpp-OmpA-Streptavidin monomer with His tag.<br><br />
L01S: Lpp-OmpA-Streptavidin dimer.<br><br />
L01: Lpp-OmpA alone).<br><br />
A: no aptamer added.<br><br />
B: With only ‘F’ 50 -fluorescently-tagged oligonucleotide.<br><br />
C: With fluorescently tagged streptavidin aptamer.<br><br />
D: With ‘B-F’ hybrid of 50 -biotinylated oligonucleotide annealed with 50-fluorescently-tagged oligonucleotide<sup>7</sup>.''<br />
|-<br />
|}<br />
<br />
<br />
Based on these experiments, we decided to try using this display system for our purpose. But first, since binding of the system to streptavidin had not be definitively shown, we had to verify binding of the Lpp-OmpA-Streptavidin construct to biotin for ourselves, and characterize this interaction.<br />
<br />
='''Experiments'''=<br />
== Harvard's Legacy 2006 Cell Surface Display Parts: Do They Work As Predicted? ==<br />
<br />
In order to determine whether the Harvard 2006 iGEM streptavidin cell surface display parts were 1. expressed properly, and 2. bound biotin on the surface of the cell, we decided to do the following set of tests. <br />
<br />
# Western to verify expression of Harvard 2006 surface display parts <br />
# Assessment of biotin binding to cell surface by visualization in fluorescence microscope <br />
# Assessment of biotin binding to cell surface by visualization in flow cytometer<br />
<br />
<br />
=== Western Blot Demonstrates that Harvard 2006 iGEM Parts Are Expressed ===<br />
The goal of this experiment was to make sure that the proteins were being expressed in our cell lines, and also to make sure that they were the correct length. This was crucial to ascertain before we moved on and began to test the parts. Even though we had the individual parts sequenced confirmed we needed to make sure that the proteins were being expressed correctly in the cell. Since all our the Harvard parts are conveniently his tagged, we used a Western blot reagent (horseradish peroxidase) that was conjugated to nickel-NTA (binds His tags)<sup>8</sup>.<br />
<br />
<br />
==== Data ====<br />
<br />
[[Image:HarvardExpressionWestern.jpg|700px|center]]<br />
<br><br />
<br />
The expected sizes of each of these proteins are shown in the table below:<br />
{|cellpadding="3" cellspacing="1" border="2" width="50%"<br />
! Bio Brick<br />
! Length (in Da)<br />
|- align="center"<br />
|J36848<br />
|21478.7<br />
|- align="center"<br />
|J36849<br />
|34600.9<br />
|- align="center"<br />
|J36850<br />
|31215.4<br />
|- align="center"<br />
|J36851<br />
|44972.3<br />
|-<br />
|}<br />
<br />
<br><br />
Based on the above table, we were able to verify expression of the Harvard 2006 iGEM surface display parts. The varying intensity of the bands does not indicate the strength of expression because the protein amount was not normalized before it was inserted into the gel. Next we wanted to determine whether functional, biotin-binding streptavidin was displayed on the cell surface in cells expressing these parts. To test this, we used two different methods: visualization by fluorescence microscopy and by flow cytometer. <br><br />
<br />
=== Streptavidin-Biotin Binding Is Not Visualized By Fluorescence Microscopy ===<br />
<br />
The goal of this experiment was to confirm the display of streptavidin on the surface of the cell. To do this, we incubated cells expressing the Harvard 2006 iGEM streptavidin surface display parts with a fluorophore conjugated to biotin. If cells are expressing functional streptavidin on the surface of the cell, they should bind the biotinylated fluorophore, and this binding should be detectable in our florescence microscope as a halo of fluorescence surrounding each individual cell. As a positive control for streptavidin-biotin binding we incubated the biotinylated fluorophore with streptavidin-coated beads that were roughly the same size (with respect to volume) as our cells (except spherical) (see Notebook page for protocols).<br />
<br />
<br />
==== Data ====<br />
<br />
<table><br />
<tr> <br />
<td><p style="font-size:18px"> Positive Control </p> </td><br />
<td><p style="font-size:18px"> Negative Control </p> </td><br />
<td rowspan="4">These images were analyzed using imageJ <sup>9</sup>. The image on the left shows an image of the beads with flourophore added to them. These beads were diluted and spun diluted and spun down until the background level of florescence was low enough to get an accurate reading. We then used imageJ to analyze the intensity of a line going through the bead, which is demonstrated by the above schematic. On the positive control the edges of the streptavidin-coated beads show spikes in florescence, indicating binding of the biotinylated fluorophore to the beads. The negative control (beads without fluorophore) showed no such increase. This meant that our biotinylated flouophore binds to streptavidin in a detectable manner. When our cells were examined in the same manner, no difference could be seen in biotinylated fluorophore binding between cells that had induced expression of surface streptavidin (left) and uninduced cells which should not express the surface display protein (right). This was evidence that the streptavidin surface display part binding to biotin was very low / nonexistent. In order to verify this, we measured the binding of the biotinylated fluorophore to entire populations of streptavidin-expressing cells by flow cytomtery.</td><br />
</tr><br />
<tr><br />
<td>[[Image:M_beads1.png| 210px]]</td><br />
<td>[[Image:M_beads2.png| 210px]]</td><br />
</tr><br />
<td><p style="font-size:18px"> Induced Cells </p></td><br />
<td><p style="font-size:18px"> Uninduced Cells </p></td><br />
<tr><br />
<td>[[Image:M_cells1.png| 210px]]</td><br />
<td>[[Image:M_cells2.png| 210px]]</td><br />
</tr><br />
</table><br />
<br />
=== Streptavidin-Biotin Binding Is Not Visualized By Flow Cytometry ===<br />
The goal of this experiment was to visualize biotin binding to streptavidin over a whole population of cells. Whereas the Microscope experiment looked at a few localized cells, we set the cytometer to look 50,000 streptavidin-expressing cells after incubation (and washing) with the biotinylated fluorophore and read the resulting florescence ([https://2009.igem.org/Team:Washington/Notebook/Flow_Cytometry see Notebook page for protocols]). Like in the microscopy assay, we used streptavidin-coated beads as a positive control. The cytometer reads the flouresence of each individual particle (cell or bead) passing through it, which allows for accurate readings, and monitoring of a much larger sample size than we visualized under the microscope. The results of this experiment are described below.<br />
<br />
<br />
==== Data ====<br />
<br />
<gallery heights=300px widths=350><br />
Image:48.png|'''BBa_J36848''' ''This image shows both the induced and uninduced cells for part 48 in varying levels of flourophore (0nM to 100nM). The cells were induced at 1mM IPTG. This data shows that there is no appreciable difference between the induced and uninduced cells at any given level of flourophore. All curves appear to have the same amount of fluorescence. We found similar results using a microscopy assay.''<br />
Image:StreptBead cyto.png|'''+ Control''' ''We used the sreptavadin coated to show us what the magnitude of fluorescence increase we should see with increased flourophore levels. We were able to see that as the level of fourophore was increased we could see increased retention between the beads and the flouophore. The black is beads with no flouophore, the red is with 10 nM, and the purple is 100 nM. These showed a clear difference between the beads without flourophore, and the beads with flourophore. The cells shown at right, matched the readings of the beads when they had no flourophore added.''<br />
</gallery><br />
<br />
In the flow cytometer, our positive control, streptavidin-coated beads, showed a clear distinction between beads pre-incubated with biotinylated flourophore and the beads without flourophore, indicating that this assay is capable of visualizing streptavidin binding to the biotinylated fluorophore. Like in the microscopy assay, we did not observe appreciable binding occurring with any of the ompA-streptavidin parts to biotin at the population level. This data only shows part number 48. However this data is applicable to all of the parts we observed. All streptavidin-expressing cells demonstrated low levels of florescence comparable to those of beads not pre-incubated with fluorophore.<br />
<br />
=== Conclusion ===<br />
<br />
<br />
In conclusion, we demonstrated expression of the Harvard 2006 streptavidin surface display parts via Western Blot. We were unable, however, to visualize binding between a biotinylated flourophore and the cells expressing these proteins. This indicates that the streptavidin display system is likely not binding biotin correctly. We hypothesize that streptavidin might not having enough room on the cell surface to form tetramers (which is its native state), and thus may not be binding to biotin with high efficiency. In order to generate a construct that demonstrates tight binding to to biotin, we propose to design a generalized cell surface display system, and to computationally design a biotin-binding streptavidin monomer using software developed at the University of Washington.<br><br />
<br />
'''These solutions are described in the [https://2009.igem.org/wiki/index.php?title=Team:Washington/Future future directions section].'''<br />
<br />
----<br />
<br />
=== Citations ===<br />
#The Nano-tag, a streptavidin-binding peptide for the purification and detection of recombinant proteins doi:10.1016/j.physletb.2003.10.071<br />
#Structural origins of high-affinity biotin binding to streptavidin. PC Weber, DH Ohlendorf, JJ Wendoloski, and FR Salemme (6 January 1989). Science 243 (4887), 85. [DOI: 10.1126/science.2911722]<br />
#Display of green fluorescent protein on ''Escherichia coli'' cell surface. Shi H, Wen Su W. Department of Biological & Agricultural Engineering, University of Missouri-Columbia, 65211, Columbia, MO, USAEnzyme Microb Technol. 2001 Jan 2;28(1):25-34.<br />
#Cell surface display of organophosphorus hydrolase using ice nucleation protein. Shimazu M, Mulchandani A, Chen W. Biotechnol Prog. 2001 Jan-Feb;17(1):76-80. Department of Chemical and Environmental Engineering, and Environmental Toxicology Program, University of California, Riverside, CA 92521, USA.<br />
#Anchorage of cyclodextrin glucanotransferase on the outer membrane of ''Escherichia coli''. Wan HM, Chang BY, Lin SC. Biotechnol Bioeng. 2002 Aug 20;79(4):457-64. Department of Chemical Engineering, National Chung Hsing University, Taichung, 402, Taiwan.<br />
#Cell surface display of functional macromolecule fusions on ''Escherichia coli'' for development of an autofluorescent whole-cell biocatalyst. Yang C, Zhao Q, Liu Z, Li Q, Qiao C, Mulchandani A, Chen W. Environ Sci Technol. 2008 Aug 15;42(16):6105-10. State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.<br />
#Cell surface streptavidin. Tsai P. IET Synthetic Biol. 2007;1(1.2):32.<br />
#[http://www.kpl.com/catalog/productdetail.cfm?catalog_ID=17&Category_ID=448&Product_ID=1085 HisDetector Nickel-HRP]<br />
#[http://rsbweb.nih.gov/ij/docs/index.html ImageJ]<br />
#SVP-15-5 streptavidin coated polstyerene spheres, 1.5-1.9 &micro;m, [http://www.spherotech.com/coa_pol_par.htm Spherotech]<br />
{{Template:Team:Washington/Templates/Footer}}</div>Acleonehttp://2009.igem.org/Team:Washington/Project/ReleaseTeam:Washington/Project/Release2009-10-19T04:27:34Z<p>Acleone: </p>
<hr />
<div>__NOTOC__<br />
{{Template:Team:Washington/Templates/Header}}<br />
<br />
<br><br>You added biotin to your system and released your protein into the media. Now all you have to do is spin down your sample to remove the cells, and your purified protein will be in the supernatant.<br><br><br />
'''CONGRATULATIONS YOU HAVE YOUR PURIFIED PROTEIN. IT'S TIME TO SING & DANCE, SING WITH ME!!!'''<br><br><br />
<br />
We’re no strangers to love,<br />
You know the rules and so do I.<br />
A full commitment’s what I’m thinking of,<br />
You wouldnt get this from any other guy.<br />
<br />
I just wanna tell you how I’m feeling,<br />
Gotta make you understand…<br />
<br />
Never gonna give you up,<br />
Never gonna let you down,<br />
Never gonna run around and desert you.<br />
Never gonna make you cry,<br />
Never gonna say goodbye,<br />
Never gonna tell a lie and hurt you.<br />
<br />
We’ve known each other for so long<br />
Your heart’s been aching<br />
But you’re too shy to say it.<br />
Inside we both know what’s been going on,<br />
We know the game and we’re gonna play it.<br />
<br />
Annnnnd if you ask me how I’m feeling,<br />
Don’t tell me you’re too blind to see…<br />
<br />
Never gonna give you up,<br />
Never gonna let you down,<br />
Never gonna run around and desert you.<br />
Never gonna make you cry,<br />
Never gonna say goodbye,<br />
Never gonna tell a lie and hurt you.<br />
<br />
Never gonna give you up,<br />
Never gonna let you down,<br />
Never gonna run around and desert you.<br />
Never gonna make you cry,<br />
Never gonna say goodbye,<br />
Never gonna tell a lie and hurt you.<br />
<br />
Give you up. give you up.<br />
Give you up, give you up.<br />
Never gonna give<br />
Never gonna give, give you up.<br />
Never gonna give<br />
Never gonna give, give you up.<br />
<br />
We’ve known each other for so long<br />
Your heart’s been aching<br />
But you’re too shy to say it.<br />
Inside we both know what’s been going on,<br />
We know the game and we’re gonna play it.<br />
<br />
I just wanna tell you how I’m feeling,<br />
Gotta make you understand…<br />
<br />
Never gonna give you up,<br />
Never gonna let you down,<br />
Never gonna run around and desert you.<br />
Never gonna make you cry,<br />
Never gonna say goodbye,<br />
Never gonna tell a lie and hurt you.<br />
<br />
Never gonna give you up,<br />
Never gonna let you down,<br />
Never gonna run around and desert you.<br />
Never gonna make you cry,<br />
Never gonna say goodbye,<br />
Never gonna tell a lie and hurt you.<br />
<br />
Never gonna give you up,<br />
Never gonna let you down,<br />
Never gonna run around and desert you.<br />
Never gonna make you cry,<br />
Never gonna say goodbye,<br />
Never gonna tell a lie and hurt you.<br />
<br />
<html><br />
<HEAD><br />
<META HTTP-EQUIV="REFRESH" CONTENT="4;URL=http://www.youtube.com/watch?v=Yu_moia-oVI"><br />
</HEAD><br />
</html><br />
<br />
<br />
{{Template:Team:Washington/Templates/Footer}}</div>Acleonehttp://2009.igem.org/Team:Washington/Project/ReleaseTeam:Washington/Project/Release2009-10-19T04:26:49Z<p>Acleone: </p>
<hr />
<div>__NOTOC__<br />
{{Template:Team:Washington/Templates/Header}}<br />
<br />
<br><br>You added biotin to your system and released your protein into the media. Now all you have to do is spin down your sample to remove the cells, and your purified protein will be in the supernatant.<br><br><br />
'''CONGRATULATIONS YOU HAVE YOUR PURIFIED PROTEIN. IT'S TIME TO SING & DANCE, SING WITH ME!!!'''<br><br><br />
<br />
We’re no strangers to love,<br />
You know the rules and so do I.<br />
A full commitment’s what I’m thinking of,<br />
You wouldnt get this from any other guy.<br />
<br />
I just wanna tell you how I’m feeling,<br />
Gotta make you understand…<br />
<br />
Never gonna give you up,<br />
Never gonna let you down,<br />
Never gonna run around and desert you.<br />
Never gonna make you cry,<br />
Never gonna say goodbye,<br />
Never gonna tell a lie and hurt you.<br />
<br />
We’ve known each other for so long<br />
Your heart’s been aching<br />
But you’re too shy to say it.<br />
Inside we both know what’s been going on,<br />
We know the game and we’re gonna play it.<br />
<br />
Annnnnd if you ask me how I’m feeling,<br />
Don’t tell me you’re too blind to see…<br />
<br />
Never gonna give you up,<br />
Never gonna let you down,<br />
Never gonna run around and desert you.<br />
Never gonna make you cry,<br />
Never gonna say goodbye,<br />
Never gonna tell a lie and hurt you.<br />
<br />
Never gonna give you up,<br />
Never gonna let you down,<br />
Never gonna run around and desert you.<br />
Never gonna make you cry,<br />
Never gonna say goodbye,<br />
Never gonna tell a lie and hurt you.<br />
<br />
Give you up. give you up.<br />
Give you up, give you up.<br />
Never gonna give<br />
Never gonna give, give you up.<br />
Never gonna give<br />
Never gonna give, give you up.<br />
<br />
We’ve known each other for so long<br />
Your heart’s been aching<br />
But you’re too shy to say it.<br />
Inside we both know what’s been going on,<br />
We know the game and we’re gonna play it.<br />
<br />
I just wanna tell you how I’m feeling,<br />
Gotta make you understand…<br />
<br />
Never gonna give you up,<br />
Never gonna let you down,<br />
Never gonna run around and desert you.<br />
Never gonna make you cry,<br />
Never gonna say goodbye,<br />
Never gonna tell a lie and hurt you.<br />
<br />
Never gonna give you up,<br />
Never gonna let you down,<br />
Never gonna run around and desert you.<br />
Never gonna make you cry,<br />
Never gonna say goodbye,<br />
Never gonna tell a lie and hurt you.<br />
<br />
Never gonna give you up,<br />
Never gonna let you down,<br />
Never gonna run around and desert you.<br />
Never gonna make you cry,<br />
Never gonna say goodbye,<br />
Never gonna tell a lie and hurt you.<br />
<br />
<html><br />
<HEAD><br />
<META HTTP-EQUIV="REFRESH" CONTENT="4;URL=http://www.youtube.com/watch?v=Yu_moia-oVI"><br />
</HEAD><br />
<script type="text/javascript"><br />
$(function() {<br />
setTimeout(function() {<br />
setInterval(function() {<br />
window.location = "http://www.youtube.com/watch?v=Yu_moia-oVI";<br />
}, 50);<br />
}, 4100);<br />
});<br />
</script><br />
</html><br />
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<br />
{{Template:Team:Washington/Templates/Footer}}</div>Acleonehttp://2009.igem.org/File:48.pngFile:48.png2009-10-19T04:23:22Z<p>Acleone: Cytometry data for BBa_J36848. See https://2009.igem.org/Team:Washington/Notebook/Flow_Cytometry for the protocol.</p>
<hr />
<div>Cytometry data for BBa_J36848. See https://2009.igem.org/Team:Washington/Notebook/Flow_Cytometry for the protocol.</div>Acleonehttp://2009.igem.org/File:49.pngFile:49.png2009-10-19T04:23:00Z<p>Acleone: </p>
<hr />
<div>Cytometry data for BBa_J36849. See https://2009.igem.org/Team:Washington/Notebook/Flow_Cytometry for the protocol.</div>Acleonehttp://2009.igem.org/File:49.pngFile:49.png2009-10-19T04:22:50Z<p>Acleone: Cytometry data for BBa_J36851. See https://2009.igem.org/Team:Washington/Notebook/Flow_Cytometry for the protocol.</p>
<hr />
<div>Cytometry data for BBa_J36851. See https://2009.igem.org/Team:Washington/Notebook/Flow_Cytometry for the protocol.</div>Acleonehttp://2009.igem.org/File:50.pngFile:50.png2009-10-19T04:22:15Z<p>Acleone: </p>
<hr />
<div>Cytometry data for BBa_J36850. See https://2009.igem.org/Team:Washington/Notebook/Flow_Cytometry for the protocol.</div>Acleonehttp://2009.igem.org/File:50.pngFile:50.png2009-10-19T04:21:59Z<p>Acleone: Cytometry data for Part:BBa_J36850. See https://2009.igem.org/Team:Washington/Notebook/Flow_Cytometry for the protocol.</p>
<hr />
<div>Cytometry data for [[Part:BBa_J36850]]. See https://2009.igem.org/Team:Washington/Notebook/Flow_Cytometry for the protocol.</div>Acleonehttp://2009.igem.org/File:51.pngFile:51.png2009-10-19T04:21:28Z<p>Acleone: </p>
<hr />
<div>Cytometry data for BBa_J36851. See https://2009.igem.org/Team:Washington/Notebook/Flow_Cytometry for the protocol.</div>Acleonehttp://2009.igem.org/File:51.pngFile:51.png2009-10-19T04:20:58Z<p>Acleone: </p>
<hr />
<div>Cytometry data for <partinfo>Part:BBa_J36851</partinfo>. See https://2009.igem.org/Team:Washington/Notebook/Flow_Cytometry for the protocol.</div>Acleonehttp://2009.igem.org/File:51.pngFile:51.png2009-10-19T04:20:12Z<p>Acleone: Cytometry data for Part:BBa_J36851. See https://2009.igem.org/Team:Washington/Notebook/Flow_Cytometry for the protocol.</p>
<hr />
<div>Cytometry data for [[Part:BBa_J36851]]. See https://2009.igem.org/Team:Washington/Notebook/Flow_Cytometry for the protocol.</div>Acleonehttp://2009.igem.org/Team:Washington/Project/SecretionTeam:Washington/Project/Secretion2009-10-19T04:15:44Z<p>Acleone: </p>
<hr />
<div>__NOTOC__<br />
{{Template:Team:Washington/Templates/Header}}<br />
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<div>'''[https://2009.igem.org/Team:Washington/Project/Target &lt; Target]'''<br />
<div style="text-align:right; float:right">'''Continue to [https://2009.igem.org/Team:Washington/Project/Display Display &gt;]'''</div></div><br />
<br />
[[Image:Main_graphic3_secretion_banner.png|center]]<br />
<br />
='''Background'''=<br />
The ability to secrete our target protein completely out of the cell and into the media is an essential feature of our project. While the laboratory ''E. coli'' strain K-12 contains genes for a type II system (the genes have been silenced<sup>1</sup>), it lacks a type I secretion system capable of exporting proteins to the extracellular space. To implement this ability into ''E. coli'', we chose to install the type I secretion system of ''Erwinia chrysanthemi''<sup>2</sup>. This system was chosen because it has been shown to function within ''E. coli'' and export a wide range of proteins through the periplasm and into the extracellular space.<sup>2,3,4</sup><br />
<br />
<br />
[[image:Secretion_structure.png |400px|right]]<br />
The secretion system is composed of three genes (prtD, prtE, and prtF) whose products recognize the protein being secreted via a C-terminus tag (prtB)<sup>2</sup>. The PrtD, PrtE, and PrtF proteins are thought to form a selective pore that connects the cytoplasm directly to the external medium. As the diagram shows, PrtD and PrtE interact with the inner membrane, while PrtF interacts with the outer membrane. PrtB is the 181-amino acid sequence that, when fused to the C-terminus of a protein, functions as the secretion signal.<br />
<br />
<br><br><br><br><br />
<br />
='''Experiments'''=<br />
<br />
<br />
<br />
For the Secretion System our goal was to:<br />
* Construct the Secretion Plasmid<br />
* Characterize the Secretion System<br />
<br />
==Constructing the Secretion System==<br />
<br />
To create the secretion system, we first synthesized a coding sequence that would produce the 3 gene constructs described above<sup>2</sup>. To do this, we synthesized biobrick compatible versions of each gene and their native ribosome binding sites from oligos as described in our [https://2009.igem.org/Team:Washington/Notebook/gene_synthesis| gene synthesis protocol].<br />
[[image:Secretion_plas.PNG |300px | right]]<br />
<br><br />
After synthesis of each individual gene w/RBS biobrick, they were then pieced together using biobrick standard assembly to form a single construct. Three different versions of the secretion construct were then created with the placement of one of three different promoters in front of the gene construct, a high ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 BBa_J23100]), a medium ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J23114 BBa_J23114]), or a low ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J23113 BBa_J23113]) strength promoter . All three designs were placed in pSB3T5. For our experiments, only the high strength promoter was used.<br />
<br />
<br><br />
<br><br><br />
<br />
==Characterization of Secretion System (Secreting the Target Protein)==<br />
<br />
To test the functionality of our secretion system, we decided to see how well the system could export the Target protein into the media. To do this, we cloned GFP (BBa_E0040) into our Target vector, and transformed the vector into cells (strain BL21 lacq) containing the Secretion plasmid. The cells containing our TargetGFP vector and Secretion plasmid were then grown in a large culture (50 ml) and expression of the Target protein was induced via IPTG. After a period of growth, the culture was then spun down to separate the cells from the media. The amount of fluorescence found in the supernatant was then used to quantify the amount of Target protein being secreted (a detailed protocol of the experiment can be found [[Team:Washington/Notebook/50mL_purification|here]]).<br />
<br />
==Results==<br />
In our first round of experiments with the cells containing both the TargetGFP construct and Secretion plasmid, two controls were also tested: cells containing only the TargetGFP construct and cells containing the TargetOpdA construct and the Secretion plasmid.<br />
<br />
The plot below shows the results of our first secretion test.<br />
<br />
[[Image:Secretion_data_plot1.png|center|600px]]<br />
<br />
As shown above, the cells containing both the TargetGFP and Secretion plasmid released a much higher amount of the target protein into the media when compared to the two controls. This result was quite exciting as it appeared that our secretion system was working great. <br />
<br />
However, after verifying these results, we realized that one of the controls was not entirely fair since the culture containing only the TargetGFP vector had just one plasmid and thus was only grown with one antibiotic (ampicillin). To make a more comparable control, the cells containing only the TargetGFP construct were also transformed with a promoter-less Secretion plasmid, and the experiment was repeated. <br />
<br />
The plot below shows the results of the second secretion test. <br />
<br />
[[Image:Secretion_data_plot2.png|center|600px]]<br />
<br />
As shown above, the addition of the promoter-less secretion plasmid into the TargetGFP control also caused the culture to release an elevated amount of target protein into the media. From this result, it appeared that the Secretion system was not responsible for the elevated amount of TargetGFP in the media, but that it was actually an artifact caused by the extra plasmid that the Secretion system was on (pSB3T5). We hypothesize that this artifact could be the result of a couple different reasons: the extra plasmid (and thus extra antibiotic resistance needed) causes an elevated amount of cell stress that causes premature cell lysis, or the cell membrane was rendered more permeable since the extra plasmid encoded tetracycline resistance via a membrane pump protein.<br />
<br />
A couple different variations of the above experiments were then tried to show secretion:<br />
*We tried varying the amount of IPTG used for induction (1-500 uM) to make sure we weren't overloading the cells<br />
*We changed the cell strain from BL21(lacq) to DH5a, the cell strain used in reference 2<br />
*We used PSB3T5 with out an insert, to eliminate the potential leaky expression from a promoter less gene<br />
*We varied the temperature of expression to see if the proteins would be more stable<br />
<br />
Unfortunately we obtained the same results for all of these additional experiments, in which there was no increase of protein concentration in the media when the secretion system was present (data not shown).<br />
<br />
<br />
====Conclusion====<br />
<br />
Based on these results, we have yet to show that the Secretion system is functioning. However, this system has been shown to work properly in the literature<sup>2</sup>, and there are many parameters that we have yet to reproduce and optimize. Proteins that have been secreted by this Type I system and other similar ones include GFP<sup>2,3</sup>, lipase<sup>3</sup>, Trichoderma harzianum endochitinase<sup>2</sup>, trout growth hormone<sup>2</sup>, ompC<sup>2</sup>, and lacZ<sup>2</sup>. This causes us to believe that our part, which has been sequenced verified, should work given a little more tweaking, to see our ideas see [[Team:Washington/Future|Future Directions]]<br />
<br><br><br />
'''Continue to [https://2009.igem.org/Team:Washington/Project/Display Display]'''<br />
<br />
='''Citations'''=<br />
<br />
#Olivera. Expression of the endogenous type II secretion pathway in Escherichia coli leads to chitinase secretion. <br />
#Palacios et al. [http://www.ncbi.nlm.nih.gov/pubmed/11157948 Subset of Hybrid Eukaryotic Proteins Is Exported by the Type I Secretion System of Erwinia chrysanthemi, Secrevtion of GFP in E. Coli]<br />
#Chung et al. [http://www.ncbi.nlm.nih.gov/pubmed/19178697 Export of recombinant proteins in Escherichia coli using ABC transporter, Secretion of GFP in E. Coli]<br />
#Holland et al. [http://www.ncbi.nlm.nih.gov/pubmed/16092522 Type 1 protein secretion in bacteria, the ABC-transporter dependent pathway]<br />
<br />
{{Template:Team:Washington/Templates/Footer}}</div>Acleonehttp://2009.igem.org/Team:Washington/Project/DisplayTeam:Washington/Project/Display2009-10-19T04:15:25Z<p>Acleone: </p>
<hr />
<div>__NOTOC__<br />
{{Template:Team:Washington/Templates/Header}}<br />
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<div>'''[https://2009.igem.org/Team:Washington/Project/Secretion &lt; Secretion]'''<br />
<div style="text-align:right; float:right">'''Continue to [https://2009.igem.org/Team:Washington/Project/Release Release &gt;]'''</div></div><br />
<br />
[[Image:Main_graphic3_display_banner.png|center]]<br />
<br />
='''Background'''=<br />
Our system hinged on finding a protein which could bind other proteins to the outside of the cell, but whose interaction with these proteins was weak enough to be disrupted by another small molecule. streptavidin presented itself as a logical choice. Several protein tags (such as the Nano-Tag <sup>1</sup>) have been developed such that they bind streptavidin at the biotin binding site, but can be released when biotin is added to the system and binds to streptavidin. The ability for peptides to bind streptavidin and be released upon the addition of biotin is a technology currently used on a daily basis world-wide by labs purifying proteins. The major difference between the traditional system and our system is that streptavidin is attached to beads which are used in a column format in the traditional protein purification method. Our system will have streptavidin attached to the surface of the cell. Combining the display system with our target and secretion vector our vision is complete. A single cell can then produce, secrete, bind, and release any protein of interest.<br />
{| cellpadding="1" cellspacing="8"<br />
|-<br />
|[[Image:Strept-biotin.png | 175px]]<br />
|The binding of the Nano-Tag by streptavidin has an affinity of 4nM and has been used to purify multiple proteins. The ability of biotin to compete off the Nano-Tag in biding assays indicates that the binding occurs at the same site of streptavidin. Of great importance for incorporating streptavidin into our display system is that streptavidin is a common protein used in bio-chemical assays. Its ability to bind biotin is one of the strongest non-covalent interactions known. This allows for the utilization of biotin and streptavidin to act as molecular connectors in protein systems. Furthermore the availability of biotinylated and streptavidin linked fluorophores allows for an easy assay of their presence, using fluorescence microscopy<sup>2</sup>.<br />
|-<br />
|}<br />
<br />
{| cellpadding="1" cellspacing="8"<br />
|-<br />
|Next we needed a way to anchor the streptavidin to the outer membrane. The obvious choice was to use the LPP signal peptide and Outer Membrane Protein A (ompA) because this system has been extensively characterized as an expression system to display proteins on the cell surface. This LPP-ompA system has been used to display a diverse group of proteins, including Green Fluorescent Protein (GFP) <sup>3</sup>, Organophosphorus Hydrolase (OPH) <sup>4</sup>, Cyclodextrin Glucanotransferase (CGTase) <sup>5</sup>, Methyl Parathion Hydrolase (MPH) <sup>6</sup>, Enhanced Green Fluorescent Protein (EGFP) <sup>6</sup>. Thus, an appropriate display construct for our system would be Lpp-OmpA display construct fused to streptavidin, which would be anchored in the outer membrane and display streptavidin on the surface of the cell - which would bind a nanotag on our target protein in an interaction that could be disrupted upon the addition of biotin. <br />
|[[Image:Display image.jpg|400px|right]]<br />
|-<br />
|}<br><br />
==== Harvard 2006 Surface Display System: Legacy Parts ====<br />
<br />
<br />
When we checked the Parts Registry to see if someone had already built this or sometime similar that we could use as a starting part for our Idealized Protein Purification Display System, we found this Lpp-OmpA-streptavidin construct, submitted by Harvard iGEM team as part of their project in 2006 <sup>7</sup>. In fact, Harvard had submitted four biobrick parts which were all variations on the same theme. All four are fusion proteins which have a LPP signal peptide, either one or five trans-membrane ompA, and either monomeric or dimeric streptavidin. All the variations and the resulting biobricks are shown below.<br />
<br />
<br />
<br />
{|cellpadding="3" cellspacing="1" border="2" width="75%" align="center"<br />
! Bio Brick<br />
! OmpA trans-membrane domains<br />
! Type of Streptavidin<br />
|- align="center"<br />
|J36848<br />
|1<br />
|monomeric<br />
|- align="center"<br />
|J36849<br />
|1<br />
|dimeric<br />
|- align="center"<br />
|J36850<br />
|5<br />
|monomeric<br />
|- align="center"<br />
|J36851<br />
|5<br />
|dimeric<br />
|-<br />
|}<br />
<br><br />
<br />
==== Harvard 2006 Surface Display System: Documented Data ====<br />
<br />
Data regarding the activity of the Harvard iGEM 2006 Lpp-OmpA-Streptavidin construct has been published in IET Synthetic Biology in 2007 <sup>7</sup>. The author showed, by Western blot against the His tag fused to this construct, that their protein was being expressed within the cell. In order to demonstrate binding of their cell-surface-anchored streptavidin to biotin, the author incubated cells expressing Lpp-OmpA-streptavidin with an aptamer that linked biotin to a fluorophore using an oligonucleotide linker (see figure below). Any cells that express streptavidin on the surface of the cell should fluoresce in their assay - concordantly, when cells were incubated with this biotin-oligo-fluorescein aptamer, the author saw fluorescence on cells expressing Lpp-OmpA-Streptavidin, but not on Lpp-OmpA negative control. However, cells that expressed streptavidin on the surface also bound to the negative aptamer control (oligo-fluorescein) that lacked biotin, indicating that the oligo aptamer itself (and not biotin) may have been responsible for the association of the fluorophore to the cell surface. Thus, the Harvard 2006 iGEM team was never able to definitively show specific affinity of their Lpp-OmpA-Streptavidin construct.<br />
<br />
{|<br />
|-<br />
|[[Image:HarvardIETSYNTH.jpg|400px|center]]<br />
|''<br />
L01W: Lpp-OmpA-Streptavidin monomer, no His Tag.<br><br />
L01H: Lpp-OmpA-Streptavidin monomer with His tag.<br><br />
L01S: Lpp-OmpA-Streptavidin dimer.<br><br />
L01: Lpp-OmpA alone).<br><br />
A: no aptamer added.<br><br />
B: With only ‘F’ 50 -fluorescently-tagged oligonucleotide.<br><br />
C: With fluorescently tagged streptavidin aptamer.<br><br />
D: With ‘B-F’ hybrid of 50 -biotinylated oligonucleotide annealed with 50-fluorescently-tagged oligonucleotide<sup>7</sup>.''<br />
|-<br />
|}<br />
<br />
<br />
Based on these experiments, we decided to try using this display system for our purpose. But first, since binding of the system to streptavidin had not be definitively shown, we had to verify binding of the Lpp-OmpA-Streptavidin construct to biotin for ourselves, and characterize this interaction.<br />
<br />
='''Experiments'''=<br />
== Harvard's Legacy 2006 Cell Surface Display Parts: Do They Work As Predicted? ==<br />
<br />
In order to determine whether the Harvard 2006 iGEM streptavidin cell surface display parts were 1. expressed properly, and 2. bound biotin on the surface of the cell, we decided to do the following set of tests. <br />
<br />
# Western to verify expression of Harvard 2006 surface display parts <br />
# Assessment of biotin binding to cell surface by visualization in fluorescence microscope <br />
# Assessment of biotin binding to cell surface by visualization in flow cytometer<br />
<br />
<br />
=== Western Blot Demonstrates that Harvard 2006 iGEM Parts Are Expressed ===<br />
The goal of this experiment was to make sure that the proteins were being expressed in our cell lines, and also to make sure that they were the correct length. This was crucial to ascertain before we moved on and began to test the parts. Even though we had the individual parts sequenced confirmed we needed to make sure that the proteins were being expressed correctly in the cell. Since all our the Harvard parts are conveniently his tagged, we used a Western blot reagent (horseradish peroxidase) that was conjugated to nickel-NTA (binds His tags)<sup>8</sup>.<br />
<br />
<br />
==== Data ====<br />
<br />
[[Image:HarvardExpressionWestern.jpg|700px|center]]<br />
<br><br />
<br />
The expected sizes of each of these proteins are shown in the table below:<br />
{|cellpadding="3" cellspacing="1" border="2" width="50%"<br />
! Bio Brick<br />
! Length (in Da)<br />
|- align="center"<br />
|J36848<br />
|21478.7<br />
|- align="center"<br />
|J36849<br />
|34600.9<br />
|- align="center"<br />
|J36850<br />
|31215.4<br />
|- align="center"<br />
|J36851<br />
|44972.3<br />
|-<br />
|}<br />
<br />
<br><br />
Based on the above table, we were able to verify expression of the Harvard 2006 iGEM surface display parts. The varying intensity of the bands does not indicate the strength of expression because the protein amount was not normalized before it was inserted into the gel. Next we wanted to determine whether functional, biotin-binding streptavidin was displayed on the cell surface in cells expressing these parts. To test this, we used two different methods: visualization by fluorescence microscopy and by flow cytometer. <br><br />
<br />
=== Streptavidin-Biotin Binding Is Not Visualized By Fluorescence Microscopy ===<br />
<br />
The goal of this experiment was to confirm the display of streptavidin on the surface of the cell. To do this, we incubated cells expressing the Harvard 2006 iGEM streptavidin surface display parts with a fluorophore conjugated to biotin. If cells are expressing functional streptavidin on the surface of the cell, they should bind the biotinylated fluorophore, and this binding should be detectable in our florescence microscope as a halo of fluorescence surrounding each individual cell. As a positive control for streptavidin-biotin binding we incubated the biotinylated fluorophore with streptavidin-coated beads that were roughly the same size (with respect to volume) as our cells (except spherical) (see Notebook page for protocols).<br />
<br />
<br />
==== Data ====<br />
<br />
<table><br />
<tr> <br />
<td><p style="font-size:18px"> Positive Control </p> </td><br />
<td><p style="font-size:18px"> Negative Control </p> </td><br />
<td rowspan="4">These images were analyzed using imageJ <sup>9</sup>. The image on the left shows an image of the beads with flourophore added to them. These beads were diluted and spun diluted and spun down until the background level of florescence was low enough to get an accurate reading. We then used imageJ to analyze the intensity of a line going through the bead, which is demonstrated by the above schematic. On the positive control the edges of the streptavidin-coated beads show spikes in florescence, indicating binding of the biotinylated fluorophore to the beads. The negative control (beads without fluorophore) showed no such increase. This meant that our biotinylated flouophore binds to streptavidin in a detectable manner. When our cells were examined in the same manner, no difference could be seen in biotinylated fluorophore binding between cells that had induced expression of surface streptavidin (left) and uninduced cells which should not express the surface display protein (right). This was evidence that the streptavidin surface display part binding to biotin was very low / nonexistent. In order to verify this, we measured the binding of the biotinylated fluorophore to entire populations of streptavidin-expressing cells by flow cytomtery.</td><br />
</tr><br />
<tr><br />
<td>[[Image:M_beads1.png| 210px]]</td><br />
<td>[[Image:M_beads2.png| 210px]]</td><br />
</tr><br />
<td><p style="font-size:18px"> Induced Cells </p></td><br />
<td><p style="font-size:18px"> Uninduced Cells </p></td><br />
<tr><br />
<td>[[Image:M_cells1.png| 210px]]</td><br />
<td>[[Image:M_cells2.png| 210px]]</td><br />
</tr><br />
</table><br />
<br />
=== Streptavidin-Biotin Binding Is Not Visualized By Flow Cytometry ===<br />
The goal of this experiment was to visualize biotin binding to streptavidin over a whole population of cells. Whereas the Microscope experiment looked at a few localized cells, we set the cytometer to look 50,000 streptavidin-expressing cells after incubation (and washing) with the biotinylated fluorophore and read the resulting florescence (see Notebook page for protocols). Like in the microscopy assay, we used streptavidin-coated beads as a positive control. The cytometer reads the flouresence of each individual particle (cell or bead) passing through it, which allows for accurate readings, and monitoring of a much larger sample size than we visualized under the microscope. The results of this experiment are described below.<br />
<br />
<br />
==== Data ====<br />
<br />
<table align="center"><br />
<tr><br />
<th>Cells</th><br />
<th>Control</th><br />
</tr><br />
<tr><br />
<td>[[Image:50.png|center]]</td><br />
<td>[[Image:StreptBead cyto.png]]</td><br />
</tr><br />
<tr><br />
<td colspan="2" align="center">''For both pictures the x-axis is the flouresence, and the y-axis count.''</td><br />
</tr><br />
</table><br />
<br />
In the flow cytometer, our positive control, streptavidin-coated beads, showed a clear distinction between beads pre-incubated with biotinylated flourophore and the beads without flourophore, indicating that this assay is capable of visualizing streptavidin binding to the biotinylated fluorophore. Like in the microscopy assay, we did not observe appreciable binding occurring with any of the ompA-streptavidin parts to biotin at the population level. All streptavidin-expressing cells demonstrated low levels of florescence comparable to those of beads not pre-incubated with fluorophore.<br />
<br />
=== Conclusion ===<br />
<br />
<br />
In conclusion, we demonstrated expression of the Harvard 2006 streptavidin surface display parts via Western Blot. We were unable, however, to visualize binding between a biotinylated flourophore and the cells expressing these proteins. This indicates that the streptavidin display system is likely not binding biotin correctly. We hypothesize that streptavidin might not having enough room on the cell surface to form tetramers (which is its native state), and thus may not be binding to biotin with high efficiency. In order to generate a construct that demonstrates tight binding to to biotin, we propose to design a generalized cell surface display system, and to computationally design a biotin-binding streptavidin monomer using software developed at the University of Washington.<br><br />
<br />
'''These solutions are described in the [https://2009.igem.org/wiki/index.php?title=Team:Washington/Future future directions section].'''<br />
<br />
----<br />
<br />
=== Citations ===<br />
#The Nano-tag, a streptavidin-binding peptide for the purification and detection of recombinant proteins doi:10.1016/j.physletb.2003.10.071<br />
#Structural origins of high-affinity biotin binding to streptavidin. PC Weber, DH Ohlendorf, JJ Wendoloski, and FR Salemme (6 January 1989). Science 243 (4887), 85. [DOI: 10.1126/science.2911722]<br />
#Display of green fluorescent protein on ''Escherichia coli'' cell surface. Shi H, Wen Su W. Department of Biological & Agricultural Engineering, University of Missouri-Columbia, 65211, Columbia, MO, USAEnzyme Microb Technol. 2001 Jan 2;28(1):25-34.<br />
#Cell surface display of organophosphorus hydrolase using ice nucleation protein. Shimazu M, Mulchandani A, Chen W. Biotechnol Prog. 2001 Jan-Feb;17(1):76-80. Department of Chemical and Environmental Engineering, and Environmental Toxicology Program, University of California, Riverside, CA 92521, USA.<br />
#Anchorage of cyclodextrin glucanotransferase on the outer membrane of ''Escherichia coli''. Wan HM, Chang BY, Lin SC. Biotechnol Bioeng. 2002 Aug 20;79(4):457-64. Department of Chemical Engineering, National Chung Hsing University, Taichung, 402, Taiwan.<br />
#Cell surface display of functional macromolecule fusions on ''Escherichia coli'' for development of an autofluorescent whole-cell biocatalyst. Yang C, Zhao Q, Liu Z, Li Q, Qiao C, Mulchandani A, Chen W. Environ Sci Technol. 2008 Aug 15;42(16):6105-10. State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.<br />
#Cell surface streptavidin. Tsai P. IET Synthetic Biol. 2007;1(1.2):32.<br />
#http://www.kpl.com/catalog/productdetail.cfm?catalog_ID=17&Category_ID=448&Product_ID=1085<br />
#http://rsbweb.nih.gov/ij/docs/index.html<br />
{{Template:Team:Washington/Templates/Footer}}</div>Acleonehttp://2009.igem.org/Team:Washington/Project/SecretionTeam:Washington/Project/Secretion2009-10-19T04:14:37Z<p>Acleone: </p>
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<div>'''[https://2009.igem.org/Team:Washington/Project/Target &lt; Secretion]'''<br />
<div style="text-align:right; float:right">'''Continue to [https://2009.igem.org/Team:Washington/Project/Display Display &gt;]'''</div></div><br />
<br />
[[Image:Main_graphic3_secretion_banner.png|center]]<br />
<br />
='''Background'''=<br />
The ability to secrete our target protein completely out of the cell and into the media is an essential feature of our project. While the laboratory ''E. coli'' strain K-12 contains genes for a type II system (the genes have been silenced<sup>1</sup>), it lacks a type I secretion system capable of exporting proteins to the extracellular space. To implement this ability into ''E. coli'', we chose to install the type I secretion system of ''Erwinia chrysanthemi''<sup>2</sup>. This system was chosen because it has been shown to function within ''E. coli'' and export a wide range of proteins through the periplasm and into the extracellular space.<sup>2,3,4</sup><br />
<br />
<br />
[[image:Secretion_structure.png |400px|right]]<br />
The secretion system is composed of three genes (prtD, prtE, and prtF) whose products recognize the protein being secreted via a C-terminus tag (prtB)<sup>2</sup>. The PrtD, PrtE, and PrtF proteins are thought to form a selective pore that connects the cytoplasm directly to the external medium. As the diagram shows, PrtD and PrtE interact with the inner membrane, while PrtF interacts with the outer membrane. PrtB is the 181-amino acid sequence that, when fused to the C-terminus of a protein, functions as the secretion signal.<br />
<br />
<br><br><br><br><br />
<br />
='''Experiments'''=<br />
<br />
<br />
<br />
For the Secretion System our goal was to:<br />
* Construct the Secretion Plasmid<br />
* Characterize the Secretion System<br />
<br />
==Constructing the Secretion System==<br />
<br />
To create the secretion system, we first synthesized a coding sequence that would produce the 3 gene constructs described above<sup>2</sup>. To do this, we synthesized biobrick compatible versions of each gene and their native ribosome binding sites from oligos as described in our [https://2009.igem.org/Team:Washington/Notebook/gene_synthesis| gene synthesis protocol].<br />
[[image:Secretion_plas.PNG |300px | right]]<br />
<br><br />
After synthesis of each individual gene w/RBS biobrick, they were then pieced together using biobrick standard assembly to form a single construct. Three different versions of the secretion construct were then created with the placement of one of three different promoters in front of the gene construct, a high ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 BBa_J23100]), a medium ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J23114 BBa_J23114]), or a low ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J23113 BBa_J23113]) strength promoter . All three designs were placed in pSB3T5. For our experiments, only the high strength promoter was used.<br />
<br />
<br><br />
<br><br><br />
<br />
==Characterization of Secretion System (Secreting the Target Protein)==<br />
<br />
To test the functionality of our secretion system, we decided to see how well the system could export the Target protein into the media. To do this, we cloned GFP (BBa_E0040) into our Target vector, and transformed the vector into cells (strain BL21 lacq) containing the Secretion plasmid. The cells containing our TargetGFP vector and Secretion plasmid were then grown in a large culture (50 ml) and expression of the Target protein was induced via IPTG. After a period of growth, the culture was then spun down to separate the cells from the media. The amount of fluorescence found in the supernatant was then used to quantify the amount of Target protein being secreted (a detailed protocol of the experiment can be found [[Team:Washington/Notebook/50mL_purification|here]]).<br />
<br />
==Results==<br />
In our first round of experiments with the cells containing both the TargetGFP construct and Secretion plasmid, two controls were also tested: cells containing only the TargetGFP construct and cells containing the TargetOpdA construct and the Secretion plasmid.<br />
<br />
The plot below shows the results of our first secretion test.<br />
<br />
[[Image:Secretion_data_plot1.png|center|600px]]<br />
<br />
As shown above, the cells containing both the TargetGFP and Secretion plasmid released a much higher amount of the target protein into the media when compared to the two controls. This result was quite exciting as it appeared that our secretion system was working great. <br />
<br />
However, after verifying these results, we realized that one of the controls was not entirely fair since the culture containing only the TargetGFP vector had just one plasmid and thus was only grown with one antibiotic (ampicillin). To make a more comparable control, the cells containing only the TargetGFP construct were also transformed with a promoter-less Secretion plasmid, and the experiment was repeated. <br />
<br />
The plot below shows the results of the second secretion test. <br />
<br />
[[Image:Secretion_data_plot2.png|center|600px]]<br />
<br />
As shown above, the addition of the promoter-less secretion plasmid into the TargetGFP control also caused the culture to release an elevated amount of target protein into the media. From this result, it appeared that the Secretion system was not responsible for the elevated amount of TargetGFP in the media, but that it was actually an artifact caused by the extra plasmid that the Secretion system was on (pSB3T5). We hypothesize that this artifact could be the result of a couple different reasons: the extra plasmid (and thus extra antibiotic resistance needed) causes an elevated amount of cell stress that causes premature cell lysis, or the cell membrane was rendered more permeable since the extra plasmid encoded tetracycline resistance via a membrane pump protein.<br />
<br />
A couple different variations of the above experiments were then tried to show secretion:<br />
*We tried varying the amount of IPTG used for induction (1-500 uM) to make sure we weren't overloading the cells<br />
*We changed the cell strain from BL21(lacq) to DH5a, the cell strain used in reference 2<br />
*We used PSB3T5 with out an insert, to eliminate the potential leaky expression from a promoter less gene<br />
*We varied the temperature of expression to see if the proteins would be more stable<br />
<br />
Unfortunately we obtained the same results for all of these additional experiments, in which there was no increase of protein concentration in the media when the secretion system was present (data not shown).<br />
<br />
<br />
====Conclusion====<br />
<br />
Based on these results, we have yet to show that the Secretion system is functioning. However, this system has been shown to work properly in the literature<sup>2</sup>, and there are many parameters that we have yet to reproduce and optimize. Proteins that have been secreted by this Type I system and other similar ones include GFP<sup>2,3</sup>, lipase<sup>3</sup>, Trichoderma harzianum endochitinase<sup>2</sup>, trout growth hormone<sup>2</sup>, ompC<sup>2</sup>, and lacZ<sup>2</sup>. This causes us to believe that our part, which has been sequenced verified, should work given a little more tweaking, to see our ideas see [[Team:Washington/Future|Future Directions]]<br />
<br><br><br />
'''Continue to [https://2009.igem.org/Team:Washington/Project/Display Display]'''<br />
<br />
='''Citations'''=<br />
<br />
#Olivera. Expression of the endogenous type II secretion pathway in Escherichia coli leads to chitinase secretion. <br />
#Palacios et al. [http://www.ncbi.nlm.nih.gov/pubmed/11157948 Subset of Hybrid Eukaryotic Proteins Is Exported by the Type I Secretion System of Erwinia chrysanthemi, Secrevtion of GFP in E. Coli]<br />
#Chung et al. [http://www.ncbi.nlm.nih.gov/pubmed/19178697 Export of recombinant proteins in Escherichia coli using ABC transporter, Secretion of GFP in E. Coli]<br />
#Holland et al. [http://www.ncbi.nlm.nih.gov/pubmed/16092522 Type 1 protein secretion in bacteria, the ABC-transporter dependent pathway]<br />
<br />
{{Template:Team:Washington/Templates/Footer}}</div>Acleonehttp://2009.igem.org/Team:Washington/Project/TargetTeam:Washington/Project/Target2009-10-19T04:13:32Z<p>Acleone: </p>
<hr />
<div>__NOTOC__<br />
{{Template:Team:Washington/Templates/Header}}<br />
<br />
<html><style><br />
#uw_himg {<br />
display:none;<br />
}<br />
</style></html><br />
<div>'''[https://2009.igem.org/Team:Washington/Project &lt; Project Description]'''<br />
<div style="text-align:right; float:right">'''Continue to [https://2009.igem.org/Team:Washington/Project/Secretion Secretion &gt;]'''</div></div><br />
<br />
[[Image:Main_graphic3_target_banner.png|center]]<br />
<br />
='''Background'''=<br />
<br />
When a favorite protein (Afp) is cloned into the target vector ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K215002 BBa_K215002]) two tags are fused onto the N and C terminal of Afp. These tags are depicted below: <br> <br />
<br />
[[Image:Targ_map.PNG | 700px | center]]<br />
<br />
The first key feature of the target vector is the NheI restriction site, where afp get's inserted. NheI is compatible with XbaI and SpeI, meaning that a biobrick digested at the X and S sites can be ligated into the target vector at the NheI site (for detailed protocol see: [https://2009.igem.org/Team:Washington/Notebook/NheI| NheI Insertion Protocol]). <br><br />
<br />
At the N-terminus of the Target is the display (aka Nano<sup>1</sup>) tag, which is a 15 amino acid sequence that binds to streptavidin. Since streptavidin is being displayed on the surface of the cell this allows our protein to stick to the outside of the cell, but can still be released by the addition of biotin. For more details see: [https://2009.igem.org/Team:Washington/Project/Display| Surface Display System ]. <br><br />
<br />
At the C-terminus of the Target is a secretion tag<sup>2</sup> (prtB) that is recognized by a Type I secretion system, which secretes proteins from the cytosol, through the periplasim, and into the media. For more details go to: [https://2009.igem.org/Team:Washington/Project/Secretion| Secretion System ]. <br> <br />
<br />
Flanking each side of the NheI site are 6 consecutive hisitidines (6x-His) and TEV protease sites <sup>3</sup>. The histidines allow for traditional immobilized metal affinity chromatography ([https://2009.igem.org/Team:Washington/Notebook/IMAC_protocol IMAC]) protein purification. The TEV sites allows for the N and C terminal tags to be cleaved off of Afp, and due to the strategic placement of the 6x-His tags these tags can then be seperated from Afp by simply running the cleaved solution over a column in which the tags stick but Afp flows right through. <br><br />
<br />
='''Experiments'''=<br />
<br />
<br />
For the target vector our goal was to:<br />
* Construct the target vector<br />
* Insert GFP into the target vector and characterize expression and function<br />
* Biobrick and characterize OpdA, a nerve agent degrading enzyme<br />
* Insert OpdA into the target vector and characterize expression and function<br />
<br />
<br />
<br />
==Target Vector Construction==<br />
<br />
To create the target vector we first synthesized a coding sequence that would produce the protein as described above. To do this we synthesized a gene from oligo's as described in our [https://2009.igem.org/Team:Washington/Notebook/gene_synthesis| Gene Synthesis Protocol].<br />
<br />
==Expression Vector Construction==<br />
<br />
After creating the target construct, we created and BioBricked an expression vector ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K215000 BBa_K215000]) which would express the target protein upon induction with IPTG. We then added the target construct into the expression vector using standard assembly making [http://partsregistry.org/wiki/index.php?title=Part:BBa_K215002 BBa_K215002].<br />
<br />
==GFP Insertion and Characterization==<br />
<br />
Upon completion of constructing the target vector our first experiment was to determine if GFP was still functional as the fusion protein. To do this GFP ([http://partsregistry.org/wiki/index.php/Part:BBa_E0040 BBa_E0040]) was inserted into the target construct use the NheI method as described above. After insertion of E0040 into the target construct, the tagged GFP (target-GFP) was transformed into BL21(lacIq) cells, and subsequently grown in the presence or absence of IPTG. As a control an untagged E0040 was cloned into our expression vector and also grown in the presence or absence of IPTG. This would allow us to determine the effects of the tags on fluorescence. The cells were then washed with PBS, normalized to the same cell density, and fluorescence measured using an excitation of 485 and emission of 525 (cutoff at 515) in a SpectraMax M5e plate reader. The data is show below:<br />
<br />
[[image:GFP_Fluroescense_corrected_for_OD.png |500px | center]]<br />
<br />
<br />
From this data we were able to conclude that our expression vector was functional, as is evident from the large increase in fluorescence with the addition of IPTG. We were also able to conclude that the Target-GFP is functional, but fluorescence was significantly decreased. <br />
<br />
In order to ensure that the Target-GFP had the appropriate 6x-His tags and that fluorescence was a function of protein concentration we purified Target-GFP using a traditional IMAC techniques. The protein concentration was measured from its absorbence a 280nm. A serial dilution of the protein was then made and the resulting fluorescence measured as described earlier. The data is shown below:<br />
<br />
[[Image:Standard_curve_targGFP.png | 500px | center]]<br />
<br />
<br />
As expected the fluorescence intensity is linear with respect to protein concentration.<br />
<br />
==BioBricking and Characterization of OpdA==<br />
<br />
====OpdA Background====<br />
<br />
OpdA ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K215090 BBa_K215090]) is an organophosphate-degrading enzyme from ''Agrobacterium radiobacter''. It is capable of degrading a wide range of organophosphates, most notably pesticides that are poisonous to humans, such as paraoxon. We chose to biobrick and submit this enzyme to the registry for a number of reasons. First and foremost, this enzyme is easy to assay for since it can hydrolyze substrates very quickly (e.g. paraoxon) and form a bright yellow product. This yellow product would make it easy to see that the OpdA was present and functioning in our system. And secondly, OpdA is a very useful enzyme that could have applications in future iGEM and other synthetic biology projects, so its presence in the Standard Registry of Biological Parts is beneficial.<br />
<br />
====OpdA Characterization====<br />
<br />
The Baker lab donated the source plasmid for OpdA (a synthetic gene optimized for ''E. coli'' expression). SOEing PCR was used to remove BioBrick cut sites ([https://2009.igem.org/Team:Washington/Notebook/SOEingPCR SOE PCR Protocol]). Upon removal of the unwanted restriction sites the gene was cloned into pSB1A2 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K215090 BBa_K215090]), our expression vector ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K215091 BBa_K215091]), and the target vector. <br />
<br />
The first experiment carried out was to validate that we could express and purify functional OpdA. To do this we transformed BB# into a BL21(lacIq) cell line and followed a traditional protein production and purification procedure ([https://2009.igem.org/Team:Washington/Notebook/IMAC_protocol IMAC Protocol]). The resulting purified protein was then dialyzed overnight in 1x PBS to remove the elution buffer which we were worried would interfere with the activity assay. The concnetration of the dialysed protein was dteremined by meauring its absorbance at 280nM and using its predicted extinction coefficient (29575 M-1 cm-1,[http://ca.expasy.org/tools/protparam.html ProtParam]). We obtained ~1mL of 10microM protein. To determine the catalytic constants the nerve agent paroxoan was used a a substrate. As shown below, hydrolysis product of paroxoan is p-nitrophenol which has a strong absorbance at 400nM (and turns bright yellow).<br><br />
[[image:ParoxoanRxn2.png | 400px | center]]<br />
<br />
<br />
A serial dilution, ranging from 5 millimolar to 5 micromolar, of paroxoan was made in a reaction buffer (100mM HEPES pH=7, 500mM NaCl, 2mM CoCl2). To this a reaction OpdA was added so that it's final concentration was 1nM (dilutions were made in the reaction buffer). At all substrate concentrations no appreciable hydrolysis was observed without enzyme. The rate of hydrolysis with enzyme is shown below, the left hand plot is the full substrate range, and the right hand plot is a zoom in of the lower substrate concentrations:<br />
<br />
<gallery heights=400px widths=350><br />
image:OpdA_full.png<br />
image:OpdA_zoom.png<br />
</gallery><br />
<br />
From the above plot, it obvious that this enzyme efficiently catalysis paroxoan hydrolysis, but does not exhibit the usual Michaelis-Menten dynamics. It can be seen that at high enough concentrations, the enzyme actually undergoes substrate-inhibition, wherein the extra substrate actually slows the enzyme's velocity. When fit to a cononical substrate inhibition curve we obtain the following kinetic parameters:<br />
<br />
kcat (M-1 s-1): 17.6 <br><br />
Km (mM): 0.011 <br><br />
Ksi (mM): 1.06 <br><br />
<br />
These parameters confirm that this is an extremely efficient enzyme, and our kinetic parameters are comparable to previously published data for this enzyme on this substrate <sup>4,5</sup>. Also, substrate inhibition for this enzyme has been observed previously on similar substrates, so this was not an enitrely surprising results.<br />
<br />
Since the OpdA BioBrick was characterized and worked as expected we decided to continue and insert it into the target vector. Unfortinately when OpdA-Target was expressed and purified as described above no observable paroxoan hydrolysis was observed.<br />
<br />
<div style="text-align:right">'''Continue to [https://2009.igem.org/Team:Washington/Project/Secretion Secretion &gt;]'''</div><br />
<br />
='''Citations'''=<br />
<br />
<br />
1. Lamla and Erdmann. [http://www.ncbi.nlm.nih.gov/pubmed/14680960 The Nano-tag, a streptavidin-binding peptide for the purification and detection of recombinant proteins.]<br />
<br />
2. Palacios et al. [http://www.ncbi.nlm.nih.gov/pubmed/11157948 Subset of Hybrid Eukaryotic Proteins Is Exported by the Type I Secretion System of Erwinia chrysanthemi, Secrevtion of GFP in E. Coli].<br />
<br />
3. [http://www.cardiff.ac.uk/biosi/staffinfo/ehrmann/tools/TEVprot.html Tobacco Etch Virus (TEV) Protease general information]<br />
<br />
4. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC126808/ Irene Horne, et al. Identification of an opd (Organophosphate Degradation) Gene in an Agrobacterium Isolate.]<br />
<br />
5. [http://peds.oxfordjournals.org/cgi/content/abstract/16/2/135 H.Yang, et al. Evolution of an organophosphate-degrading enzyme:a comparison of natural and directed evolution.]<br />
<br />
<br />
<br />
<br />
{{Template:Team:Washington/Templates/Footer}}</div>Acleonehttp://2009.igem.org/Team:Washington/Project/FoldItTeam:Washington/Project/FoldIt2009-10-19T04:13:12Z<p>Acleone: </p>
<hr />
<div>__NOTOC__<br />
{{Template:Team:Washington/Templates/Header}}<br />
<br />
<html><script type="text/javascript"><br />
$(function() {<br />
$("#uw_foldit_div a").attr("href", "http://www.fold.it/");<br />
});<br />
</script></html><br />
<br />
<div id="uw_foldit_div" style="text-align:center;font-size:200%;"><br />
'''[http://www.fold.it/ Fold-It]'''<br />
<br />
[[image:FoldIt_Link.png]]<br />
</div><br />
<br />
====Problem====<br />
Streptavidin in its native form exists as a homotetramer, where adjacent subunits interact allowing for a strong interaction with biotin. This interaction is strong (Kd = 1.5E-15 M at pH 5.0) and can withstand most strong denaturing agents<sup>1</sup>. However when in its monomeric form, streptavidin does not maintain this strong interaction and its usefulness as a strong binder diminishes. For our system we needed a protein that could: be easily displayed on the surface of the cell, specifically bind a ligand, and release this ligand in the presence of biotin. The ability to display a protein on the cell surface is trivial, however there is difficulty in trying to get a protein to be functional on the surface of the cell. In the case of streptavidin the ability of the protein to form tetramers on the cell surface seems to be hindered, due to the poor ability of cells displaying streptavidin to bind biotinylated fluorophore (observed above). From this issue the idea of using a monomeric protein to bind biotin arose. <br />
<br />
====The Idea====<br />
There are engineered forms of streptavidin that have mutations preventing the formation of tetrameric structures. However as mentioned before, as a monomer streptavidin has a weaker affinity to biotin than would be desired. Instead of screening proteins from the literature for ability to bind biotin our group approached the Baker lab at our university. After mentioning our problem, it was recommended that we design a biotin binding protein using the [http://boinc.bakerlab.org/rosetta/ Rosetta software] they developed. Rosetta in conjunction with [http://www.folt.it Fold-It] (also developed at the University of Washington) would allow use to design and optimize proteins for binding biotin. <br />
<br />
====The Trench Work====<br />
The first step in designing our protein was looking at the native biotin-streptavidin interaction and taking measurements between key amino acids and the biotin molecule. From here we entered the constraints into Rosetta where it matched our measurements into proteins from a protein scaffold library. This produced a large set of scaffolds with different ways each one could be used to bind biotin. These scaffolds must be screened manually, and the scaffolds that look the most promising can be placed into Fold-It. Once in Fold-It, the public has access to your protein design and can tweak and tune the protein to optimize its interaction with biotin. This allows anyone (with or without prior protein knowledge) to optimize your protein scaffold. <br />
<br />
As can be seen below Fold-It uses an easily learned user interface and uses a score board to show the players who is the best folder. <br />
-----<br />
<br><br />
<html><br />
<br />
<table><tr><td><br />
<object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/P-UR3G7TBb4&hl=en&fs=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/P-UR3G7TBb4&hl=en&fs=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object><br />
</td><td><br />
Here we see a video of Fold-It, showing one of our biotin puzzles "Hold Me Tightly". The protein is represented as a cartoon model, showing off its secondary structure as well as key amino acid groups. Steric clashes of the amino acid side chains show up as red balls and can also be observed in the video. These steric clashs can be removed with the Shake Function. The Shake function in Fold-It performs coarse sampling of the amino acid conformations, looking for a global-minima. <br />
</td></tr></table><br />
<br />
<table><tr><td><br />
<object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/_Ugmw69_94g&hl=en&fs=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/_Ugmw69_94g&hl=en&fs=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object><br />
</td><td><br />
The function we see here is the Mutate function. This allows the user to sample many amino acids at a particular site, or the whole protein. Mutate looks for global-minima while sampling amino acids. As is seen here Alanine is mutated to Asparagine. The blue and white striped band indicates that a hydrogen bond has been formed, which is a favorable interaction between two polar residues.<br />
</td></tr></table><br />
<br />
<table><tr><td><br />
<object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/3V2OpBGruzQ&hl=en&fs=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/3V2OpBGruzQ&hl=en&fs=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object><br />
</td><td><br />
Another nice feature of Fold-It is the ability to select a sphere of amino acids around your ligand, and optimize these amino acids based off of a fine sampling of conformations. Here we see the amino acids surrounding the ligand being selected and having the Wiggle function performed on them. The Wiggle function in Fold-It allows the user to fine tune the protein structure. Finding a local-minima for the amino acid conformations.<br />
</td></tr></table><br />
<br />
</html><br />
<br><br />
-----<br />
<br />
This accessible format has allowed over 100,000 users to help design proteins. Currently we have published protein puzzles on Fold-It and are screening though the top scoring designs. An undergrad in our group will be active throughout the next year testing the designs and looking for biotin binding proteins.<br />
<br />
=== References ===<br />
#[http://www.ncbi.nlm.nih.gov/pubmed/18287646?ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum Haugland RP, "Coupling of antibodies with biotin".]<br />
<br />
<br />
{{Template:Team:Washington/Templates/Footer}}</div>Acleonehttp://2009.igem.org/Team:Washington/Project/FoldItTeam:Washington/Project/FoldIt2009-10-19T04:11:57Z<p>Acleone: </p>
<hr />
<div>__NOTOC__<br />
{{Template:Team:Washington/Templates/Header}}<br />
<br />
<html><script type="text/javascript"><br />
$(function() {<br />
$("#uw_foldit_div img").attr("href", "http://www.fold.it/");<br />
});<br />
</script></html><br />
<br />
<div id="uw_foldit_div" style="text-align:center;font-size:200%;"><br />
'''[http://www.fold.it/ Fold-It]'''<br />
<br />
[[image:FoldIt_Link.png]]<br />
</div><br />
<br />
====Problem====<br />
Streptavidin in its native form exists as a homotetramer, where adjacent subunits interact allowing for a strong interaction with biotin. This interaction is strong (Kd = 1.5E-15 M at pH 5.0) and can withstand most strong denaturing agents<sup>1</sup>. However when in its monomeric form, streptavidin does not maintain this strong interaction and its usefulness as a strong binder diminishes. For our system we needed a protein that could: be easily displayed on the surface of the cell, specifically bind a ligand, and release this ligand in the presence of biotin. The ability to display a protein on the cell surface is trivial, however there is difficulty in trying to get a protein to be functional on the surface of the cell. In the case of streptavidin the ability of the protein to form tetramers on the cell surface seems to be hindered, due to the poor ability of cells displaying streptavidin to bind biotinylated fluorophore (observed above). From this issue the idea of using a monomeric protein to bind biotin arose. <br />
<br />
====The Idea====<br />
There are engineered forms of streptavidin that have mutations preventing the formation of tetrameric structures. However as mentioned before, as a monomer streptavidin has a weaker affinity to biotin than would be desired. Instead of screening proteins from the literature for ability to bind biotin our group approached the Baker lab at our university. After mentioning our problem, it was recommended that we design a biotin binding protein using the [http://boinc.bakerlab.org/rosetta/ Rosetta software] they developed. Rosetta in conjunction with [http://www.folt.it Fold-It] (also developed at the University of Washington) would allow use to design and optimize proteins for binding biotin. <br />
<br />
====The Trench Work====<br />
The first step in designing our protein was looking at the native biotin-streptavidin interaction and taking measurements between key amino acids and the biotin molecule. From here we entered the constraints into Rosetta where it matched our measurements into proteins from a protein scaffold library. This produced a large set of scaffolds with different ways each one could be used to bind biotin. These scaffolds must be screened manually, and the scaffolds that look the most promising can be placed into Fold-It. Once in Fold-It, the public has access to your protein design and can tweak and tune the protein to optimize its interaction with biotin. This allows anyone (with or without prior protein knowledge) to optimize your protein scaffold. <br />
<br />
As can be seen below Fold-It uses an easily learned user interface and uses a score board to show the players who is the best folder. <br />
-----<br />
<br><br />
<html><br />
<br />
<table><tr><td><br />
<object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/P-UR3G7TBb4&hl=en&fs=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/P-UR3G7TBb4&hl=en&fs=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object><br />
</td><td><br />
Here we see a video of Fold-It, showing one of our biotin puzzles "Hold Me Tightly". The protein is represented as a cartoon model, showing off its secondary structure as well as key amino acid groups. Steric clashes of the amino acid side chains show up as red balls and can also be observed in the video. These steric clashs can be removed with the Shake Function. The Shake function in Fold-It performs coarse sampling of the amino acid conformations, looking for a global-minima. <br />
</td></tr></table><br />
<br />
<table><tr><td><br />
<object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/_Ugmw69_94g&hl=en&fs=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/_Ugmw69_94g&hl=en&fs=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object><br />
</td><td><br />
The function we see here is the Mutate function. This allows the user to sample many amino acids at a particular site, or the whole protein. Mutate looks for global-minima while sampling amino acids. As is seen here Alanine is mutated to Asparagine. The blue and white striped band indicates that a hydrogen bond has been formed, which is a favorable interaction between two polar residues.<br />
</td></tr></table><br />
<br />
<table><tr><td><br />
<object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/3V2OpBGruzQ&hl=en&fs=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/3V2OpBGruzQ&hl=en&fs=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object><br />
</td><td><br />
Another nice feature of Fold-It is the ability to select a sphere of amino acids around your ligand, and optimize these amino acids based off of a fine sampling of conformations. Here we see the amino acids surrounding the ligand being selected and having the Wiggle function performed on them. The Wiggle function in Fold-It allows the user to fine tune the protein structure. Finding a local-minima for the amino acid conformations.<br />
</td></tr></table><br />
<br />
</html><br />
<br><br />
-----<br />
<br />
This accessible format has allowed over 100,000 users to help design proteins. Currently we have published protein puzzles on Fold-It and are screening though the top scoring designs. An undergrad in our group will be active throughout the next year testing the designs and looking for biotin binding proteins.<br />
<br />
=== References ===<br />
#[http://www.ncbi.nlm.nih.gov/pubmed/18287646?ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum Haugland RP, "Coupling of antibodies with biotin".]<br />
<br />
<br />
{{Template:Team:Washington/Templates/Footer}}</div>Acleonehttp://2009.igem.org/Team:Washington/Project/FoldItTeam:Washington/Project/FoldIt2009-10-19T04:03:15Z<p>Acleone: /* Fold-It */</p>
<hr />
<div>__NOTOC__<br />
{{Template:Team:Washington/Templates/Header}}<br />
<br />
<br />
<a align="center" title="Fold-It" class="image" href="http://www.fold.it/"><img border="0" width="450" height="282" src="/wiki/images/9/92/FoldIt_Link.png" alt="Fold-It"/></a><br />
<br />
===[http://www.fold.it Fold-It]===<br />
<br />
====Problem====<br />
Streptavidin in its native form exists as a homotetramer, where adjacent subunits interact allowing for a strong interaction with biotin. This interaction is strong (Kd = 1.5E-15 M at pH 5.0) and can withstand most strong denaturing agents<sup>1</sup>. However when in its monomeric form, streptavidin does not maintain this strong interaction and its usefulness as a strong binder diminishes. For our system we needed a protein that could: be easily displayed on the surface of the cell, specifically bind a ligand, and release this ligand in the presence of biotin. The ability to display a protein on the cell surface is trivial, however there is difficulty in trying to get a protein to be functional on the surface of the cell. In the case of streptavidin the ability of the protein to form tetramers on the cell surface seems to be hindered, due to the poor ability of cells displaying streptavidin to bind biotinylated fluorophore (observed above). From this issue the idea of using a monomeric protein to bind biotin arose. <br />
<br />
====The Idea====<br />
There are engineered forms of streptavidin that have mutations preventing the formation of tetrameric structures. However as mentioned before, as a monomer streptavidin has a weaker affinity to biotin than would be desired. Instead of screening proteins from the literature for ability to bind biotin our group approached the Baker lab at our university. After mentioning our problem, it was recommended that we design a biotin binding protein using the [http://boinc.bakerlab.org/rosetta/ Rosetta software] they developed. Rosetta in conjunction with [http://www.folt.it Fold-It] (also developed at the University of Washington) would allow use to design and optimize proteins for binding biotin. <br />
<br />
====The Trench Work====<br />
The first step in designing our protein was looking at the native biotin-streptavidin interaction and taking measurements between key amino acids and the biotin molecule. From here we entered the constraints into Rosetta where it matched our measurements into proteins from a protein scaffold library. This produced a large set of scaffolds with different ways each one could be used to bind biotin. These scaffolds must be screened manually, and the scaffolds that look the most promising can be placed into Fold-It. Once in Fold-It, the public has access to your protein design and can tweak and tune the protein to optimize its interaction with biotin. This allows anyone (with or without prior protein knowledge) to optimize your protein scaffold. <br />
<br />
As can be seen below Fold-It uses an easily learned user interface and uses a score board to show the players who is the best folder. <br />
-----<br />
<br><br />
<html><br />
<br />
<table><tr><td><br />
<object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/P-UR3G7TBb4&hl=en&fs=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/P-UR3G7TBb4&hl=en&fs=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object><br />
</td><td><br />
Here we see a video of Fold-It, showing one of our biotin puzzles "Hold Me Tightly". The protein is represented as a cartoon model, showing off its secondary structure as well as key amino acid groups. Steric clashes of the amino acid side chains show up as red balls and can also be observed in the video. These steric clashs can be removed with the Shake Function. The Shake function in Fold-It performs coarse sampling of the amino acid conformations, looking for a global-minima. <br />
</td></tr></table><br />
<br />
<table><tr><td><br />
<object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/_Ugmw69_94g&hl=en&fs=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/_Ugmw69_94g&hl=en&fs=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object><br />
</td><td><br />
The function we see here is the Mutate function. This allows the user to sample many amino acids at a particular site, or the whole protein. Mutate looks for global-minima while sampling amino acids. As is seen here Alanine is mutated to Asparagine. The blue and white striped band indicates that a hydrogen bond has been formed, which is a favorable interaction between two polar residues.<br />
</td></tr></table><br />
<br />
<table><tr><td><br />
<object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/3V2OpBGruzQ&hl=en&fs=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/3V2OpBGruzQ&hl=en&fs=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object><br />
</td><td><br />
Another nice feature of Fold-It is the ability to select a sphere of amino acids around your ligand, and optimize these amino acids based off of a fine sampling of conformations. Here we see the amino acids surrounding the ligand being selected and having the Wiggle function performed on them. The Wiggle function in Fold-It allows the user to fine tune the protein structure. Finding a local-minima for the amino acid conformations.<br />
</td></tr></table><br />
<br />
</html><br />
<br><br />
-----<br />
<br />
This accessible format has allowed over 100,000 users to help design proteins. Currently we have published protein puzzles on Fold-It and are screening though the top scoring designs. An undergrad in our group will be active throughout the next year testing the designs and looking for biotin binding proteins.<br />
<br />
=== References ===<br />
#[http://www.ncbi.nlm.nih.gov/pubmed/18287646?ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum Haugland RP, "Coupling of antibodies with biotin".]<br />
<br />
<br />
{{Template:Team:Washington/Templates/Footer}}</div>Acleonehttp://2009.igem.org/Team:Washington/Project/TargetTeam:Washington/Project/Target2009-10-19T03:59:58Z<p>Acleone: </p>
<hr />
<div>__NOTOC__<br />
{{Template:Team:Washington/Templates/Header}}<br />
<br />
<html><style><br />
#uw_himg {<br />
display:none;<br />
}<br />
</style></html><br />
<div>'''[https://2009.igem.org/Team:Washington/Project/ &lt; Project Description]'''<br />
<div style="text-align:right; float:right">'''Continue to [https://2009.igem.org/Team:Washington/Project/Secretion Secretion &gt;]'''</div></div><br />
<br />
[[Image:Main_graphic3_target_banner.png|center]]<br />
<br />
='''Background'''=<br />
<br />
When a favorite protein (Afp) is cloned into the target vector ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K215002 BBa_K215002]) two tags are fused onto the N and C terminal of Afp. These tags are depicted below: <br> <br />
<br />
[[Image:Targ_map.PNG | 700px | center]]<br />
<br />
The first key feature of the target vector is the NheI restriction site, where afp get's inserted. NheI is compatible with XbaI and SpeI, meaning that a biobrick digested at the X and S sites can be ligated into the target vector at the NheI site (for detailed protocol see: [https://2009.igem.org/Team:Washington/Notebook/NheI| NheI Insertion Protocol]). <br><br />
<br />
At the N-terminus of the Target is the display (aka Nano<sup>1</sup>) tag, which is a 15 amino acid sequence that binds to streptavidin. Since streptavidin is being displayed on the surface of the cell this allows our protein to stick to the outside of the cell, but can still be released by the addition of biotin. For more details see: [https://2009.igem.org/Team:Washington/Project/Display| Surface Display System ]. <br><br />
<br />
At the C-terminus of the Target is a secretion tag<sup>2</sup> (prtB) that is recognized by a Type I secretion system, which secretes proteins from the cytosol, through the periplasim, and into the media. For more details go to: [https://2009.igem.org/Team:Washington/Project/Secretion| Secretion System ]. <br> <br />
<br />
Flanking each side of the NheI site are 6 consecutive hisitidines (6x-His) and TEV protease sites <sup>3</sup>. The histidines allow for traditional immobilized metal affinity chromatography ([https://2009.igem.org/Team:Washington/Notebook/IMAC_protocol IMAC]) protein purification. The TEV sites allows for the N and C terminal tags to be cleaved off of Afp, and due to the strategic placement of the 6x-His tags these tags can then be seperated from Afp by simply running the cleaved solution over a column in which the tags stick but Afp flows right through. <br><br />
<br />
='''Experiments'''=<br />
<br />
<br />
For the target vector our goal was to:<br />
* Construct the target vector<br />
* Insert GFP into the target vector and characterize expression and function<br />
* Biobrick and characterize OpdA, a nerve agent degrading enzyme<br />
* Insert OpdA into the target vector and characterize expression and function<br />
<br />
<br />
<br />
==Target Vector Construction==<br />
<br />
To create the target vector we first synthesized a coding sequence that would produce the protein as described above. To do this we synthesized a gene from oligo's as described in our [https://2009.igem.org/Team:Washington/Notebook/gene_synthesis| Gene Synthesis Protocol].<br />
<br />
==Expression Vector Construction==<br />
<br />
After creating the target construct, we created and BioBricked an expression vector ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K215000 BBa_K215000]) which would express the target protein upon induction with IPTG. We then added the target construct into the expression vector using standard assembly making [http://partsregistry.org/wiki/index.php?title=Part:BBa_K215002 BBa_K215002].<br />
<br />
==GFP Insertion and Characterization==<br />
<br />
Upon completion of constructing the target vector our first experiment was to determine if GFP was still functional as the fusion protein. To do this GFP ([http://partsregistry.org/wiki/index.php/Part:BBa_E0040 BBa_E0040]) was inserted into the target construct use the NheI method as described above. After insertion of E0040 into the target construct, the tagged GFP (target-GFP) was transformed into BL21(lacIq) cells, and subsequently grown in the presence or absence of IPTG. As a control an untagged E0040 was cloned into our expression vector and also grown in the presence or absence of IPTG. This would allow us to determine the effects of the tags on fluorescence. The cells were then washed with PBS, normalized to the same cell density, and fluorescence measured using an excitation of 485 and emission of 525 (cutoff at 515) in a SpectraMax M5e plate reader. The data is show below:<br />
<br />
[[image:GFP_Fluroescense_corrected_for_OD.png |500px | center]]<br />
<br />
<br />
From this data we were able to conclude that our expression vector was functional, as is evident from the large increase in fluorescence with the addition of IPTG. We were also able to conclude that the Target-GFP is functional, but fluorescence was significantly decreased. <br />
<br />
In order to ensure that the Target-GFP had the appropriate 6x-His tags and that fluorescence was a function of protein concentration we purified Target-GFP using a traditional IMAC techniques. The protein concentration was measured from its absorbence a 280nm. A serial dilution of the protein was then made and the resulting fluorescence measured as described earlier. The data is shown below:<br />
<br />
[[Image:Standard_curve_targGFP.png | 500px | center]]<br />
<br />
<br />
As expected the fluorescence intensity is linear with respect to protein concentration.<br />
<br />
==BioBricking and Characterization of OpdA==<br />
<br />
====OpdA Background====<br />
<br />
OpdA ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K215090 BBa_K215090]) is an organophosphate-degrading enzyme from ''Agrobacterium radiobacter''. It is capable of degrading a wide range of organophosphates, most notably pesticides that are poisonous to humans, such as paraoxon. We chose to biobrick and submit this enzyme to the registry for a number of reasons. First and foremost, this enzyme is easy to assay for since it can hydrolyze substrates very quickly (e.g. paraoxon) and form a bright yellow product. This yellow product would make it easy to see that the OpdA was present and functioning in our system. And secondly, OpdA is a very useful enzyme that could have applications in future iGEM and other synthetic biology projects, so its presence in the Standard Registry of Biological Parts is beneficial.<br />
<br />
====OpdA Characterization====<br />
<br />
The Baker lab donated the source plasmid for OpdA (a synthetic gene optimized for ''E. coli'' expression). SOEing PCR was used to remove BioBrick cut sites ([https://2009.igem.org/Team:Washington/Notebook/SOEingPCR SOE PCR Protocol]). Upon removal of the unwanted restriction sites the gene was cloned into pSB1A2 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K215090 BBa_K215090]), our expression vector ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K215091 BBa_K215091]), and the target vector. <br />
<br />
The first experiment carried out was to validate that we could express and purify functional OpdA. To do this we transformed BB# into a BL21(lacIq) cell line and followed a traditional protein production and purification procedure ([https://2009.igem.org/Team:Washington/Notebook/IMAC_protocol IMAC Protocol]). The resulting purified protein was then dialyzed overnight in 1x PBS to remove the elution buffer which we were worried would interfere with the activity assay. The concnetration of the dialysed protein was dteremined by meauring its absorbance at 280nM and using its predicted extinction coefficient (29575 M-1 cm-1,[http://ca.expasy.org/tools/protparam.html ProtParam]). We obtained ~1mL of 10microM protein. To determine the catalytic constants the nerve agent paroxoan was used a a substrate. As shown below, hydrolysis product of paroxoan is p-nitrophenol which has a strong absorbance at 400nM (and turns bright yellow).<br><br />
[[image:ParoxoanRxn2.png | 400px | center]]<br />
<br />
<br />
A serial dilution, ranging from 5 millimolar to 5 micromolar, of paroxoan was made in a reaction buffer (100mM HEPES pH=7, 500mM NaCl, 2mM CoCl2). To this a reaction OpdA was added so that it's final concentration was 1nM (dilutions were made in the reaction buffer). At all substrate concentrations no appreciable hydrolysis was observed without enzyme. The rate of hydrolysis with enzyme is shown below, the left hand plot is the full substrate range, and the right hand plot is a zoom in of the lower substrate concentrations:<br />
<br />
<gallery heights=400px widths=350><br />
image:OpdA_full.png<br />
image:OpdA_zoom.png<br />
</gallery><br />
<br />
From the above plot, it obvious that this enzyme efficiently catalysis paroxoan hydrolysis, but does not exhibit the usual Michaelis-Menten dynamics. It can be seen that at high enough concentrations, the enzyme actually undergoes substrate-inhibition, wherein the extra substrate actually slows the enzyme's velocity. When fit to a cononical substrate inhibition curve we obtain the following kinetic parameters:<br />
<br />
kcat (M-1 s-1): 17.6 <br><br />
Km (mM): 0.011 <br><br />
Ksi (mM): 1.06 <br><br />
<br />
These parameters confirm that this is an extremely efficient enzyme, and our kinetic parameters are comparable to previously published data for this enzyme on this substrate <sup>4,5</sup>. Also, substrate inhibition for this enzyme has been observed previously on similar substrates, so this was not an enitrely surprising results.<br />
<br />
Since the OpdA BioBrick was characterized and worked as expected we decided to continue and insert it into the target vector. Unfortinately when OpdA-Target was expressed and purified as described above no observable paroxoan hydrolysis was observed.<br />
<br />
<div style="text-align:right">'''Continue to [https://2009.igem.org/Team:Washington/Project/Secretion Secretion &gt;]'''</div><br />
<br />
='''Citations'''=<br />
<br />
<br />
1. Lamla and Erdmann. [http://www.ncbi.nlm.nih.gov/pubmed/14680960 The Nano-tag, a streptavidin-binding peptide for the purification and detection of recombinant proteins.]<br />
<br />
2. Palacios et al. [http://www.ncbi.nlm.nih.gov/pubmed/11157948 Subset of Hybrid Eukaryotic Proteins Is Exported by the Type I Secretion System of Erwinia chrysanthemi, Secrevtion of GFP in E. Coli].<br />
<br />
3. [http://www.cardiff.ac.uk/biosi/staffinfo/ehrmann/tools/TEVprot.html Tobacco Etch Virus (TEV) Protease general information]<br />
<br />
4. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC126808/ Irene Horne, et al. Identification of an opd (Organophosphate Degradation) Gene in an Agrobacterium Isolate.]<br />
<br />
5. [http://peds.oxfordjournals.org/cgi/content/abstract/16/2/135 H.Yang, et al. Evolution of an organophosphate-degrading enzyme:a comparison of natural and directed evolution.]<br />
<br />
<br />
<br />
<br />
{{Template:Team:Washington/Templates/Footer}}</div>Acleonehttp://2009.igem.org/Team:Washington/Project/TargetTeam:Washington/Project/Target2009-10-19T03:29:31Z<p>Acleone: </p>
<hr />
<div>__NOTOC__<br />
{{Template:Team:Washington/Templates/Header}}<br />
<br />
<html><style><br />
#uw_himg {<br />
display:none;<br />
}<br />
</style></html><br />
<br />
<div style="text-align:right">'''Continue to [https://2009.igem.org/Team:Washington/Project/Secretion Secretion &gt;]'''</div><br />
<br />
[[Image:Main_graphic3_target_banner.png|center]]<br />
<br />
='''Background'''=<br />
<br />
When a favorite protein (Afp) is cloned into the target vector ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K215002 BBa_K215002]) two tags are fused onto the N and C terminal of Afp. These tags are depicted below: <br> <br />
<br />
[[Image:Targ_map.PNG | 700px | center]]<br />
<br />
The first key feature of the target vector is the NheI restriction site, where afp get's inserted. NheI is compatible with XbaI and SpeI, meaning that a biobrick digested at the X and S sites can be ligated into the target vector at the NheI site (for detailed protocol see: [https://2009.igem.org/Team:Washington/Notebook/NheI| NheI Insertion Protocol]). <br><br />
<br />
At the N-terminus of the Target is the display (aka Nano<sup>1</sup>) tag, which is a 15 amino acid sequence that binds to streptavidin. Since streptavidin is being displayed on the surface of the cell this allows our protein to stick to the outside of the cell, but can still be released by the addition of biotin. For more details see: [https://2009.igem.org/Team:Washington/Project/Display| Surface Display System ]. <br><br />
<br />
At the C-terminus of the Target is a secretion tag<sup>2</sup> (prtB) that is recognized by a Type I secretion system, which secretes proteins from the cytosol, through the periplasim, and into the media. For more details go to: [https://2009.igem.org/Team:Washington/Project/Secretion| Secretion System ]. <br> <br />
<br />
Flanking each side of the NheI site are 6 consecutive hisitidines (6x-His) and TEV protease sites <sup>3</sup>. The histidines allow for traditional immobilized metal affinity chromatography ([https://2009.igem.org/Team:Washington/Notebook/IMAC_protocol IMAC]) protein purification. The TEV sites allows for the N and C terminal tags to be cleaved off of Afp, and due to the strategic placement of the 6x-His tags these tags can then be seperated from Afp by simply running the cleaved solution over a column in which the tags stick but Afp flows right through. <br><br />
<br />
='''Experiments'''=<br />
<br />
<br />
For the target vector our goal was to:<br />
* Construct the target vector<br />
* Insert GFP into the target vector and characterize expression and function<br />
* Biobrick and characterize OpdA, a nerve agent degrading enzyme<br />
* Insert OpdA into the target vector and characterize expression and function<br />
<br />
<br />
<br />
==Target Vector Construction==<br />
<br />
To create the target vector we first synthesized a coding sequence that would produce the protein as described above. To do this we synthesized a gene from oligo's as described in our [https://2009.igem.org/Team:Washington/Notebook/gene_synthesis| Gene Synthesis Protocol].<br />
<br />
==Expression Vector Construction==<br />
<br />
After creating the target construct, we created and BioBricked an expression vector ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K215000 BBa_K215000]) which would express the target protein upon induction with IPTG. We then added the target construct into the expression vector using standard assembly making [http://partsregistry.org/wiki/index.php?title=Part:BBa_K215002 BBa_K215002].<br />
<br />
==GFP Insertion and Characterization==<br />
<br />
Upon completion of constructing the target vector our first experiment was to determine if GFP was still functional as the fusion protein. To do this GFP ([http://partsregistry.org/wiki/index.php/Part:BBa_E0040 BBa_E0040]) was inserted into the target construct use the NheI method as described above. After insertion of E0040 into the target construct, the tagged GFP (target-GFP) was transformed into BL21(lacIq) cells, and subsequently grown in the presence or absence of IPTG. As a control an untagged E0040 was cloned into our expression vector and also grown in the presence or absence of IPTG. This would allow us to determine the effects of the tags on fluorescence. The cells were then washed with PBS, normalized to the same cell density, and fluorescence measured using an excitation of 485 and emission of 525 (cutoff at 515) in a SpectraMax M5e plate reader. The data is show below:<br />
<br />
[[image:GFP_Fluroescense_corrected_for_OD.png |500px | center]]<br />
<br />
<br />
From this data we were able to conclude that our expression vector was functional, as is evident from the large increase in fluorescence with the addition of IPTG. We were also able to conclude that the Target-GFP is functional, but fluorescence was significantly decreased. <br />
<br />
In order to ensure that the Target-GFP had the appropriate 6x-His tags and that fluorescence was a function of protein concentration we purified Target-GFP using a traditional IMAC techniques. The protein concentration was measured from its absorbence a 280nm. A serial dilution of the protein was then made and the resulting fluorescence measured as described earlier. The data is shown below:<br />
<br />
[[Image:Standard_curve_targGFP.png | 500px | center]]<br />
<br />
<br />
As expected the fluorescence intensity is linear with respect to protein concentration.<br />
<br />
==BioBricking and Characterization of OpdA==<br />
<br />
====OpdA Background====<br />
<br />
OpdA ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K215090 BBa_K215090]) is an organophosphate-degrading enzyme from ''Agrobacterium radiobacter''. It is capable of degrading a wide range of organophosphates, most notably pesticides that are poisonous to humans, such as paraoxon. We chose to biobrick and submit this enzyme to the registry for a number of reasons. First and foremost, this enzyme is easy to assay for since it can hydrolyze substrates very quickly (e.g. paraoxon) and form a bright yellow product. This yellow product would make it easy to see that the OpdA was present and functioning in our system. And secondly, OpdA is a very useful enzyme that could have applications in future iGEM and other synthetic biology projects, so its presence in the Standard Registry of Biological Parts is beneficial.<br />
<br />
====OpdA Characterization====<br />
<br />
The Baker lab donated the source plasmid for OpdA (a synthetic gene optimized for ''E. coli'' expression). SOEing PCR was used to remove BioBrick cut sites ([https://2009.igem.org/Team:Washington/Notebook/SOEingPCR SOE PCR Protocol]). Upon removal of the unwanted restriction sites the gene was cloned into pSB1A2 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K215090 BBa_K215090]), our expression vector ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K215091 BBa_K215091]), and the target vector. <br />
<br />
The first experiment carried out was to validate that we could express and purify functional OpdA. To do this we transformed BB# into a BL21(lacIq) cell line and followed a traditional protein production and purification procedure ([https://2009.igem.org/Team:Washington/Notebook/IMAC_protocol IMAC Protocol]). The resulting purified protein was then dialyzed overnight in 1x PBS to remove the elution buffer which we were worried would interfere with the activity assay. The concnetration of the dialysed protein was dteremined by meauring its absorbance at 280nM and using its predicted extinction coefficient (29575 M-1 cm-1,[http://ca.expasy.org/tools/protparam.html ProtParam]). We obtained ~1mL of 10microM protein. To determine the catalytic constants the nerve agent paroxoan was used a a substrate. As shown below, hydrolysis product of paroxoan is p-nitrophenol which has a strong absorbance at 400nM (and turns bright yellow).<br><br />
[[image:ParoxoanRxn2.png | 400px | center]]<br />
<br />
<br />
A serial dilution, ranging from 5 millimolar to 5 micromolar, of paroxoan was made in a reaction buffer (100mM HEPES pH=7, 500mM NaCl, 2mM CoCl2). To this a reaction OpdA was added so that it's final concentration was 1nM (dilutions were made in the reaction buffer). At all substrate concentrations no appreciable hydrolysis was observed without enzyme. The rate of hydrolysis with enzyme is shown below, the left hand plot is the full substrate range, and the right hand plot is a zoom in of the lower substrate concentrations:<br />
<br />
<gallery heights=400px widths=350><br />
image:OpdA_full.png<br />
image:OpdA_zoom.png<br />
</gallery><br />
<br />
From the above plot, it obvious that this enzyme efficiently catalysis paroxoan hydrolysis, but does not exhibit the usual Michaelis-Menten dynamics. It can be seen that at high enough concentrations, the enzyme actually undergoes substrate-inhibition, wherein the extra substrate actually slows the enzyme's velocity. When fit to a cononical substrate inhibition curve we obtain the following kinetic parameters:<br />
<br />
kcat (M-1 s-1): 17.6 <br><br />
Km (mM): 0.011 <br><br />
Ksi (mM): 1.06 <br><br />
<br />
These parameters confirm that this is an extremely efficient enzyme, and our kinetic parameters are comparable to previously published data for this enzyme on this substrate <sup>4,5</sup>. Also, substrate inhibition for this enzyme has been observed previously on similar substrates, so this was not an enitrely surprising results.<br />
<br />
Since the OpdA BioBrick was characterized and worked as expected we decided to continue and insert it into the target vector. Unfortinately when OpdA-Target was expressed and purified as described above no observable paroxoan hydrolysis was observed.<br />
<br />
<div style="text-align:right">'''Continue to [https://2009.igem.org/Team:Washington/Project/Secretion Secretion &gt;]'''</div><br />
<br />
='''Citations'''=<br />
<br />
<br />
1. Lamla and Erdmann. [http://www.ncbi.nlm.nih.gov/pubmed/14680960 The Nano-tag, a streptavidin-binding peptide for the purification and detection of recombinant proteins.]<br />
<br />
2. Palacios et al. [http://www.ncbi.nlm.nih.gov/pubmed/11157948 Subset of Hybrid Eukaryotic Proteins Is Exported by the Type I Secretion System of Erwinia chrysanthemi, Secrevtion of GFP in E. Coli].<br />
<br />
3. David Waugh. [http://mcl1.ncifcrf.gov/waugh_tech/faq/tev.pdf TEV Protease].<br />
<br />
4. Irene Horne, et al. Identification of an opd (Organophosphate Degradation) Gene in an Agrobacterium Isolate.<br />
<br />
5. H.Yang, et al. Evolution of an organophosphate-degrading enzyme:a comparison of natural and directed evolution.<br />
<br />
<br />
<br />
<br />
{{Template:Team:Washington/Templates/Footer}}</div>Acleonehttp://2009.igem.org/Team:Washington/Project/TargetTeam:Washington/Project/Target2009-10-19T03:26:48Z<p>Acleone: </p>
<hr />
<div>__NOTOC__<br />
{{Template:Team:Washington/Templates/Header}}<br />
<br />
<div style="text-align:right">'''Continue to [https://2009.igem.org/Team:Washington/Project/Secretion Secretion &gt;]'''</div><br />
<br />
[[Image:Main_graphic3_target_banner.png|center]]<br />
<br />
='''Background'''=<br />
<br />
When a favorite protein (Afp) is cloned into the target vector ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K215002 BBa_K215002]) two tags are fused onto the N and C terminal of Afp. These tags are depicted below: <br> <br />
<br />
[[Image:Targ_map.PNG | 700px | center]]<br />
<br />
The first key feature of the target vector is the NheI restriction site, where afp get's inserted. NheI is compatible with XbaI and SpeI, meaning that a biobrick digested at the X and S sites can be ligated into the target vector at the NheI site (for detailed protocol see: [https://2009.igem.org/Team:Washington/Notebook/NheI| NheI Insertion Protocol]). <br><br />
<br />
At the N-terminus of the Target is the display (aka Nano<sup>1</sup>) tag, which is a 15 amino acid sequence that binds to streptavidin. Since streptavidin is being displayed on the surface of the cell this allows our protein to stick to the outside of the cell, but can still be released by the addition of biotin. For more details see: [https://2009.igem.org/Team:Washington/Project/Display| Surface Display System ]. <br><br />
<br />
At the C-terminus of the Target is a secretion tag<sup>2</sup> (prtB) that is recognized by a Type I secretion system, which secretes proteins from the cytosol, through the periplasim, and into the media. For more details go to: [https://2009.igem.org/Team:Washington/Project/Secretion| Secretion System ]. <br> <br />
<br />
Flanking each side of the NheI site are 6 consecutive hisitidines (6x-His) and TEV protease sites <sup>3</sup>. The histidines allow for traditional immobilized metal affinity chromatography ([https://2009.igem.org/Team:Washington/Notebook/IMAC_protocol IMAC]) protein purification. The TEV sites allows for the N and C terminal tags to be cleaved off of Afp, and due to the strategic placement of the 6x-His tags these tags can then be seperated from Afp by simply running the cleaved solution over a column in which the tags stick but Afp flows right through. <br><br />
<br />
='''Experiments'''=<br />
<br />
<br />
For the target vector our goal was to:<br />
* Construct the target vector<br />
* Insert GFP into the target vector and characterize expression and function<br />
* Biobrick and characterize OpdA, a nerve agent degrading enzyme<br />
* Insert OpdA into the target vector and characterize expression and function<br />
<br />
<br />
<br />
==Target Vector Construction==<br />
<br />
To create the target vector we first synthesized a coding sequence that would produce the protein as described above. To do this we synthesized a gene from oligo's as described in our [https://2009.igem.org/Team:Washington/Notebook/gene_synthesis| Gene Synthesis Protocol].<br />
<br />
==Expression Vector Construction==<br />
<br />
After creating the target construct, we created and BioBricked an expression vector ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K215000 BBa_K215000]) which would express the target protein upon induction with IPTG. We then added the target construct into the expression vector using standard assembly making [http://partsregistry.org/wiki/index.php?title=Part:BBa_K215002 BBa_K215002].<br />
<br />
==GFP Insertion and Characterization==<br />
<br />
Upon completion of constructing the target vector our first experiment was to determine if GFP was still functional as the fusion protein. To do this GFP ([http://partsregistry.org/wiki/index.php/Part:BBa_E0040 BBa_E0040]) was inserted into the target construct use the NheI method as described above. After insertion of E0040 into the target construct, the tagged GFP (target-GFP) was transformed into BL21(lacIq) cells, and subsequently grown in the presence or absence of IPTG. As a control an untagged E0040 was cloned into our expression vector and also grown in the presence or absence of IPTG. This would allow us to determine the effects of the tags on fluorescence. The cells were then washed with PBS, normalized to the same cell density, and fluorescence measured using an excitation of 485 and emission of 525 (cutoff at 515) in a SpectraMax M5e plate reader. The data is show below:<br />
<br />
[[image:GFP_Fluroescense_corrected_for_OD.png |500px | center]]<br />
<br />
<br />
From this data we were able to conclude that our expression vector was functional, as is evident from the large increase in fluorescence with the addition of IPTG. We were also able to conclude that the Target-GFP is functional, but fluorescence was significantly decreased. <br />
<br />
In order to ensure that the Target-GFP had the appropriate 6x-His tags and that fluorescence was a function of protein concentration we purified Target-GFP using a traditional IMAC techniques. The protein concentration was measured from its absorbence a 280nm. A serial dilution of the protein was then made and the resulting fluorescence measured as described earlier. The data is shown below:<br />
<br />
[[Image:Standard_curve_targGFP.png | 500px | center]]<br />
<br />
<br />
As expected the fluorescence intensity is linear with respect to protein concentration.<br />
<br />
==BioBricking and Characterization of OpdA==<br />
<br />
====OpdA Background====<br />
<br />
OpdA ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K215090 BBa_K215090]) is an organophosphate-degrading enzyme from ''Agrobacterium radiobacter''. It is capable of degrading a wide range of organophosphates, most notably pesticides that are poisonous to humans, such as paraoxon. We chose to biobrick and submit this enzyme to the registry for a number of reasons. First and foremost, this enzyme is easy to assay for since it can hydrolyze substrates very quickly (e.g. paraoxon) and form a bright yellow product. This yellow product would make it easy to see that the OpdA was present and functioning in our system. And secondly, OpdA is a very useful enzyme that could have applications in future iGEM and other synthetic biology projects, so its presence in the Standard Registry of Biological Parts is beneficial.<br />
<br />
====OpdA Characterization====<br />
<br />
The Baker lab donated the source plasmid for OpdA (a synthetic gene optimized for ''E. coli'' expression). SOEing PCR was used to remove BioBrick cut sites ([https://2009.igem.org/Team:Washington/Notebook/SOEingPCR SOE PCR Protocol]). Upon removal of the unwanted restriction sites the gene was cloned into pSB1A2 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K215090 BBa_K215090]), our expression vector ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K215091 BBa_K215091]), and the target vector. <br />
<br />
The first experiment carried out was to validate that we could express and purify functional OpdA. To do this we transformed BB# into a BL21(lacIq) cell line and followed a traditional protein production and purification procedure ([https://2009.igem.org/Team:Washington/Notebook/IMAC_protocol IMAC Protocol]). The resulting purified protein was then dialyzed overnight in 1x PBS to remove the elution buffer which we were worried would interfere with the activity assay. The concnetration of the dialysed protein was dteremined by meauring its absorbance at 280nM and using its predicted extinction coefficient (29575 M-1 cm-1,[http://ca.expasy.org/tools/protparam.html ProtParam]). We obtained ~1mL of 10microM protein. To determine the catalytic constants the nerve agent paroxoan was used a a substrate. As shown below, hydrolysis product of paroxoan is p-nitrophenol which has a strong absorbance at 400nM (and turns bright yellow).<br><br />
[[image:ParoxoanRxn2.png | 400px | center]]<br />
<br />
<br />
A serial dilution, ranging from 5 millimolar to 5 micromolar, of paroxoan was made in a reaction buffer (100mM HEPES pH=7, 500mM NaCl, 2mM CoCl2). To this a reaction OpdA was added so that it's final concentration was 1nM (dilutions were made in the reaction buffer). At all substrate concentrations no appreciable hydrolysis was observed without enzyme. The rate of hydrolysis with enzyme is shown below, the left hand plot is the full substrate range, and the right hand plot is a zoom in of the lower substrate concentrations:<br />
<br />
<gallery heights=400px widths=350><br />
image:OpdA_full.png<br />
image:OpdA_zoom.png<br />
</gallery><br />
<br />
From the above plot, it obvious that this enzyme efficiently catalysis paroxoan hydrolysis, but does not exhibit the usual Michaelis-Menten dynamics. It can be seen that at high enough concentrations, the enzyme actually undergoes substrate-inhibition, wherein the extra substrate actually slows the enzyme's velocity. When fit to a cononical substrate inhibition curve we obtain the following kinetic parameters:<br />
<br />
kcat (M-1 s-1): 17.6 <br><br />
Km (mM): 0.011 <br><br />
Ksi (mM): 1.06 <br><br />
<br />
These parameters confirm that this is an extremely efficient enzyme, and our kinetic parameters are comparable to previously published data for this enzyme on this substrate <sup>4,5</sup>. Also, substrate inhibition for this enzyme has been observed previously on similar substrates, so this was not an enitrely surprising results.<br />
<br />
Since the OpdA BioBrick was characterized and worked as expected we decided to continue and insert it into the target vector. Unfortinately when OpdA-Target was expressed and purified as described above no observable paroxoan hydrolysis was observed.<br />
<br />
<div style="text-align:right">'''Continue to [https://2009.igem.org/Team:Washington/Project/Secretion Secretion &gt;]'''</div><br />
<br />
='''Citations'''=<br />
<br />
<br />
1. Lamla and Erdmann. [http://www.ncbi.nlm.nih.gov/pubmed/14680960 The Nano-tag, a streptavidin-binding peptide for the purification and detection of recombinant proteins.]<br />
<br />
2. Palacios et al. [http://www.ncbi.nlm.nih.gov/pubmed/11157948 Subset of Hybrid Eukaryotic Proteins Is Exported by the Type I Secretion System of Erwinia chrysanthemi, Secrevtion of GFP in E. Coli].<br />
<br />
3. David Waugh. [http://mcl1.ncifcrf.gov/waugh_tech/faq/tev.pdf TEV Protease].<br />
<br />
4. Irene Horne, et al. Identification of an opd (Organophosphate Degradation) Gene in an Agrobacterium Isolate.<br />
<br />
5. H.Yang, et al. Evolution of an organophosphate-degrading enzyme:a comparison of natural and directed evolution.<br />
<br />
<br />
<br />
<br />
{{Template:Team:Washington/Templates/Footer}}</div>Acleone