http://2014.igem.org/wiki/index.php?title=Team:SJTU-BioX-Shanghai/Part2_Extension&feed=atom&action=historyTeam:SJTU-BioX-Shanghai/Part2 Extension - Revision history2024-03-28T09:36:17ZRevision history for this page on the wikiMediaWiki 1.16.5http://2014.igem.org/wiki/index.php?title=Team:SJTU-BioX-Shanghai/Part2_Extension&diff=370946&oldid=prevHorizonP at 23:51, 17 October 20142014-10-17T23:51:54Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2 id=References>References</h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2 id=References>References</h2></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><ol></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ol <ins class="diffchange diffchange-inline">style="font-style: italic;"</ins>></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li>Pailler, Jérémy, et al. "Phosphatidylglycerol:: prolipoprotein diacylglyceryl transferase (Lgt) of Escherichia coli has seven transmembrane segments, and its essential residues are embedded in the membrane." Journal of bacteriology 194.9 (2012): 2142-2151.</li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li>Pailler, Jérémy, et al. "Phosphatidylglycerol:: prolipoprotein diacylglyceryl transferase (Lgt) of Escherichia coli has seven transmembrane segments, and its essential residues are embedded in the membrane." Journal of bacteriology 194.9 (2012): 2142-2151.</li></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li>Schierle, Clark F., et al. "The DsbA signal sequence directs efficient, cotranslational export of passenger proteins to the Escherichia coli periplasm via the signal recognition particle pathway." Journal of bacteriology 185.19 (2003): 5706-5713.</li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li>Schierle, Clark F., et al. "The DsbA signal sequence directs efficient, cotranslational export of passenger proteins to the Escherichia coli periplasm via the signal recognition particle pathway." Journal of bacteriology 185.19 (2003): 5706-5713.</li></div></td></tr>
</table>HorizonPhttp://2014.igem.org/wiki/index.php?title=Team:SJTU-BioX-Shanghai/Part2_Extension&diff=369124&oldid=prevJustin maria at 23:36, 17 October 20142014-10-17T23:36:30Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><strong><em>ssDsbA</em></strong>: SsDsbA is the signal recognition particle (SRP)-dependent signaling sequence of DsbA. SsDsbA-tagged proteins are exported to the periplasm through the SRP pathway. With ssDsbA fused to the N-terminus, fusion proteins with Lgt are expected to be anchored onto inner membrane of <i>E.coli</i>.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><strong><em>ssDsbA</em></strong>: SsDsbA is the signal recognition particle (SRP)-dependent signaling sequence of DsbA. SsDsbA-tagged proteins are exported to the periplasm through the SRP pathway. With ssDsbA fused to the N-terminus, fusion proteins with Lgt are expected to be anchored onto inner membrane of <i>E.coli</i>.</p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p><strong><em>FP</em></strong>: To visualize the localization of fusion protein with fluorescence test , we added FP in the <strong><em>Connectee1</strong></em> and placed it just after the ssDsbA. We chose mRFP, CFP<del class="diffchange diffchange-inline">, </del>YFP in our system.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p><strong><em>FP</em></strong>: To visualize the localization of fusion protein with fluorescence test , we added FP in the <strong><em>Connectee1</strong></em> and placed it just after the ssDsbA. We chose mRFP, CFP <ins class="diffchange diffchange-inline">and </ins>YFP in our system.</p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><strong><em>Lgt</em></strong>: Phosphatidylglycerol prolipoprotein diacylglyceryl transferase (Lgt) is an inner membrane protein that acts as a membrane anchor of <i>E.coli</i> with seven transmembrane segments and has been successfully overexpressed in E. coli without causing harm to cells.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><strong><em>Lgt</em></strong>: Phosphatidylglycerol prolipoprotein diacylglyceryl transferase (Lgt) is an inner membrane protein that acts as a membrane anchor of <i>E.coli</i> with seven transmembrane segments and has been successfully overexpressed in E. coli without causing harm to cells.</p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p><strong><em>TAL effectors</em></strong>:As mentioned <del class="diffchange diffchange-inline">earlier,we choose </del>three kinds of combinations to build three different TAL proteins, <del class="diffchange diffchange-inline">which is </del>based on the parts that <del class="diffchange diffchange-inline">the </del>team <del class="diffchange diffchange-inline">of Freiburg </del>offered <del class="diffchange diffchange-inline">in 2012</del>. These three TAL proteins can identify three different 14bp nucleotide sequences on a <strong><em>Connector</strong></em>. Note that we added a His Tag at the end of TAL protein to facilitate separation and purification.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p><strong><em>TAL effectors</em></strong>:As mentioned <ins class="diffchange diffchange-inline">earlier, we chose </ins>three kinds of combinations to build three different TAL proteins, based on the parts that <ins class="diffchange diffchange-inline">2012 Freiburg iGEM </ins>team offered. These three TAL proteins can identify three different 14bp nucleotide sequences on a <strong><em>Connector</strong></em>. Note that we added a His Tag at the end of TAL protein to facilitate separation and purification.</p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>For the three kinds of corresponding <strong><em>Connectee2</strong></em>, we did not <del class="diffchange diffchange-inline">introduced </del>ssDsbA-Lgt section to keep them in a free intracellular state.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>For the three kinds of corresponding <strong><em>Connectee2</strong></em>, we did not <ins class="diffchange diffchange-inline">introduce </ins>ssDsbA-Lgt section to keep them in a free intracellular state.</p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>In the final production of our construction, we <del class="diffchange diffchange-inline">add </del>an easy-to-hand interface sequence between the Lgt and TAL protein in <strong><em>Connectee1</strong></em> or just before the TAL protein in <strong><em>Connectee2</strong></em>, as sites to adding enzymes.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>In the final production of our construction, we <ins class="diffchange diffchange-inline">added </ins>an easy-to-hand interface sequence between the Lgt and TAL protein in <strong><em>Connectee1</strong></em> or just before the TAL protein in <strong><em>Connectee2</strong></em>, as sites to adding enzymes.</p></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><img src="https://static.igem.org/mediawiki/2014/d/dd/SJTU14_EnzymeUCB%2BTAL_USB.jpg"></img></center></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><img src="https://static.igem.org/mediawiki/2014/d/dd/SJTU14_EnzymeUCB%2BTAL_USB.jpg"></img></center></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><img src="https://static.igem.org/mediawiki/2014/8/81/Pks_ii_original.png"></img></center></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><img src="https://static.igem.org/mediawiki/2014/8/81/Pks_ii_original.png"></img></center></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></br><center><small><strong>Figure 1.3.5 pBluescript II KS(+)</strong></small></center></br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></br><center><small><strong>Figure 1.3.5 pBluescript II KS(+)</strong></small></center></br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>We used the four reformed plasmids to co-transform and induced the <strong><em>Connectors</strong></em> to express. Now the structure of the crown appears in the <i>E.coli</i> cell membrane or cytoplasm. Here we <del class="diffchange diffchange-inline">use </del>the fusion proteins with two kinds of enzymes and TAL1 as examples. </p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>We used the four reformed plasmids to co-transform and induced the <strong><em>Connectors</strong></em> to express. Now the structure of the crown appears in the <i>E.coli</i> cell membrane or cytoplasm. Here we <ins class="diffchange diffchange-inline">used </ins>the fusion proteins with two kinds of enzymes and TAL1 as examples. </p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><img src="https://static.igem.org/mediawiki/2014/0/0d/SJTU14_Enzyme%2BTAL1.jpg"></img></center></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><img src="https://static.igem.org/mediawiki/2014/0/0d/SJTU14_Enzyme%2BTAL1.jpg"></img></center></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></br><center><small><strong>Figure 1.3.6 ssDsbA-Lgt-pykF/poxB-TAL1-His Tag</strong></small></center></br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></br><center><small><strong>Figure 1.3.6 ssDsbA-Lgt-pykF/poxB-TAL1-His Tag</strong></small></center></br></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><h3 id="Testmethods">Test <del class="diffchange diffchange-inline">methods</del></h3></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><h3 id="Testmethods">Test <ins class="diffchange diffchange-inline">Methods</ins></h3></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>We <del class="diffchange diffchange-inline">use </del>formaldehyde to stabilize the connected fusion proteins and <strong><em>Connector</strong></em>, then <del class="diffchange diffchange-inline">separate </del>the <del class="diffchange diffchange-inline">proteins</del>-Connector complexes and <del class="diffchange diffchange-inline">digest </del>the protein. Finally <del class="diffchange diffchange-inline">use </del>PCR to detect the three sequences of the <strong><em>Connector</strong></em> which TAL proteins <del class="diffchange diffchange-inline">are </del>designed to bind to.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>We <ins class="diffchange diffchange-inline">used </ins>formaldehyde to stabilize the connected fusion proteins and <strong><em>Connector</strong></em>, then <ins class="diffchange diffchange-inline">separated </ins>the <ins class="diffchange diffchange-inline">protein</ins>-Connector complexes and <ins class="diffchange diffchange-inline">digested </ins>the protein. Finally <ins class="diffchange diffchange-inline">we used </ins>PCR to detect the three sequences of the <strong><em>Connector</strong></em> which TAL proteins <ins class="diffchange diffchange-inline">were </ins>designed to bind to.</p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>Then, we <del class="diffchange diffchange-inline">can </del>test the yield of different <del class="diffchange diffchange-inline">product </del>to determine the efficiency of selective combination.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>Then, we <ins class="diffchange diffchange-inline">were able to </ins>test the yield of different <ins class="diffchange diffchange-inline">products </ins>to determine the efficiency of selective combination.</p></div></td></tr>
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</table>Justin mariahttp://2014.igem.org/wiki/index.php?title=Team:SJTU-BioX-Shanghai/Part2_Extension&diff=367839&oldid=prevJustin maria at 23:25, 17 October 20142014-10-17T23:25:40Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></br><center><small><strong>Figure 1.3.1 Principle of selectivity</strong></small></center></br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></br><center><small><strong>Figure 1.3.1 Principle of selectivity</strong></small></center></br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br/></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br/></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>All the three enzymes involved are expressed in the bacteria. Each fusion protein contains ssDsbA, Lgt, FL3, enzyme, HL and TAL; different TAL parts can recognize different sites. <strong><em>Connector</strong></em> is originally designed with three recognition sequences(RS), which are combined to form two different connectors, one with RSⅠ and RSⅡ while the other one with RSⅠ and RSⅢ (<del class="diffchange diffchange-inline">Show </del>in the figure below). <strong><em>Connector</strong></em> with RSⅠand RSⅡ can bind to Connectee-enzymeⅠ and Connectee-enzymeⅡ then get production A in the end, while <strong><em>Connector</strong></em> with RSⅠ and RSⅢ can bind to Connectee-enzymeⅠ and Connectee-enzymeⅢ then get production B.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>All the three enzymes involved are expressed in the bacteria. Each fusion protein contains ssDsbA, Lgt, FL3, enzyme, HL and TAL; different TAL parts can recognize different sites. <strong><em>Connector</strong></em> is originally designed with three recognition sequences(RS), which are combined to form two different connectors, one with RSⅠ and RSⅡ while the other one with RSⅠ and RSⅢ (<ins class="diffchange diffchange-inline">shown </ins>in the figure below). <strong><em>Connector</strong></em> with RSⅠand RSⅡ can bind to Connectee-enzymeⅠ and Connectee-enzymeⅡ then get production A in the end, while <strong><em>Connector</strong></em> with RSⅠ and RSⅢ can bind to Connectee-enzymeⅠ and Connectee-enzymeⅢ then get production B.</p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><img src="https://static.igem.org/mediawiki/2014/0/08/SJTU14_pBdeletion.jpg" id="dingweidian3"></img></center></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><img src="https://static.igem.org/mediawiki/2014/0/08/SJTU14_pBdeletion.jpg" id="dingweidian3"></img></center></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></br><center><small><strong>Figure 1.3.2 pBluescript II KS(+) ScaI/EcoRV deletion</strong></small></center></br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></br><center><small><strong>Figure 1.3.2 pBluescript II KS(+) ScaI/EcoRV deletion</strong></small></center></br></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3 id="ConstructionMethod">Construction Method</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3 id="ConstructionMethod">Construction Method</h3></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>Above all, we <del class="diffchange diffchange-inline">use </del>3 sets of <strong><em>Connectee1</strong></em> to build polymerization system. There are four main parts in a <strong><em>Connectee1</strong></em> for testing.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>Above all, we <ins class="diffchange diffchange-inline">used </ins>3 sets of <strong><em>Connectee1</strong></em> to build polymerization system. There are four main parts in a <strong><em>Connectee1</strong></em> for testing.</p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><img src="https://static.igem.org/mediawiki/2014/1/1a/SJTU14_3_fusion_proteins.jpg"></img></center></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><img src="https://static.igem.org/mediawiki/2014/1/1a/SJTU14_3_fusion_proteins.jpg"></img></center></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><strong><em>ssDsbA</em></strong>: SsDsbA is the signal recognition particle (SRP)-dependent signaling sequence of DsbA. SsDsbA-tagged proteins are exported to the periplasm through the SRP pathway. With ssDsbA fused to the N-terminus, fusion proteins with Lgt are expected to be anchored onto inner membrane of <i>E.coli</i>.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><strong><em>ssDsbA</em></strong>: SsDsbA is the signal recognition particle (SRP)-dependent signaling sequence of DsbA. SsDsbA-tagged proteins are exported to the periplasm through the SRP pathway. With ssDsbA fused to the N-terminus, fusion proteins with Lgt are expected to be anchored onto inner membrane of <i>E.coli</i>.</p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p><strong><em>FP</em></strong>: To visualize the localization of fusion protein with fluorescence test , we added FP in the <strong><em>Connectee1</strong></em> and placed it just after the ssDsbA. We chose mRFP,CFP,YFP in our system.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p><strong><em>FP</em></strong>: To visualize the localization of fusion protein with fluorescence test , we added FP in the <strong><em>Connectee1</strong></em> and placed it just after the ssDsbA. We chose mRFP, CFP, YFP in our system.</p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p><strong><em>Lgt</em></strong>: Phosphatidylglycerol prolipoprotein diacylglyceryl transferase (Lgt) is an inner membrane protein <del class="diffchange diffchange-inline">act </del>as <del class="diffchange diffchange-inline">an </del>membrane anchor of E.coli with seven transmembrane segments and has been successfully overexpressed in E. coli without causing harm to cells.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p><strong><em>Lgt</em></strong>: Phosphatidylglycerol prolipoprotein diacylglyceryl transferase (Lgt) is an inner membrane protein <ins class="diffchange diffchange-inline">that acts </ins>as <ins class="diffchange diffchange-inline">a </ins>membrane anchor of <ins class="diffchange diffchange-inline"><i></ins>E.coli<ins class="diffchange diffchange-inline"></i> </ins>with seven transmembrane segments and has been successfully overexpressed in E. coli without causing harm to cells.</p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><strong><em>TAL effectors</em></strong>:As mentioned earlier,we choose three kinds of combinations to build three different TAL proteins, which is based on the parts that the team of Freiburg offered in 2012. These three TAL proteins can identify three different 14bp nucleotide sequences on a <strong><em>Connector</strong></em>. Note that we added a His Tag at the end of TAL protein to facilitate separation and purification.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><strong><em>TAL effectors</em></strong>:As mentioned earlier,we choose three kinds of combinations to build three different TAL proteins, which is based on the parts that the team of Freiburg offered in 2012. These three TAL proteins can identify three different 14bp nucleotide sequences on a <strong><em>Connector</strong></em>. Note that we added a His Tag at the end of TAL protein to facilitate separation and purification.</p></div></td></tr>
</table>Justin mariahttp://2014.igem.org/wiki/index.php?title=Team:SJTU-BioX-Shanghai/Part2_Extension&diff=366316&oldid=prevJustin maria at 23:12, 17 October 20142014-10-17T23:12:35Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p><strong>Gearbox</strong></em> is the model of selectivity. Our <del class="diffchange diffchange-inline">fused </del>protein can not only be used to polymerize enzymes but also be used to control selective combinations. We can control the direction of pathway by simply transforming different <strong><em>Connector</strong></em> plasmids. </p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p><strong>Gearbox</strong></em> is the model of selectivity. Our <ins class="diffchange diffchange-inline">fusion </ins>protein can not only be used to polymerize enzymes but also be used to control selective combinations. We can control the direction of pathway by simply transforming different <strong><em>Connector</strong></em> plasmids. </p></div></td></tr>
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</table>Justin mariahttp://2014.igem.org/wiki/index.php?title=Team:SJTU-BioX-Shanghai/Part2_Extension&diff=364028&oldid=prevWsyhdyjn at 22:49, 17 October 20142014-10-17T22:49:31Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></br><center><small><strong>Figure 1.3.1 Principle of selectivity</strong></small></center></br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></br><center><small><strong>Figure 1.3.1 Principle of selectivity</strong></small></center></br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br/></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br/></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p <del class="diffchange diffchange-inline">id="dingweidian3"</del>>All the three enzymes involved are expressed in the bacteria. Each fusion protein contains ssDsbA, Lgt, FL3, enzyme, HL and TAL; different TAL parts can recognize different sites. <strong><em>Connector</strong></em> is originally designed with three recognition sequences(RS), which are combined to form two different connectors, one with RSⅠ and RSⅡ while the other one with RSⅠ and RSⅢ (Show in the figure below). <strong><em>Connector</strong></em> with RSⅠand RSⅡ can bind to Connectee-enzymeⅠ and Connectee-enzymeⅡ then get production A in the end, while <strong><em>Connector</strong></em> with RSⅠ and RSⅢ can bind to Connectee-enzymeⅠ and Connectee-enzymeⅢ then get production B.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>All the three enzymes involved are expressed in the bacteria. Each fusion protein contains ssDsbA, Lgt, FL3, enzyme, HL and TAL; different TAL parts can recognize different sites. <strong><em>Connector</strong></em> is originally designed with three recognition sequences(RS), which are combined to form two different connectors, one with RSⅠ and RSⅡ while the other one with RSⅠ and RSⅢ (Show in the figure below). <strong><em>Connector</strong></em> with RSⅠand RSⅡ can bind to Connectee-enzymeⅠ and Connectee-enzymeⅡ then get production A in the end, while <strong><em>Connector</strong></em> with RSⅠ and RSⅢ can bind to Connectee-enzymeⅠ and Connectee-enzymeⅢ then get production B.</p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><center><img src="https://static.igem.org/mediawiki/2014/0/08/SJTU14_pBdeletion.jpg"></img></center></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><center><img src="https://static.igem.org/mediawiki/2014/0/08/SJTU14_pBdeletion.jpg<ins class="diffchange diffchange-inline">" id="dingweidian3</ins>"></img></center></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></br><center><small><strong>Figure 1.3.2 pBluescript II KS(+) ScaI/EcoRV deletion</strong></small></center></br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></br><center><small><strong>Figure 1.3.2 pBluescript II KS(+) ScaI/EcoRV deletion</strong></small></center></br></div></td></tr>
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</table>Wsyhdyjnhttp://2014.igem.org/wiki/index.php?title=Team:SJTU-BioX-Shanghai/Part2_Extension&diff=363844&oldid=prevWsyhdyjn at 22:47, 17 October 20142014-10-17T22:47:59Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <div class="projtile"></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <div class="projtile"></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <a href="#<del class="diffchange diffchange-inline">Booster</del>" title="Gearbox"></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <a href="#<ins class="diffchange diffchange-inline">dingweidian16</ins>" title="Gearbox"></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <center> <h2>Gearbox</h2></center></a></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <center> <h2>Gearbox</h2></center></a></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><strong><em>Booster</strong></em> is the model of polymerization and maximization. Polymerized enzymes, different from normal scattered ones, have much more opportunities to contact with the substrates. Therefore, our project aims to increase as well as to control the efficiency of complex reactions. We have built a framework that makes multi-enzyme complex easy to form, which we call a <strong><em>Crown</strong></em>.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><strong><em>Booster</strong></em> is the model of polymerization and maximization. Polymerized enzymes, different from normal scattered ones, have much more opportunities to contact with the substrates. Therefore, our project aims to increase as well as to control the efficiency of complex reactions. We have built a framework that makes multi-enzyme complex easy to form, which we call a <strong><em>Crown</strong></em>.</p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><img src="https://static.igem.org/mediawiki/2014/2/2e/SJTU14_Part2%281-3%29.jpg"></img></center></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><img src="https://static.igem.org/mediawiki/2014/2/2e/SJTU14_Part2%281-3%29.jpg"></img></center></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>And based on the success of the individual fusion protein(The first jewel) expression, we tried to add more jewels on the <strong><em>Crown</strong></em>.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p <ins class="diffchange diffchange-inline">id="dingweidian16"</ins>>And based on the success of the individual fusion protein(The first jewel) expression, we tried to add more jewels on the <strong><em>Crown</strong></em>.</p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>In order to build a polymerization system, we made more fusion proteins bind to the same <strong><em>Connector</strong></em>. Then with more enzymes added, it has the practical effect of accelerating the reactions and enhancing the production efficiency. So far, the <strong><em>Crown</strong></em> is built with more than one jewel shining on it.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>In order to build a polymerization system, we made more fusion proteins bind to the same <strong><em>Connector</strong></em>. Then with more enzymes added, it has the practical effect of accelerating the reactions and enhancing the production efficiency. So far, the <strong><em>Crown</strong></em> is built with more than one jewel shining on it.</p></div></td></tr>
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</table>Wsyhdyjnhttp://2014.igem.org/wiki/index.php?title=Team:SJTU-BioX-Shanghai/Part2_Extension&diff=359158&oldid=prevCorrine at 21:59, 17 October 20142014-10-17T21:59:49Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><img src="https://static.igem.org/mediawiki/2014/d/dd/SJTU14_EnzymeUCB%2BTAL_USB.jpg"></img></center></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><img src="https://static.igem.org/mediawiki/2014/d/dd/SJTU14_EnzymeUCB%2BTAL_USB.jpg"></img></center></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div></br><center><small><strong>Figure 1.<del class="diffchange diffchange-inline">2</del>.4 ssDsbA-Lgt-Enzyme USB(AarI/BsmAI)-TAL USB-His Tag</strong></small></center></br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></br><center><small><strong>Figure 1.<ins class="diffchange diffchange-inline">3</ins>.4 ssDsbA-Lgt-Enzyme USB(AarI/BsmAI)-TAL USB-His Tag</strong></small></center></br></div></td></tr>
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</table>Corrinehttp://2014.igem.org/wiki/index.php?title=Team:SJTU-BioX-Shanghai/Part2_Extension&diff=359058&oldid=prevCorrine at 21:58, 17 October 20142014-10-17T21:58:49Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><img src="https://static.igem.org/mediawiki/2014/7/75/SJTU14_Selectivity.jpg"></img></center></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><img src="https://static.igem.org/mediawiki/2014/7/75/SJTU14_Selectivity.jpg"></img></center></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div></br><center><small><strong>Figure 1.<del class="diffchange diffchange-inline">2</del>.1 Principle of selectivity</strong></small></center></br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></br><center><small><strong>Figure 1.<ins class="diffchange diffchange-inline">3</ins>.1 Principle of selectivity</strong></small></center></br></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p id="dingweidian3">All the three enzymes involved are expressed in the bacteria. Each fusion protein contains ssDsbA, Lgt, FL3, enzyme, HL and TAL; different TAL parts can recognize different sites. <strong><em>Connector</strong></em> is originally designed with three recognition sequences(RS), which are combined to form two different connectors, one with RSⅠ and RSⅡ while the other one with RSⅠ and RSⅢ (Show in the figure below). <strong><em>Connector</strong></em> with RSⅠand RSⅡ can bind to Connectee-enzymeⅠ and Connectee-enzymeⅡ then get production A in the end, while <strong><em>Connector</strong></em> with RSⅠ and RSⅢ can bind to Connectee-enzymeⅠ and Connectee-enzymeⅢ then get production B.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p id="dingweidian3">All the three enzymes involved are expressed in the bacteria. Each fusion protein contains ssDsbA, Lgt, FL3, enzyme, HL and TAL; different TAL parts can recognize different sites. <strong><em>Connector</strong></em> is originally designed with three recognition sequences(RS), which are combined to form two different connectors, one with RSⅠ and RSⅡ while the other one with RSⅠ and RSⅢ (Show in the figure below). <strong><em>Connector</strong></em> with RSⅠand RSⅡ can bind to Connectee-enzymeⅠ and Connectee-enzymeⅡ then get production A in the end, while <strong><em>Connector</strong></em> with RSⅠ and RSⅢ can bind to Connectee-enzymeⅠ and Connectee-enzymeⅢ then get production B.</p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><center><img src="https://static.igem.org/mediawiki/2014/<del class="diffchange diffchange-inline">e</del>/<del class="diffchange diffchange-inline">e8</del>/SJTU14_pBdeletion.<del class="diffchange diffchange-inline">png</del>"></img></center></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><center><img src="https://static.igem.org/mediawiki/2014/<ins class="diffchange diffchange-inline">0</ins>/<ins class="diffchange diffchange-inline">08</ins>/SJTU14_pBdeletion.<ins class="diffchange diffchange-inline">jpg</ins>"></img></center></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div></br><center><small><strong>Figure 1.<del class="diffchange diffchange-inline">2</del>.2 pBluescript II KS(+) ScaI/EcoRV deletion</strong></small></center></br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></br><center><small><strong>Figure 1.<ins class="diffchange diffchange-inline">3</ins>.2 pBluescript II KS(+) ScaI/EcoRV deletion</strong></small></center></br></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div></br><center><small><strong>Figure 1.<del class="diffchange diffchange-inline">2</del>.3 ssDsbA-FP-Lgt-TAL-His Tag</strong></small></center></br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></br><center><small><strong>Figure 1.<ins class="diffchange diffchange-inline">3</ins>.3 ssDsbA-FP-Lgt-TAL-His Tag</strong></small></center></br></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><strong><em>ssDsbA</em></strong>: SsDsbA is the signal recognition particle (SRP)-dependent signaling sequence of DsbA. SsDsbA-tagged proteins are exported to the periplasm through the SRP pathway. With ssDsbA fused to the N-terminus, fusion proteins with Lgt are expected to be anchored onto inner membrane of <i>E.coli</i>.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><strong><em>ssDsbA</em></strong>: SsDsbA is the signal recognition particle (SRP)-dependent signaling sequence of DsbA. SsDsbA-tagged proteins are exported to the periplasm through the SRP pathway. With ssDsbA fused to the N-terminus, fusion proteins with Lgt are expected to be anchored onto inner membrane of <i>E.coli</i>.</p></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>The <strong><em>Connector</strong></em> is pBluescript II KS(+) .</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>The <strong><em>Connector</strong></em> is pBluescript II KS(+) .</p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><img src="https://static.igem.org/mediawiki/2014/8/81/Pks_ii_original.png"></img></center></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><img src="https://static.igem.org/mediawiki/2014/8/81/Pks_ii_original.png"></img></center></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div></br><center><small><strong>Figure 1.<del class="diffchange diffchange-inline">2</del>.5 pBluescript II KS(+)</strong></small></center></br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></br><center><small><strong>Figure 1.<ins class="diffchange diffchange-inline">3</ins>.5 pBluescript II KS(+)</strong></small></center></br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>We used the four reformed plasmids to co-transform and induced the <strong><em>Connectors</strong></em> to express. Now the structure of the crown appears in the <i>E.coli</i> cell membrane or cytoplasm. Here we use the fusion proteins with two kinds of enzymes and TAL1 as examples. </p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>We used the four reformed plasmids to co-transform and induced the <strong><em>Connectors</strong></em> to express. Now the structure of the crown appears in the <i>E.coli</i> cell membrane or cytoplasm. Here we use the fusion proteins with two kinds of enzymes and TAL1 as examples. </p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><img src="https://static.igem.org/mediawiki/2014/0/0d/SJTU14_Enzyme%2BTAL1.jpg"></img></center></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><img src="https://static.igem.org/mediawiki/2014/0/0d/SJTU14_Enzyme%2BTAL1.jpg"></img></center></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div></br><center><small><strong>Figure 1.<del class="diffchange diffchange-inline">2</del>.6 ssDsbA-Lgt-pykF/poxB-TAL1-His Tag</strong></small></center></br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></br><center><small><strong>Figure 1.<ins class="diffchange diffchange-inline">3</ins>.6 ssDsbA-Lgt-pykF/poxB-TAL1-His Tag</strong></small></center></br></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3 id="Testmethods">Test methods</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3 id="Testmethods">Test methods</h3></div></td></tr>
</table>Corrinehttp://2014.igem.org/wiki/index.php?title=Team:SJTU-BioX-Shanghai/Part2_Extension&diff=358600&oldid=prevFlyFreedom at 21:54, 17 October 20142014-10-17T21:54:10Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><h2 id=<del class="diffchange diffchange-inline">Reference</del>><del class="diffchange diffchange-inline">Reference</del></h2></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><h2 id=<ins class="diffchange diffchange-inline">References</ins>><ins class="diffchange diffchange-inline">References</ins></h2></div></td></tr>
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</table>FlyFreedomhttp://2014.igem.org/wiki/index.php?title=Team:SJTU-BioX-Shanghai/Part2_Extension&diff=358124&oldid=prevCorrine at 21:48, 17 October 20142014-10-17T21:48:30Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><strong>Gearbox</strong></em> is the model of selectivity. Our fused protein can not only be used to polymerize enzymes but also be used to control selective combinations. We can control the direction of pathway by simply transforming different <strong><em>Connector</strong></em> plasmids. </p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><strong>Gearbox</strong></em> is the model of selectivity. Our fused protein can not only be used to polymerize enzymes but also be used to control selective combinations. We can control the direction of pathway by simply transforming different <strong><em>Connector</strong></em> plasmids. </p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br/></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br/></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"><center><img src="https://static.igem.org/mediawiki/2014/7/75/SJTU14_Selectivity.jpg"></img></center></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></br><center><small><strong>Figure 1.2.1 Principle of selectivity</strong></small></center></br></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"><br/></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"><p id="dingweidian3">All the three enzymes involved are expressed in the bacteria. Each fusion protein contains ssDsbA, Lgt, FL3, enzyme, HL and TAL; different TAL parts can recognize different sites. <strong><em>Connector</strong></em> is originally designed with three recognition sequences(RS), which are combined to form two different connectors, one with RSⅠ and RSⅡ while the other one with RSⅠ and RSⅢ (Show in the figure below). <strong><em>Connector</strong></em> with RSⅠand RSⅡ can bind to Connectee-enzymeⅠ and Connectee-enzymeⅡ then get production A in the end, while <strong><em>Connector</strong></em> with RSⅠ and RSⅢ can bind to Connectee-enzymeⅠ and Connectee-enzymeⅢ then get production B.</p></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><img src="https://static.igem.org/mediawiki/2014/e/e8/SJTU14_pBdeletion.png"></img></center></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><img src="https://static.igem.org/mediawiki/2014/e/e8/SJTU14_pBdeletion.png"></img></center></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div></br><center><small><strong>Figure 1.2.<del class="diffchange diffchange-inline">1 </del>pBluescript II KS(+) ScaI/EcoRV deletion</strong></small></center></br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></br><center><small><strong>Figure 1.2.<ins class="diffchange diffchange-inline">2 </ins>pBluescript II KS(+) ScaI/EcoRV deletion</strong></small></center></br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"><br/></del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"><p id="dingweidian3">All the three enzymes involved are expressed in the bacteria. Each fusion protein contains ssDsbA, Lgt, FL3, enzyme, HL and TAL; different TAL parts can recognize different sites. <strong><em>Connector</strong></em> is originally designed with three recognition sequences(RS), which are combined to form two different connectors, one with RSⅠ and RSⅡ while the other one with RSⅠ and RSⅢ (Show in the figure above). <strong><em>Connector</strong></em> with RSⅠand RSⅡ can bind to Connectee-enzymeⅠ and Connectee-enzymeⅡ then get production A in the end, while <strong><em>Connector</strong></em> with RSⅠ and RSⅢ can bind to Connectee-enzymeⅠ and Connectee-enzymeⅢ then get production B.</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">.</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><img src="https://static.igem.org/mediawiki/2014/1/1a/SJTU14_3_fusion_proteins.jpg"></img></center></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><img src="https://static.igem.org/mediawiki/2014/1/1a/SJTU14_3_fusion_proteins.jpg"></img></center></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div></br><center><small><strong>Figure 1.2.<del class="diffchange diffchange-inline">2 </del>ssDsbA-FP-Lgt-TAL-His Tag</strong></small></center></br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></br><center><small><strong>Figure 1.2.<ins class="diffchange diffchange-inline">3 </ins>ssDsbA-FP-Lgt-TAL-His Tag</strong></small></center></br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><strong><em>ssDsbA</em></strong>: SsDsbA is the signal recognition particle (SRP)-dependent signaling sequence of DsbA. SsDsbA-tagged proteins are exported to the periplasm through the SRP pathway. With ssDsbA fused to the N-terminus, fusion proteins with Lgt are expected to be anchored onto inner membrane of <i>E.coli</i>.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><strong><em>ssDsbA</em></strong>: SsDsbA is the signal recognition particle (SRP)-dependent signaling sequence of DsbA. SsDsbA-tagged proteins are exported to the periplasm through the SRP pathway. With ssDsbA fused to the N-terminus, fusion proteins with Lgt are expected to be anchored onto inner membrane of <i>E.coli</i>.</p></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><img src="https://static.igem.org/mediawiki/2014/d/dd/SJTU14_EnzymeUCB%2BTAL_USB.jpg"></img></center></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><img src="https://static.igem.org/mediawiki/2014/d/dd/SJTU14_EnzymeUCB%2BTAL_USB.jpg"></img></center></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div></br><center><small><strong>Figure 1.2.<del class="diffchange diffchange-inline">3 </del>ssDsbA-Lgt-Enzyme USB(AarI/BsmAI)-TAL USB-His Tag</strong></small></center></br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></br><center><small><strong>Figure 1.2.<ins class="diffchange diffchange-inline">4 </ins>ssDsbA-Lgt-Enzyme USB(AarI/BsmAI)-TAL USB-His Tag</strong></small></center></br></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>The <strong><em>Connector</strong></em> is pBluescript II KS(+) .</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>The <strong><em>Connector</strong></em> is pBluescript II KS(+) .</p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><img src="https://static.igem.org/mediawiki/2014/8/81/Pks_ii_original.png"></img></center></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><img src="https://static.igem.org/mediawiki/2014/8/81/Pks_ii_original.png"></img></center></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div></br><center><small><strong>Figure 1.2.<del class="diffchange diffchange-inline">4 </del>pBluescript II KS(+)</strong></small></center></br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></br><center><small><strong>Figure 1.2.<ins class="diffchange diffchange-inline">5 </ins>pBluescript II KS(+)</strong></small></center></br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>We used the four reformed plasmids to co-transform and induced the <strong><em>Connectors</strong></em> to express. Now the structure of the crown appears in the <i>E.coli</i> cell membrane or cytoplasm. Here we use the fusion proteins with two kinds of enzymes and TAL1 as examples. </p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>We used the four reformed plasmids to co-transform and induced the <strong><em>Connectors</strong></em> to express. Now the structure of the crown appears in the <i>E.coli</i> cell membrane or cytoplasm. Here we use the fusion proteins with two kinds of enzymes and TAL1 as examples. </p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><img src="https://static.igem.org/mediawiki/2014/0/0d/SJTU14_Enzyme%2BTAL1.jpg"></img></center></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><img src="https://static.igem.org/mediawiki/2014/0/0d/SJTU14_Enzyme%2BTAL1.jpg"></img></center></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div></br><center><small><strong>Figure 1.2.<del class="diffchange diffchange-inline">5 </del>ssDsbA-Lgt-pykF/poxB-TAL1-His Tag</strong></small></center></br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></br><center><small><strong>Figure 1.2.<ins class="diffchange diffchange-inline">6 </ins>ssDsbA-Lgt-pykF/poxB-TAL1-His Tag</strong></small></center></br></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3 id="Testmethods">Test methods</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3 id="Testmethods">Test methods</h3></div></td></tr>
</table>Corrine