http://2009.igem.org/wiki/index.php?title=Team:HKU-HKBU/Polar_Expression_Results&feed=atom&action=historyTeam:HKU-HKBU/Polar Expression Results - Revision history2024-03-29T13:31:16ZRevision history for this page on the wikiMediaWiki 1.16.5http://2009.igem.org/wiki/index.php?title=Team:HKU-HKBU/Polar_Expression_Results&diff=167300&oldid=prevYubin: /* Fluorescence Microscopy */2009-10-22T03:06:59Z<p><span class="autocomment">Fluorescence Microscopy</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The surface expression of '''Lpp-ompA-GFP-Strp''' was consistent; however the polar expression of GFP-strp may not be achieved. Therefore, this plasmid was transformed into various strains of ''E.coli'' and ''S.typhimurium''. We finally found that in the stationary phase of YBS01, polar expression of the GFP-Strp was observed. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The surface expression of '''Lpp-ompA-GFP-Strp''' was consistent; however the polar expression of GFP-strp may not be achieved. Therefore, this plasmid was transformed into various strains of ''E.coli'' and ''S.typhimurium''. We finally found that in the stationary phase of YBS01, polar expression of the GFP-Strp was observed. </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>In the figure below showed the polar expression of the plasmid ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K283000 '''BBa_K283000''']) in the stationary phase of ''YBS01''. The right panel showed the merge picture of the bright field and FITC field under the fluorescence microscope with a magnification of 600 times. The result indicated that polar expression of proteins was achieved by this system in <del class="diffchange diffchange-inline">''</del>YBS01<del class="diffchange diffchange-inline">''</del>. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>In the figure below showed the polar expression of the plasmid ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K283000 '''BBa_K283000''']) in the stationary phase of ''YBS01''. The right panel showed the merge picture of the bright field and FITC field under the fluorescence microscope with a magnification of 600 times. The result indicated that polar expression of proteins was achieved by this system in YBS01. </div></td></tr>
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</table>Yubinhttp://2009.igem.org/wiki/index.php?title=Team:HKU-HKBU/Polar_Expression_Results&diff=167238&oldid=prevYubin: /* SDS-PAGE and Western Blot */2009-10-22T03:05:36Z<p><span class="autocomment">SDS-PAGE and Western Blot</span></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[Image:HKU-BU-AIDA-west3.png| center|thumb|300px|'''Figure 3.''' SDS-PAGE and Western Blot of GFP-Strp-AIDA in BL21. Bacteria were induced by 0.5mM IPTG for <del class="diffchange diffchange-inline">2h</del>; '''Cn,''' 0h control; '''TE,''' total cell extract; '''TM,''' total membrane; '''OM,''' outer membrane.]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[Image:HKU-BU-AIDA-west3.png| center|thumb|300px|'''Figure 3.''' SDS-PAGE and Western Blot of GFP-Strp-AIDA in BL21. Bacteria were induced by 0.5mM IPTG for <ins class="diffchange diffchange-inline">2 h</ins>; '''Cn,''' 0h control; '''TE,''' total cell extract; '''TM,''' total membrane; '''OM,''' outer membrane.]]</div></td></tr>
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</table>Yubinhttp://2009.igem.org/wiki/index.php?title=Team:HKU-HKBU/Polar_Expression_Results&diff=167218&oldid=prevYubin: /* Fluorescence Microscopy */2009-10-22T03:05:09Z<p><span class="autocomment">Fluorescence Microscopy</span></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[Image:HKU-HKBU polar expression results E coli fluorescent3.png | center|thumb|300px|'''Figure 2.''' Polar Expression of GFP-Strp-AIDA in BL21. ''E.coli'' BL21 with plasmid pGFP-Strp-AIDA were induced by 0.5 mM IPTG for <del class="diffchange diffchange-inline">2h</del>.]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[Image:HKU-HKBU polar expression results E coli fluorescent3.png | center|thumb|300px|'''Figure 2.''' Polar Expression of GFP-Strp-AIDA in BL21. ''E.coli'' BL21 with plasmid pGFP-Strp-AIDA were induced by 0.5 mM IPTG for <ins class="diffchange diffchange-inline">2 h</ins>.]]</div></td></tr>
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</table>Yubinhttp://2009.igem.org/wiki/index.php?title=Team:HKU-HKBU/Polar_Expression_Results&diff=167207&oldid=prevYubin: /* Fluorescence Microscopy */2009-10-22T03:04:53Z<p><span class="autocomment">Fluorescence Microscopy</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Fluorescence Microscopy===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Fluorescence Microscopy===</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>For AIDA system, the requirements for strains were critical, such as LPS completeness, strong expression, etc. Another polar expression system was constructed as our back-up plan. '''Lpp''' consists of 9 amino acids of signal peptide for targeting the inner membrane of the bacteria. '''OmpA (<del class="diffchange diffchange-inline">91</del>-<del class="diffchange diffchange-inline">261</del>)''' is a 5-cross trans-membrane protein which could bring cargos to the outer membrane of the bacteria. We constructed a plasmid ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K283000 '''BBa_K283000''']) by fusing GFP-strp with '''Lpp-OmpA''' system. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>For AIDA system, the requirements for strains were critical, such as LPS completeness, strong expression, etc. Another polar expression system was constructed as our back-up plan. '''Lpp''' consists of 9 amino acids of signal peptide for targeting the inner membrane of the bacteria. '''OmpA (<ins class="diffchange diffchange-inline">46</ins>-<ins class="diffchange diffchange-inline">159</ins>)''' is a 5-cross trans-membrane protein which could bring cargos to the outer membrane of the bacteria. We constructed a plasmid ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K283000 '''BBa_K283000''']) by fusing GFP-strp with '''Lpp-OmpA''' system. </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>The surface expression of '''Lpp-ompA-GFP-Strp''' was consistent; however the polar expression of GFP-strp may not be achieved. Therefore, this plasmid was transformed into various strains of ''E.coli'' and ''S.typhimurium''. We finally found that in the stationary phase of YBS01, polar expression of the GFP-Strp was observed. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The surface expression of '''Lpp-ompA-GFP-Strp''' was consistent; however the polar expression of GFP-strp may not be achieved. Therefore, this plasmid was transformed into various strains of ''E.coli'' and ''S.typhimurium''. We finally found that in the stationary phase of YBS01, polar expression of the GFP-Strp was observed. </div></td></tr>
</table>Yubinhttp://2009.igem.org/wiki/index.php?title=Team:HKU-HKBU/Polar_Expression_Results&diff=167172&oldid=prevYubin: /* Fluorescence Microscopy */2009-10-22T03:04:09Z<p><span class="autocomment">Fluorescence Microscopy</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For AIDA system, the requirements for strains were critical, such as LPS completeness, strong expression, etc. Another polar expression system was constructed as our back-up plan. '''Lpp''' consists of 9 amino acids of signal peptide for targeting the inner membrane of the bacteria. '''OmpA (91-261)''' is a 5-cross trans-membrane protein which could bring cargos to the outer membrane of the bacteria. We constructed a plasmid ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K283000 '''BBa_K283000''']) by fusing GFP-strp with '''Lpp-OmpA''' system. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For AIDA system, the requirements for strains were critical, such as LPS completeness, strong expression, etc. Another polar expression system was constructed as our back-up plan. '''Lpp''' consists of 9 amino acids of signal peptide for targeting the inner membrane of the bacteria. '''OmpA (91-261)''' is a 5-cross trans-membrane protein which could bring cargos to the outer membrane of the bacteria. We constructed a plasmid ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K283000 '''BBa_K283000''']) by fusing GFP-strp with '''Lpp-OmpA''' system. </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>The surface expression of '''Lpp-ompA-GFP-Strp''' was consistent; however the polar expression of GFP-strp may not be achieved. Therefore, this plasmid was transformed into various strains of ''E.coli'' and ''S.typhimurium''. We finally found that in the stationary phase of <del class="diffchange diffchange-inline">''</del>YBS01<del class="diffchange diffchange-inline">''</del>, polar expression of the GFP-Strp was observed. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The surface expression of '''Lpp-ompA-GFP-Strp''' was consistent; however the polar expression of GFP-strp may not be achieved. Therefore, this plasmid was transformed into various strains of ''E.coli'' and ''S.typhimurium''. We finally found that in the stationary phase of YBS01, polar expression of the GFP-Strp was observed. </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>In the figure below showed the polar expression of the plasmid ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K283000 '''BBa_K283000''']) in the stationary phase of ''YBS01''. The right panel showed the merge picture of the bright field and FITC field under the fluorescence microscope with a magnification of 600 times. The result indicated that polar expression of proteins was achieved by this system in ''YBS01''. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In the figure below showed the polar expression of the plasmid ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K283000 '''BBa_K283000''']) in the stationary phase of ''YBS01''. The right panel showed the merge picture of the bright field and FITC field under the fluorescence microscope with a magnification of 600 times. The result indicated that polar expression of proteins was achieved by this system in ''YBS01''. </div></td></tr>
</table>Yubinhttp://2009.igem.org/wiki/index.php?title=Team:HKU-HKBU/Polar_Expression_Results&diff=165848&oldid=prevYinanZhang at 02:36, 22 October 20092009-10-22T02:36:42Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===LPS Completeness Search===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===LPS Completeness Search===</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>LPS takes vital part in AIDA polar expression system. Due to many mutations may exist in the strains of ''Escherichia coli'' or ''Salmonella typhimurium'', some of these mutations may cause the <del class="diffchange diffchange-inline">defciency </del>in <del class="diffchange diffchange-inline">there </del>LPS layers. <del class="diffchange diffchange-inline">There </del>mutants could survive in the culture. But for AIDA expression, when the <del class="diffchange diffchange-inline">host</del>'s LPS is incomplete, the AIDA will express all over the bacteria surface; while the <del class="diffchange diffchange-inline">host's </del>LPS is complete, the AIDA will express only on one side of the bacteria. After literature review, ''E. coli''-YBE01 [[#Reference | [1]]] and ''S.typhimurium''-YBS01 [[#Reference | [2]]] are identified to possess complete LPS layer. Their ability to express desirable proteins on the head is examined in later experiment.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>LPS takes vital part in AIDA polar expression system. Due to many mutations may exist in the strains of ''Escherichia coli'' or ''Salmonella typhimurium'', some of these mutations may cause the <ins class="diffchange diffchange-inline">deficiency </ins>in <ins class="diffchange diffchange-inline">the </ins>LPS layers. <ins class="diffchange diffchange-inline">These </ins>mutants could survive in the culture. But for AIDA expression, when the <ins class="diffchange diffchange-inline">microorganism</ins>'s LPS is incomplete, the AIDA will express all over the bacteria surface; while the <ins class="diffchange diffchange-inline">bacterial </ins>LPS is complete, the AIDA will express only on one side of the bacteria. After literature review, ''E. coli''-YBE01 [[#Reference | [1]]] and ''S.typhimurium''-YBS01 [[#Reference | [2]]] are identified to possess complete LPS layer. Their ability to express desirable proteins on the head is examined in later experiment.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=Polar Expression=</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=Polar Expression=</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Fluorescence Microscopy===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Fluorescence Microscopy===</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>After literature review, we found the ''E.coli'' BL21 was also LPS complete <del class="diffchange diffchange-inline">strains</del>. Therefore, we used the plasmid ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K283001 '''BBa_K283001''']) containing T7 promoter which controlled the expression of '''GFP-Strp-AIDA'''. BL21 strain also <del class="diffchange diffchange-inline">contained </del>T7 polymerase<del class="diffchange diffchange-inline">, therefore when the </del>IPTG was added into the culture medium, '''GFP-Strp-AIDA''' would be strongly induced. In the figure below, it <del class="diffchange diffchange-inline">showed </del>the expression of '''GFP'''. This expression was observed under the fluorescent microscope using oil lens with the <del class="diffchange diffchange-inline">manification </del>of 600 times. In the <del class="diffchange diffchange-inline">merge </del>picture of dark field and FITC field, the fluorescent proteins were showed at one end of the bacteria.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>After literature review, we found the ''E. coli'' BL21 was also <ins class="diffchange diffchange-inline">an </ins>LPS complete <ins class="diffchange diffchange-inline">strain</ins>. Therefore, we used the plasmid ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K283001 '''BBa_K283001''']) containing T7 promoter which controlled the expression of '''GFP-Strp-AIDA''' <ins class="diffchange diffchange-inline">to test the polar expression</ins>. BL21 strain also <ins class="diffchange diffchange-inline">contains </ins>T7 polymerase <ins class="diffchange diffchange-inline">which expression is induced by IPTG. When </ins>IPTG was added into the culture medium, '''GFP-Strp-AIDA''' would be strongly induced. In the figure below, it <ins class="diffchange diffchange-inline">showes </ins>the expression of '''GFP'''. This expression was observed under the fluorescent microscope using oil lens with the <ins class="diffchange diffchange-inline">magnification </ins>of 600 times. In the <ins class="diffchange diffchange-inline">merged </ins>picture of dark field and FITC field, the fluorescent proteins were showed at one end of the bacteria.</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>For this project, we required a strain that expresses specific proteins only in one side, and has swimming ability at the same time. However, the BL21 strain couldn't swim. Therefore we need to apply one side expression system into our new candidate YBE01. <del class="diffchange diffchange-inline">While </del>YBE01 <del class="diffchange diffchange-inline">didn't </del>contain T7 polymerase<del class="diffchange diffchange-inline">, being </del>not able to activate T7 promoter in <del class="diffchange diffchange-inline">this </del>plasmid ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K283001 '''BBa_K283001''']). Therefore, a noval plasmid with strongly inducible or constitutive expression system <del class="diffchange diffchange-inline">should be </del>constructed. Attempts were made to use '''<del class="diffchange diffchange-inline">Plac</del>''' without '''LacI''' as a strong constitutive promoter. However, only weak and unstable expression of '''GFP''' could be observed. One of the possible explanation could be that these systems were not strong or stable enough to ensure the consistent expression of our required proteins.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>For this project, we required a strain that expresses specific proteins only in one side, and has swimming ability at the same time. However, the BL21 strain couldn't swim. Therefore we need to apply one side expression system into our new candidate YBE01. <ins class="diffchange diffchange-inline">Unfortunately </ins>YBE01 <ins class="diffchange diffchange-inline">does not </ins>contain T7 polymerase <ins class="diffchange diffchange-inline">and is </ins>not able to activate <ins class="diffchange diffchange-inline">the </ins>T7 promoter in <ins class="diffchange diffchange-inline">the '''GFP-Strp-AIDA''' expressing </ins>plasmid ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K283001 '''BBa_K283001''']). Therefore, a noval plasmid with strongly inducible or constitutive expression system <ins class="diffchange diffchange-inline">was </ins>constructed. Attempts were made to use '''<ins class="diffchange diffchange-inline">PLac</ins>''' without '''LacI''' as a strong constitutive promoter. However, only weak and unstable expression of '''GFP''' could be observed. One of the possible explanation could be that these systems were not strong or stable enough to ensure the consistent expression of our required proteins.</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>===SDS-PAGE and Western Blot===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===SDS-PAGE and Western Blot===</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>The surface expression of streptavidin was essential <del class="diffchange diffchange-inline">to specifically bind </del>to biotin-coated motor. To prove that the '''GFP-strp''' was expressed on the outer membrane of the bacteria through''' AIDA''' system, different contents of the bacteria were separated by ultracentrifuge. After separation, the outer membrane, inner membrane, total membrane and cytoplasm were collected as different samples. SDS-PAGE and Western Blot were conducted for verification. Each sample was quantified by BCA assay to make sure the equal loading of proteins. The western blotting results showed that most '''GFP''' proteins were localized on the outer membrane of the bacteria, which corresponded with the expected result that the specific expressions of proteins were on the outer membrane.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The surface expression of streptavidin was essential <ins class="diffchange diffchange-inline">for the specific binding of the bacteria </ins>to biotin-coated motor. To prove that the '''GFP-strp''' was expressed on the outer membrane of the bacteria through''' AIDA''' system, different contents of the bacteria were separated by ultracentrifuge. After separation, the outer membrane, inner membrane, total membrane and cytoplasm were collected as different samples. SDS-PAGE and Western Blot were conducted for verification. Each sample was quantified by BCA assay to make sure the equal loading of proteins. The western blotting results showed that most '''GFP''' proteins were localized on the outer membrane of the bacteria, which corresponded with the expected result that the specific expressions of proteins were on the outer membrane.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For AIDA system, the requirements for strains were critical, such as LPS completeness, strong expression, etc. Another polar expression system was constructed as our back-up plan. '''Lpp''' consists of 9 amino acids of signal peptide for targeting the inner membrane of the bacteria. '''OmpA (91-261)''' is a 5-cross trans-membrane protein which could bring cargos to the outer membrane of the bacteria. We constructed a plasmid ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K283000 '''BBa_K283000''']) by fusing GFP-strp with '''Lpp-OmpA''' system. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For AIDA system, the requirements for strains were critical, such as LPS completeness, strong expression, etc. Another polar expression system was constructed as our back-up plan. '''Lpp''' consists of 9 amino acids of signal peptide for targeting the inner membrane of the bacteria. '''OmpA (91-261)''' is a 5-cross trans-membrane protein which could bring cargos to the outer membrane of the bacteria. We constructed a plasmid ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K283000 '''BBa_K283000''']) by fusing GFP-strp with '''Lpp-OmpA''' system. </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>The surface expression of '''Lpp-ompA-GFP-Strp''' was consistent<del class="diffchange diffchange-inline">, </del>however the polar expression of GFP-strp may not be achieved. Therefore, this plasmid was transformed into various strains of ''E.coli'' and ''S.typhimurium''. We finally found that in the <del class="diffchange diffchange-inline">stage </del>phase of ''YBS01'', polar expression of the <del class="diffchange diffchange-inline">plamid </del>was observed. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The surface expression of '''Lpp-ompA-GFP-Strp''' was consistent<ins class="diffchange diffchange-inline">; </ins>however the polar expression of GFP-strp may not be achieved. Therefore, this plasmid was transformed into various strains of ''E.coli'' and ''S.typhimurium''. We finally found that in the <ins class="diffchange diffchange-inline">stationary </ins>phase of ''YBS01'', polar expression of the <ins class="diffchange diffchange-inline">GFP-Strp </ins>was observed. </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>In the figure below showed the polar expression of the plasmid ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K283000 '''BBa_K283000''']) in the stationary phase of ''YBS01''. The right panel showed the merge picture of the bright field and FITC field under the fluorescence microscope with a magnification of 600 times. The result indicated that polar expression of proteins was achieved by this system in ''YBS01''. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In the figure below showed the polar expression of the plasmid ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K283000 '''BBa_K283000''']) in the stationary phase of ''YBS01''. The right panel showed the merge picture of the bright field and FITC field under the fluorescence microscope with a magnification of 600 times. The result indicated that polar expression of proteins was achieved by this system in ''YBS01''. </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Western Blot===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Western Blot===</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>To verify our experimental result in a more convincing way, Western Blot was conducted, which could reflect the surface expression of proteins. By studying the relative concentrations of expressed protein GFP in different samples, an obvious darker band was observed in the outer membrane samples. <del class="diffchange diffchange-inline">This </del>results verified our hypothesis that the GFP proteins are particularly expressed on the outer membrane.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>To verify our experimental result in a more convincing way, Western Blot was conducted, which could reflect the surface expression of proteins. By studying the relative concentrations of expressed protein GFP in different samples, an obvious darker band was observed in the outer membrane samples. <ins class="diffchange diffchange-inline">These </ins>results verified our hypothesis that the GFP proteins are particularly expressed on the outer membrane.</div></td></tr>
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</table>YinanZhanghttp://2009.igem.org/wiki/index.php?title=Team:HKU-HKBU/Polar_Expression_Results&diff=165368&oldid=prevCarlosX at 02:25, 22 October 20092009-10-22T02:25:34Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{Team:HKU-HKBU/header}}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{Team:HKU-HKBU/header}}</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>=Strain <del class="diffchange diffchange-inline">Selecion</del>=</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>=Strain <ins class="diffchange diffchange-inline">Selection</ins>=</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Swimming Ability Assay===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Swimming Ability Assay===</div></td></tr>
</table>CarlosXhttp://2009.igem.org/wiki/index.php?title=Team:HKU-HKBU/Polar_Expression_Results&diff=165327&oldid=prevYinanZhang at 02:24, 22 October 20092009-10-22T02:24:39Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{Team:HKU-HKBU/header}}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{Team:HKU-HKBU/header}}</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>=<del class="diffchange diffchange-inline">'''</del>Strain Selecion<del class="diffchange diffchange-inline">'''</del>=</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>=Strain Selecion=</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Swimming Ability Assay===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Swimming Ability Assay===</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>The purpose of this part is to find one or several strains of bacteria which could <del class="diffchange diffchange-inline">swim</del>. <del class="diffchange diffchange-inline">Therefore, we </del>tested ''Escherichia coli'' and ''Salmonella typhimurium''. <del class="diffchange diffchange-inline">The swimming plates of </del>BL21, NCM3722 and YBE03 <del class="diffchange diffchange-inline">yielded negative results. We found </del>a ''Salmonella typhimurium'' strain - YBS01 is a bacteria with high swimming abilities (~4.5 mm/<del class="diffchange diffchange-inline">h</del>)<del class="diffchange diffchange-inline">, and a strain of </del>''Escherichia coli'' <del class="diffchange diffchange-inline">- </del>YBE01 showed <del class="diffchange diffchange-inline">a stronger swim ability</del>, with a speed of <del class="diffchange diffchange-inline">approximate </del>5.5 mm increase in radius <del class="diffchange diffchange-inline">every hour </del>at the end of eight-hour-experiment.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The purpose of this part is to find one or several strains of bacteria which could <ins class="diffchange diffchange-inline">propel the motor efficiently</ins>. <ins class="diffchange diffchange-inline">Swimming Ability Assay was used to identify the best bacterial strain. We </ins>tested <ins class="diffchange diffchange-inline">several strains of </ins>''Escherichia coli'' and ''Salmonella typhimurium''. <ins class="diffchange diffchange-inline">We found that </ins>BL21, NCM3722 and YBE03 <ins class="diffchange diffchange-inline">cannot swim whilst </ins>a ''Salmonella typhimurium'' strain - <ins class="diffchange diffchange-inline">we named it </ins>YBS01 <ins class="diffchange diffchange-inline">- </ins>is a bacteria with high swimming abilities (~4.5 mm/<ins class="diffchange diffchange-inline">hr</ins>)<ins class="diffchange diffchange-inline">. </ins>''Escherichia coli''<ins class="diffchange diffchange-inline">, we named it </ins>YBE01<ins class="diffchange diffchange-inline">, </ins>showed <ins class="diffchange diffchange-inline">even more impressive performance</ins>, with a speed of <ins class="diffchange diffchange-inline">~</ins>5.5 mm<ins class="diffchange diffchange-inline">/hr </ins>increase in radius at the end of eight-hour-experiment.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>LPS takes vital part in AIDA polar expression system. Due to many mutations may exist in the strains of ''Escherichia coli'' or ''Salmonella typhimurium'', some of these mutations may cause the defciency in there LPS layers. There mutants could survive in the culture. But for AIDA expression, when the host's LPS is incomplete, the AIDA will express all over the bacteria surface; while the host's LPS is complete, the AIDA will express only on one side of the bacteria. After literature review, ''E. coli''-YBE01 [[#Reference | [1]]] and ''S.typhimurium''-YBS01 [[#Reference | [2]]] are identified to possess complete LPS layer. Their ability to express desirable proteins on the head is examined in later experiment.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>LPS takes vital part in AIDA polar expression system. Due to many mutations may exist in the strains of ''Escherichia coli'' or ''Salmonella typhimurium'', some of these mutations may cause the defciency in there LPS layers. There mutants could survive in the culture. But for AIDA expression, when the host's LPS is incomplete, the AIDA will express all over the bacteria surface; while the host's LPS is complete, the AIDA will express only on one side of the bacteria. After literature review, ''E. coli''-YBE01 [[#Reference | [1]]] and ''S.typhimurium''-YBS01 [[#Reference | [2]]] are identified to possess complete LPS layer. Their ability to express desirable proteins on the head is examined in later experiment.</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>=<del class="diffchange diffchange-inline">'''</del>Polar Expression<del class="diffchange diffchange-inline">'''</del>=</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>=Polar Expression=</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==AIDA Polar Expression System==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==AIDA Polar Expression System==</div></td></tr>
</table>YinanZhanghttp://2009.igem.org/wiki/index.php?title=Team:HKU-HKBU/Polar_Expression_Results&diff=148950&oldid=prevCarlosX at 17:08, 21 October 20092009-10-21T17:08:21Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[Image:HKU-BU-Swimming assay second3.png| center|thumb|<del class="diffchange diffchange-inline">400px</del>|'''Figure 1.''' Swimming ability of different bacteria strains. '''a,''' BL21; '''b,''' NCM3722; '''c,''' YBE03; '''d,''' YBE01; '''e,''' YBS01; '''f,''' MG1655.]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[Image:HKU-BU-Swimming assay second3.png| center|thumb|<ins class="diffchange diffchange-inline">300px</ins>|'''Figure 1.''' Swimming ability of different bacteria strains. '''a,''' BL21; '''b,''' NCM3722; '''c,''' YBE03; '''d,''' YBE01; '''e,''' YBS01; '''f,''' MG1655.]]</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===LPS Completeness Search===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===LPS Completeness Search===</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </div></td><td class='diff-marker'> </td><td style="background: #eee; 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>[[Image:HKU-HKBU polar expression results E coli fluorescent3.png | center|thumb|<del class="diffchange diffchange-inline">400px</del>|'''Figure 2.''' Polar Expression of GFP-Strp-AIDA in BL21. ''E.coli'' BL21 with plasmid pGFP-Strp-AIDA were induced by 0.5 mM IPTG for 2h.]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[Image:HKU-HKBU polar expression results E coli fluorescent3.png | center|thumb|<ins class="diffchange diffchange-inline">300px</ins>|'''Figure 2.''' Polar Expression of GFP-Strp-AIDA in BL21. ''E.coli'' BL21 with plasmid pGFP-Strp-AIDA were induced by 0.5 mM IPTG for 2h.]]</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[Image:HKU-BU-AIDA-west3.png| center|thumb|<del class="diffchange diffchange-inline">400px</del>|'''Figure 3.''' SDS-PAGE and Western Blot of GFP-Strp-AIDA in BL21. Bacteria were induced by 0.5mM IPTG for 2h; '''Cn,''' 0h control; '''TE,''' total cell extract; '''TM,''' total membrane; '''OM,''' outer membrane.]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[Image:HKU-BU-AIDA-west3.png| center|thumb|<ins class="diffchange diffchange-inline">300px</ins>|'''Figure 3.''' SDS-PAGE and Western Blot of GFP-Strp-AIDA in BL21. Bacteria were induced by 0.5mM IPTG for 2h; '''Cn,''' 0h control; '''TE,''' total cell extract; '''TM,''' total membrane; '''OM,''' outer membrane.]]</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[Image:Polar.png| center|thumb|<del class="diffchange diffchange-inline">500px</del>|'''Figure 4.''' Polar Expression of Lpp-ompA-GFP-Strp in YBS01. Bacteria were harvested through over-night culture.]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[Image:Polar.png| center|thumb|<ins class="diffchange diffchange-inline">300px</ins>|'''Figure 4.''' Polar Expression of Lpp-ompA-GFP-Strp in YBS01. Bacteria were harvested through over-night culture.]]</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Western Blot===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Western Blot===</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[Image:HKU-HKBU polar expression results Salmonella western4.png| center|thumb|<del class="diffchange diffchange-inline">400px</del>|'''Figure 5.''' Western Blot of Lpp-OmpA-GFP-Strp in YBS01. Bacteria were harvested through over-night culture ; '''OM,''' outer membrane; '''TM,''' total membrane; '''IM,''' inter membrane.]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[Image:HKU-HKBU polar expression results Salmonella western4.png| center|thumb|<ins class="diffchange diffchange-inline">300px</ins>|'''Figure 5.''' Western Blot of Lpp-OmpA-GFP-Strp in YBS01. Bacteria were harvested through over-night culture ; '''OM,''' outer membrane; '''TM,''' total membrane; '''IM,''' inter membrane.]]</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=='''Conclusion'''==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=='''Conclusion'''==</div></td></tr>
</table>CarlosXhttp://2009.igem.org/wiki/index.php?title=Team:HKU-HKBU/Polar_Expression_Results&diff=148930&oldid=prevCarlosX: /* Western Blot */2009-10-21T17:07:38Z<p><span class="autocomment">Western Blot</span></p>
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<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 17:07, 21 October 2009</td>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[Image:HKU-HKBU polar expression results Salmonella western4.png| center|thumb|400px|'''Figure 5.''' Western Blot of Lpp-OmpA-GFP-Strp in YBS01. Bacteria were harvested through over-night culture ; '''OM,''' outer membrane; '''TM,''' total membrane; '''IM,''' inter membrane]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[Image:HKU-HKBU polar expression results Salmonella western4.png| center|thumb|400px|'''Figure 5.''' Western Blot of Lpp-OmpA-GFP-Strp in YBS01. Bacteria were harvested through over-night culture ; '''OM,''' outer membrane; '''TM,''' total membrane; '''IM,''' inter membrane<ins class="diffchange diffchange-inline">.</ins>]]</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=='''Conclusion'''==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=='''Conclusion'''==</div></td></tr>
</table>CarlosX