http://2012.igem.org/wiki/index.php?title=Team:NTU-Taida/Result/Secretion-GLP1&feed=atom&action=historyTeam:NTU-Taida/Result/Secretion-GLP1 - Revision history2024-03-29T14:28:35ZRevision history for this page on the wikiMediaWiki 1.16.0http://2012.igem.org/wiki/index.php?title=Team:NTU-Taida/Result/Secretion-GLP1&diff=292771&oldid=prevPopo: /* Method */2012-10-27T00:06:15Z<p><span class="autocomment">Method</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Method==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Method==</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>To check whether our bacteria successfully secrete GLP-1 to the environment, we collected the bacterial media and measured its GLP-1 concentration. After centrifugation of bacterial culture, we collected the supernatant and passed it through 0.22 μm filter to exclude bacteria. Since the secreted GLP-1 is too diluted by the large volume of media, we concentrated the solution by 5 kDa ultra centrifugal filter tubes. We also collected the bacterial pellet and add lyze it by ''E. coli'' lysis buffer and freeze-thaw to measure GLP-1 inside bacteria. Both Immunoblotting and ELISA are used to detect GLP-1 concentration inside and outside bacteria.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><p style="text-indent: 2em;"></ins>To check whether our bacteria successfully secrete GLP-1 to the environment, we collected the bacterial media and measured its GLP-1 concentration. After centrifugation of bacterial culture, we collected the supernatant and passed it through 0.22 μm filter to exclude bacteria. Since the secreted GLP-1 is too diluted by the large volume of media, we concentrated the solution by 5 kDa ultra centrifugal filter tubes. We also collected the bacterial pellet and add lyze it by ''E. coli'' lysis buffer and freeze-thaw to measure GLP-1 inside bacteria. Both Immunoblotting and ELISA are used to detect GLP-1 concentration inside and outside bacteria.<ins class="diffchange diffchange-inline"></p></ins></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Protocol==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Protocol==</div></td></tr>
</table>Popohttp://2012.igem.org/wiki/index.php?title=Team:NTU-Taida/Result/Secretion-GLP1&diff=284386&oldid=prevEdmund0610: /* Method */2012-10-26T13:46:55Z<p><span class="autocomment">Method</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Method==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Method==</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>To check whether our bacteria successfully secrete GLP-1 to the environment, we collected the bacterial media and <del class="diffchange diffchange-inline">measure </del>its GLP-1 concentration. After centrifugation of bacterial culture, we collected the supernatant and passed it through 0.22 μm filter to exclude bacteria. Since the secreted GLP-1 is too diluted by the large volume of media, we concentrated the solution by 5 kDa ultra centrifugal filter tubes. We also collected the bacterial pellet and add lyze it by ''E. coli'' lysis buffer and freeze-thaw to measure GLP-1 inside bacteria. Both Immunoblotting and ELISA are used to detect GLP-1 concentration inside and outside bacteria.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>To check whether our bacteria successfully secrete GLP-1 to the environment, we collected the bacterial media and <ins class="diffchange diffchange-inline">measured </ins>its GLP-1 concentration. After centrifugation of bacterial culture, we collected the supernatant and passed it through 0.22 μm filter to exclude bacteria. Since the secreted GLP-1 is too diluted by the large volume of media, we concentrated the solution by 5 kDa ultra centrifugal filter tubes. We also collected the bacterial pellet and add lyze it by ''E. coli'' lysis buffer and freeze-thaw to measure GLP-1 inside bacteria. Both Immunoblotting and ELISA are used to detect GLP-1 concentration inside and outside bacteria.</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>==Protocol==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Protocol==</div></td></tr>
</table>Edmund0610http://2012.igem.org/wiki/index.php?title=Team:NTU-Taida/Result/Secretion-GLP1&diff=220565&oldid=prevDarlisereno at 22:21, 26 September 20122012-09-26T22:21:04Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Method==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Method==</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>To check whether our bacteria successfully <del class="diffchange diffchange-inline">secret </del>GLP-1 to the environment, we <del class="diffchange diffchange-inline">collect </del>the bacterial media and measure its GLP-1 concentration. After centrifugation of bacterial culture, we <del class="diffchange diffchange-inline">collect </del>supernatant and <del class="diffchange diffchange-inline">pass </del>it through 0.22 <del class="diffchange diffchange-inline">um </del>filter to exclude bacteria. <del class="diffchange diffchange-inline">We then concentrate </del>the solution by 5 kDa ultra centrifugal filter tubes. We also <del class="diffchange diffchange-inline">collect </del>the bacterial pellet and lyze ''E. coli'' <del class="diffchange diffchange-inline">delicately by adding </del>lysis buffer<del class="diffchange diffchange-inline">, followed by freeze </del>and thaw <del class="diffchange diffchange-inline">the media </del>to <del class="diffchange diffchange-inline">free </del>GLP-1 inside bacteria. Both Immunoblotting and ELISA are used to detect GLP-1 concentration inside and outside bacteria.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>To check whether our bacteria successfully <ins class="diffchange diffchange-inline">secrete </ins>GLP-1 to the environment, we <ins class="diffchange diffchange-inline">collected </ins>the bacterial media and measure its GLP-1 concentration. After centrifugation of bacterial culture, we <ins class="diffchange diffchange-inline">collected the </ins>supernatant and <ins class="diffchange diffchange-inline">passed </ins>it through 0.22 <ins class="diffchange diffchange-inline">μm </ins>filter to exclude bacteria. <ins class="diffchange diffchange-inline">Since the secreted GLP-1 is too diluted by the large volume of media, we concentrated </ins>the solution by 5 kDa ultra centrifugal filter tubes. We also <ins class="diffchange diffchange-inline">collected </ins>the bacterial pellet and <ins class="diffchange diffchange-inline">add </ins>lyze <ins class="diffchange diffchange-inline">it by </ins>''E. coli'' lysis buffer and <ins class="diffchange diffchange-inline">freeze-</ins>thaw to <ins class="diffchange diffchange-inline">measure </ins>GLP-1 inside bacteria. Both Immunoblotting and ELISA are used to detect GLP-1 concentration inside and outside bacteria.</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>==Protocol==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Protocol==</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===A. Sample Preparation===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===A. Sample Preparation===</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>#<del class="diffchange diffchange-inline">10 </del>μL <del class="diffchange diffchange-inline">bacterial </del>culture was cultured in 5mL LB at 37℃, with suitable concentration of antibiotics shaking till 600 nm absorbance in cuvette is 1.5 (usually it takes about <del class="diffchange diffchange-inline">18hr</del>).</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>#<ins class="diffchange diffchange-inline">310 </ins>μL <ins class="diffchange diffchange-inline">Bacterial </ins>culture was cultured in 5mL LB at 37℃, with suitable concentration of antibiotics shaking till 600 nm absorbance in cuvette is 1.5 (usually it takes about <ins class="diffchange diffchange-inline">18 hr</ins>).</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Centrifuge the bacterial culture at 12000 rpm for 3 min.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Centrifuge the bacterial culture at 12000 rpm for 3 min.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Pass the supernatant through 0.22 μm filter. Transfer the supernatant to 5 kDa ultra centrifugal filter tubes and centrifuge at 10000 rpm for 40 min.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Pass the supernatant through 0.22 μm filter. Transfer the supernatant to 5 kDa ultra centrifugal filter tubes and centrifuge at 10000 rpm for 40 min.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>#Add <del class="diffchange diffchange-inline">250μL </del>of ''E. coli'' lysis buffer to the pellet and use liquid Nitrogen to freeze-thaw for three cycles.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>#Add <ins class="diffchange diffchange-inline">250 μL </ins>of ''E. coli'' lysis buffer to the pellet and use liquid Nitrogen to freeze-thaw for three cycles.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>#Heat the samples at <del class="diffchange diffchange-inline">95℃ </del>for 10 min before immune-blotting.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>#Heat the samples at <ins class="diffchange diffchange-inline">95。C </ins>for 10 min before immune-blotting.</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>===B. Dot-Blotting===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===B. Dot-Blotting===</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>#Soak the PVDF membrane in ethanol for <del class="diffchange diffchange-inline">5min </del>to activate the membrane.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>#Soak the PVDF membrane in ethanol for <ins class="diffchange diffchange-inline">5 min </ins>to activate the membrane.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Drop every 2 μL sample in a very small area on the membrane.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Drop every 2 μL sample in a very small area on the membrane.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>#Blocking with 5% de-fat milk (in PBST) for <del class="diffchange diffchange-inline">1hr</del>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>#Blocking with 5% de-fat milk (in PBST) for <ins class="diffchange diffchange-inline">1 hr</ins>.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Wash with PBST for 10 min, repeat 3 times.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Wash with PBST for 10 min, repeat 3 times.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>#Add primary antibody (1:1000 dilution in PBST) and incubate at <del class="diffchange diffchange-inline">4℃overnight</del>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>#Add primary antibody (1:1000 dilution in PBST) and incubate at <ins class="diffchange diffchange-inline">4℃ overnight</ins>.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Wash with PBST for 10 min, repeat 3 times.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Wash with PBST for 10 min, repeat 3 times.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>#Add secondary antibody (1:5000 dilution in PBST) and incubate at 25℃ <del class="diffchange diffchange-inline">2hr</del>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>#Add secondary antibody (1:5000 dilution in PBST) and incubate at 25℃ <ins class="diffchange diffchange-inline">2 hr</ins>.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Wash with PBST for 10 min, repeat 3 times.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Wash with PBST for 10 min, repeat 3 times.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Mix 2 mL ECL with 2mL substrate, add to the membrane and let stand for 5 min. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Mix 2 mL ECL with 2mL substrate, add to the membrane and let stand for 5 min. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Expose the membrane to the film in dark room.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Expose the membrane to the film in dark room.</div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===C. ELISA===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===C. ELISA===</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Decant liquid from plate.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Decant liquid from plate.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Wash 5 times with 250 μL Wash Buffer.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Wash 5 times with 250 μL Wash Buffer.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>#Add 200 μL Detection Conjugate in each well. Incubate <del class="diffchange diffchange-inline">2hr </del>at room temperature.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>#Add 200 μL Detection Conjugate in each well. Incubate <ins class="diffchange diffchange-inline">2 hr </ins>at room temperature.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Wash 3 times with 250 μL Wash Buffer.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Wash 3 times with 250 μL Wash Buffer.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Add 200 μL diluted substrate and incubate for 20 min.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Add 200 μL diluted substrate and incubate for 20 min.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Read plate on fluorescence plate reader with excitation/emission wavelength of 355 nm/460 nm.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Read plate on fluorescence plate reader with excitation/emission wavelength of 355 nm/460 nm.</div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Data==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Data==</div></td></tr>
</table>Darliserenohttp://2012.igem.org/wiki/index.php?title=Team:NTU-Taida/Result/Secretion-GLP1&diff=215484&oldid=prevDarlisereno at 20:42, 26 September 20122012-09-26T20:42:16Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Read plate on fluorescence plate reader with excitation/emission wavelength of 355 nm/460 nm.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Read plate on fluorescence plate reader with excitation/emission wavelength of 355 nm/460 nm.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">==Data==</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;">===A. Dot-Blotting===</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td 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;">[[FIle:NTU-Taida-Result-GLP-fig1.png|450px|thumb|center|TEST]]</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td 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;">The upper dot is from the lyzed bacterial pellet, which represents the GLP-1 inside the bacteria. The middle part is from bacterial culture supernatant, which means the secreted GLP-1, and the left dot is the media we did 10X concentration, the right dot is the one we did 100X concentration. The lower dot is from pure GLP-1 solution as positive control.</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td 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;">===B. ELISA===</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td 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;">[[FIle:NTU-Taida-Result-GLP-fig2.png|450px|thumb|center|TEST]]</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td 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;">The left bar is from the lyzed bacterial pellet, which represents the GLP-1 inside the bacteria. The right bar is from 10X concentrated bacterial culture supernatant, which means the secreted GLP-1.</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td 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;">==Conclusion==</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;">Our data indicates that although there is much GLP-1 inside the bacteria, some of GLP-1 is secreted by our signal peptide design. The secreted GLP-1 can be delivered to intestinal lumen and carry out its physiological function once absorbed.</ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><!-- EOF --></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><!-- EOF --></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:NTU-Taida/Templates/ContentEnd}}{{:Team:NTU-Taida/Templates/Footer|ActiveNavbar=Result, #nav-Result-Secetion-GLP1}}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:NTU-Taida/Templates/ContentEnd}}{{:Team:NTU-Taida/Templates/Footer|ActiveNavbar=Result, #nav-Result-Secetion-GLP1}}</div></td></tr>
</table>Darliserenohttp://2012.igem.org/wiki/index.php?title=Team:NTU-Taida/Result/Secretion-GLP1&diff=211522&oldid=prevDarlisereno at 19:17, 26 September 20122012-09-26T19:17:25Z<p></p>
<table style="background-color: white; color:black;">
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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 19:17, 26 September 2012</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Protocol==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Protocol==</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>===A. Sample Preparation==</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>===A. Sample Preparation<ins class="diffchange diffchange-inline">=</ins>==</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#10 μL bacterial culture was cultured in 5mL LB at 37℃, with suitable concentration of antibiotics shaking till 600 nm absorbance in cuvette is 1.5 (usually it takes about 18hr).</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#10 μL bacterial culture was cultured in 5mL LB at 37℃, with suitable concentration of antibiotics shaking till 600 nm absorbance in cuvette is 1.5 (usually it takes about 18hr).</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Centrifuge the bacterial culture at 12000 rpm for 3 min.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Centrifuge the bacterial culture at 12000 rpm for 3 min.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Wash 3 times with 250 μL Wash Buffer.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Wash 3 times with 250 μL Wash Buffer.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Add 200 μL diluted substrate and incubate for 20 min.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Add 200 μL diluted substrate and incubate for 20 min.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Read plate on fluorescence plate reader with excitation/emission wavelength of 355 nm/460 nm.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">#</ins>Read plate on fluorescence plate reader with excitation/emission wavelength of 355 nm/460 nm.</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>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><!-- EOF --></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><!-- EOF --></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:NTU-Taida/Templates/ContentEnd}}{{:Team:NTU-Taida/Templates/Footer|ActiveNavbar=Result, #nav-Result-Secetion-GLP1}}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:NTU-Taida/Templates/ContentEnd}}{{:Team:NTU-Taida/Templates/Footer|ActiveNavbar=Result, #nav-Result-Secetion-GLP1}}</div></td></tr>
</table>Darliserenohttp://2012.igem.org/wiki/index.php?title=Team:NTU-Taida/Result/Secretion-GLP1&diff=211409&oldid=prevDarlisereno at 19:14, 26 September 20122012-09-26T19:14:55Z<p></p>
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<td colspan='2' style="background-color: white; color:black;">Revision as of 19:14, 26 September 2012</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:NTU-Taida/Templates/BSHero|Title=Secretion: GLP-1|Content=}}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:NTU-Taida/Templates/BSHero|Title=Secretion: GLP-1|Content=}}</div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">==Method==</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">To check whether our bacteria successfully secret GLP-1 to the environment, we collect the bacterial media and measure its GLP-1 concentration. After centrifugation of bacterial culture, we collect supernatant and pass it through 0.22 um filter to exclude bacteria. We then concentrate the solution by 5 kDa ultra centrifugal filter tubes. We also collect the bacterial pellet and lyze ''E. coli'' delicately by adding lysis buffer, followed by freeze and thaw the media to free GLP-1 inside bacteria. Both Immunoblotting and ELISA are used to detect GLP-1 concentration inside and outside bacteria.</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td 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;">==Protocol==</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td 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;">===A. Sample Preparation==</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;">#10 μL bacterial culture was cultured in 5mL LB at 37℃, with suitable concentration of antibiotics shaking till 600 nm absorbance in cuvette is 1.5 (usually it takes about 18hr).</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;">#Centrifuge the bacterial culture at 12000 rpm for 3 min.</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;">#Pass the supernatant through 0.22 μm filter. Transfer the supernatant to 5 kDa ultra centrifugal filter tubes and centrifuge at 10000 rpm for 40 min.</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;">#Add 250μL of ''E. coli'' lysis buffer to the pellet and use liquid Nitrogen to freeze-thaw for three cycles.</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;">#Heat the samples at 95℃ for 10 min before immune-blotting.</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td 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;">===B. Dot-Blotting===</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;">#Soak the PVDF membrane in ethanol for 5min to activate the membrane.</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;">#Drop every 2 μL sample in a very small area on the membrane.</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;">#Blocking with 5% de-fat milk (in PBST) for 1hr.</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;">#Wash with PBST for 10 min, repeat 3 times.</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;">#Add primary antibody (1:1000 dilution in PBST) and incubate at 4℃overnight.</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;">#Wash with PBST for 10 min, repeat 3 times.</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;">#Add secondary antibody (1:5000 dilution in PBST) and incubate at 25℃ 2hr.</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;">#Wash with PBST for 10 min, repeat 3 times.</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;">#Mix 2 mL ECL with 2mL substrate, add to the membrane and let stand for 5 min. </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;">#Expose the membrane to the film in dark room.</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td 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;">===C. ELISA===</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;">#Wash the wells with 250 μL Wash Buffer.</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;">#Add 100 μL Assay Buffer to each well, and add 100 μL standards in ascending orders to wells, and add samples in the remaining wells.</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;">#Incubate at 4℃ overnight.</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;">#Decant liquid from plate.</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;">#Wash 5 times with 250 μL Wash Buffer.</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;">#Add 200 μL Detection Conjugate in each well. Incubate 2hr at room temperature.</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;">#Wash 3 times with 250 μL Wash Buffer.</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;">#Add 200 μL diluted substrate and incubate for 20 min.</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;">Read plate on fluorescence plate reader with excitation/emission wavelength of 355 nm/460 nm.</ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><!-- EOF --></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><!-- EOF --></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:NTU-Taida/Templates/ContentEnd}}{{:Team:NTU-Taida/Templates/Footer|ActiveNavbar=Result, #nav-Result-Secetion-GLP1}}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:NTU-Taida/Templates/ContentEnd}}{{:Team:NTU-Taida/Templates/Footer|ActiveNavbar=Result, #nav-Result-Secetion-GLP1}}</div></td></tr>
</table>Darliserenohttp://2012.igem.org/wiki/index.php?title=Team:NTU-Taida/Result/Secretion-GLP1&diff=209194&oldid=prevLbwang: Created page with "__FORCETOC__{{:Team:NTU-Taida/Templates/Header}}{{:Team:NTU-Taida/Templates/Navbar}}{{:Team:NTU-Taida/Templates/Sidebar|Title=Secretion: GLP-1}}{{:Team:NTU-Taida/Templates/Conten..."2012-09-26T18:30:05Z<p>Created page with "__FORCETOC__{{:Team:NTU-Taida/Templates/Header}}{{:Team:NTU-Taida/Templates/Navbar}}{{:Team:NTU-Taida/Templates/Sidebar|Title=Secretion: GLP-1}}{{:Team:NTU-Taida/Templates/Conten..."</p>
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