http://2014hs.igem.org/wiki/index.php?title=Team:WalthamHS_BioHawks/Project/Procedure&feed=atom&action=historyTeam:WalthamHS BioHawks/Project/Procedure - Revision history2024-03-29T09:55:28ZRevision history for this page on the wikiMediaWiki 1.16.5http://2014hs.igem.org/wiki/index.php?title=Team:WalthamHS_BioHawks/Project/Procedure&diff=27293&oldid=prevRutviBio at 03:08, 21 June 20142014-06-21T03:08:51Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Procedure==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Procedure==</div></td></tr>
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</table>RutviBiohttp://2014hs.igem.org/wiki/index.php?title=Team:WalthamHS_BioHawks/Project/Procedure&diff=25958&oldid=prevMaddoxm: /* Procedure */2014-06-21T01:10:45Z<p><span class="autocomment">Procedure</span></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>2: Complete <del class="diffchange diffchange-inline">Biological </del>Control Test</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>2: Complete <ins class="diffchange diffchange-inline">Subtilisin </ins>Control Test</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We then wanted to confirm that subtilisin had protein-degrading capabilities, and not ''E. coli''. We also wanted to eliminate liquid dispersion as a source of error. So, we repeated the experiment with three plates, each with 2ml of skim milk applied on top. The first control plate had 10 drops of 10ul of water, the second with 10 drops of 10ul of subtilisin, and the third with 10 drops of 10ul of ''E. coli'', which was grown in LB overnight. Similar to the previous experiment with only milk, the control with water was unchanged and the water diffused into the milk without modifying it. When ''E. coli'' was applied to the milk, there were no significant changes. Similar to the last experiment, milk treated with subtilisin was degraded. This proved that subtilisin was solely responsible for protein degradation. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We then wanted to confirm that subtilisin had protein-degrading capabilities, and not ''E. coli''. We also wanted to eliminate liquid dispersion as a source of error. So, we repeated the experiment with three plates, each with 2ml of skim milk applied on top. The first control plate had 10 drops of 10ul of water, the second with 10 drops of 10ul of subtilisin, and the third with 10 drops of 10ul of ''E. coli'', which was grown in LB overnight. Similar to the previous experiment with only milk, the control with water was unchanged and the water diffused into the milk without modifying it. When ''E. coli'' was applied to the milk, there were no significant changes. Similar to the last experiment, milk treated with subtilisin was degraded. This proved that subtilisin was solely responsible for protein degradation. </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[https://2014hs.igem.org/Team:WalthamHS_BioHawks/Project/Results4 View Our Results]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[https://2014hs.igem.org/Team:WalthamHS_BioHawks/Project/Results4 View Our Results]</div></td></tr>
</table>Maddoxmhttp://2014hs.igem.org/wiki/index.php?title=Team:WalthamHS_BioHawks/Project/Procedure&diff=25776&oldid=prevMaddoxm: /* Procedure */2014-06-21T00:40:02Z<p><span class="autocomment">Procedure</span></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>2: Running <del class="diffchange diffchange-inline">The </del>Gel</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>2: Running <ins class="diffchange diffchange-inline">the </ins>Gel</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>After performing the restriction digest for BBa_J04500, the subtilisin gene, the plasmid, and the RFP control, a gel was run to identify the fragment sizes and verify the digest occurred properly. The gel lanes were loaded using the DNA and loading dye, totaling 5 ul. The gel ran for 6 mins under 275v. If the digestion worked correctly, and the gel supplied evidence to support it, then the transformation could continue. However, after running the gel electrophoresis of all digested sequences, no visible results were found. We then repeated the digest and gel and still we were unsuccessful. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>After performing the restriction digest for BBa_J04500, the subtilisin gene, the plasmid, and the RFP control, a gel was run to identify the fragment sizes and verify the digest occurred properly. The gel lanes were loaded using the DNA and loading dye, totaling 5 ul. The gel ran for 6 mins under 275v. If the digestion worked correctly, and the gel supplied evidence to support it, then the transformation could continue. However, after running the gel electrophoresis of all digested sequences, no visible results were found. We then repeated the digest and gel and still we were unsuccessful. </div></td></tr>
</table>Maddoxmhttp://2014hs.igem.org/wiki/index.php?title=Team:WalthamHS_BioHawks/Project/Procedure&diff=25772&oldid=prevMaddoxm: /* Procedure */2014-06-21T00:39:26Z<p><span class="autocomment">Procedure</span></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>1: Initial <del class="diffchange diffchange-inline">subtilisin control test</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>1: Initial <ins class="diffchange diffchange-inline">Subtilisin Control Test</ins></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We added 1ml and 2ml of skim milk to agar plates, and placed 10 drops of 10ul of subtilisin enzyme onto each experimental dish. Subtilisin's enzymatic decomposition was successfully observed. The control plates with only milk were relatively unchanged. This experiment supported the functionality of subtilisin in the decomposition of the proteins. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We added 1ml and 2ml of skim milk to agar plates, and placed 10 drops of 10ul of subtilisin enzyme onto each experimental dish. Subtilisin's enzymatic decomposition was successfully observed. The control plates with only milk were relatively unchanged. This experiment supported the functionality of subtilisin in the decomposition of the proteins. </div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>2: Complete <del class="diffchange diffchange-inline">biological control test</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>2: Complete <ins class="diffchange diffchange-inline">Biological Control Test</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>We then wanted to confirm that subtilisin had protein-degrading capabilities, and not ''E. coli''. We also wanted to eliminate liquid dispersion as a source of error. So, we repeated the experiment with three plates, each with 2ml of skim milk applied on top. The first control plate had 10 drops of 10ul of water, the second with 10 drops of 10ul of subtilisin, and the third with 10 drops of 10ul of ''E. coli'', which was grown in LB overnight. Similar to the previous experiment with only milk, the control with water was unchanged and the water diffused into the milk without modifying it. When ''E. coli'' was applied to the milk, there were no significant changes. Similar to the last experiment, milk treated with subtilisin was degraded. This proved that subtilisin was solely responsible for protein degradation. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We then wanted to confirm that subtilisin had protein-degrading capabilities, and not ''E. coli''. We also wanted to eliminate liquid dispersion as a source of error. So, we repeated the experiment with three plates, each with 2ml of skim milk applied on top. The first control plate had 10 drops of 10ul of water, the second with 10 drops of 10ul of subtilisin, and the third with 10 drops of 10ul of ''E. coli'', which was grown in LB overnight. Similar to the previous experiment with only milk, the control with water was unchanged and the water diffused into the milk without modifying it. When ''E. coli'' was applied to the milk, there were no significant changes. Similar to the last experiment, milk treated with subtilisin was degraded. This proved that subtilisin was solely responsible for protein degradation. </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>[https://2014hs.igem.org/Team:WalthamHS_BioHawks/Project/Results4 View Our Results]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[https://2014hs.igem.org/Team:WalthamHS_BioHawks/Project/Results4 View Our Results]</div></td></tr>
</table>Maddoxmhttp://2014hs.igem.org/wiki/index.php?title=Team:WalthamHS_BioHawks/Project/Procedure&diff=25770&oldid=prevMaddoxm: /* Procedure */2014-06-21T00:38:46Z<p><span class="autocomment">Procedure</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:proc5.jpg|right|wrap|]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:proc5.jpg|right|wrap|]]</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>1: Initial subtilisin control test</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>1: Initial subtilisin control test</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[https://2014hs.igem.org/Team:WalthamHS_BioHawks/Project/Results3 View Our Results]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[https://2014hs.igem.org/Team:WalthamHS_BioHawks/Project/Results3 View Our Results]</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>2: Complete biological control test</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>2: Complete biological control test</div></td></tr>
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</table>Maddoxmhttp://2014hs.igem.org/wiki/index.php?title=Team:WalthamHS_BioHawks/Project/Procedure&diff=25766&oldid=prevMaddoxm: /* Procedure */2014-06-21T00:38:11Z<p><span class="autocomment">Procedure</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>4: Prokaryotic Transformation</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>4: Prokaryotic Transformation</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>The competent E.coli would be incubated on ice for half an hour. The vectors would then be forced in by heating the cells in a water<del class="diffchange diffchange-inline">-</del>bath at 47 degrees Celsius for 1 minute, then put back on ice for 5 minutes.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The competent <ins class="diffchange diffchange-inline">''</ins>E. coli<ins class="diffchange diffchange-inline">'' </ins>would be incubated on ice for half an hour. The vectors would then be forced in by heating the cells in a water bath at 47 degrees Celsius for 1 minute, then put back on ice for 5 minutes.</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 bacteria are then grown at 37 degrees Celsius in an incubator for 24 hours on agar plates. Following initial incubation, they would then undergo another 24<del class="diffchange diffchange-inline">-</del>hour growing period at room temperature in a controlled environment. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The bacteria are then grown at 37 degrees Celsius in an incubator for 24 hours on agar plates. Following initial incubation, they would then undergo another 24 hour growing period at room temperature in a controlled environment. </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>An antibiotic would be applied, and all bacteria not properly transformed would not grow, leaving only the subtilisin-producing colonies. The colonies are then placed in 5ml tubes, where the subtilisin will be secreted into solution and then extracted from the cells.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>An antibiotic would be applied, and all bacteria not properly transformed would not grow, leaving only the subtilisin-producing colonies. The colonies are then placed in 5ml tubes, where the subtilisin will be secreted into solution and then extracted from the cells.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Testing the Stain Remover'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Testing the Stain Remover'''</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>Even though our transformation of E.coli failed, we still wanted to simulate how our project idea would have worked if the experiment was successful. Since our goal was to transform the subtilisin gene into E.coli to produce enzymes that digest proteins, we re-hydrated subtilisin enzyme powder (Sigma Aldrich P5380-25 mg) and tested its effectiveness on milk. We chose skim milk because it had a high concentration of proteins.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Even though our transformation of <ins class="diffchange diffchange-inline">''</ins>E. coli<ins class="diffchange diffchange-inline">'' </ins>failed, we still wanted to simulate how our project idea would have worked if the experiment was successful. Since our goal was to transform the subtilisin gene into <ins class="diffchange diffchange-inline">''</ins>E. coli<ins class="diffchange diffchange-inline">'' </ins>to produce enzymes that digest proteins, we re-hydrated subtilisin enzyme powder (Sigma Aldrich P5380-25 mg) and tested its effectiveness on milk. We chose skim milk because it had a high concentration of proteins.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:proc5.jpg|right|wrap|]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:proc5.jpg|right|wrap|]]</div></td></tr>
</table>Maddoxmhttp://2014hs.igem.org/wiki/index.php?title=Team:WalthamHS_BioHawks/Project/Procedure&diff=25757&oldid=prevMaddoxm: /* Procedure */2014-06-21T00:35:00Z<p><span class="autocomment">Procedure</span></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>3: Ligation <del class="diffchange diffchange-inline">of </del>Plasmid</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>3: Ligation <ins class="diffchange diffchange-inline">into </ins>Plasmid</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>If digestion worked properly, the parts would be combined in solution and ligated. Although some variations of ligation would be arranged incorrectly, there should be sufficient correct vectors that <del class="diffchange diffchange-inline">can </del>could be inserted into ''E. coli''. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>If digestion worked properly, the parts would be combined in solution and ligated. Although some variations of ligation would be arranged incorrectly, there should be sufficient correct vectors that could be inserted into ''E. coli''. </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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</table>Maddoxmhttp://2014hs.igem.org/wiki/index.php?title=Team:WalthamHS_BioHawks/Project/Procedure&diff=25740&oldid=prevMaddoxm: /* Procedure */2014-06-21T00:31:56Z<p><span class="autocomment">Procedure</span></p>
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<td colspan='2' style="background-color: white; color:black;">Revision as of 00:31, 21 June 2014</td>
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<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>As with the 3A assembly practice, there were 4 fundamental steps to the process. The first was to use the restriction enzymes to digest the component parts, in this case BBa_J04500 and the synthesized subtilisin DNA. Our subtilisin gene and terminator were synthesized by BioBasics, and delivered in a plasmid with compatible BioBrick restriction sites. Second, a gel is run prior to ligation to verify the fragment sizes match the calculated predictions.The third was to ligate the two parts into a plasmid. And the fourth was to transform the E.coli with the plasmid. The engineered bacteria can then be incubated, allowing it to secrete subtilisin, which can then be extracted for potential stain removal.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>As with the 3A assembly practice, there were 4 fundamental steps to the process. The first was to use the restriction enzymes to digest the component parts, in this case BBa_J04500 and the synthesized subtilisin DNA. Our subtilisin gene and terminator were synthesized by BioBasics, and delivered in a plasmid with compatible BioBrick restriction sites. Second, a gel is run prior to ligation to verify the fragment sizes match the calculated predictions.The third was to ligate the two parts into a plasmid. And the fourth was to transform the E.coli with the plasmid. The engineered bacteria can then be incubated, allowing it to secrete subtilisin, which can then be extracted for potential stain removal.</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>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:proc3.jpg|left|wrap|]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:proc3.jpg|left|wrap|]]</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>
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<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>If digestion worked properly, the parts would be combined in solution and ligated. Although some variations of ligation would be arranged incorrectly, there should be sufficient correct vectors that can could be inserted into ''E. coli''. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>If digestion worked properly, the parts would be combined in solution and ligated. Although some variations of ligation would be arranged incorrectly, there should be sufficient correct vectors that can could be inserted into ''E. coli''. </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 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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</table>Maddoxmhttp://2014hs.igem.org/wiki/index.php?title=Team:WalthamHS_BioHawks/Project/Procedure&diff=25736&oldid=prevMaddoxm: /* Procedure */2014-06-21T00:31:31Z<p><span class="autocomment">Procedure</span></p>
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<td colspan='2' style="background-color: white; color:black;">Revision as of 00:31, 21 June 2014</td>
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<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>As with the 3A assembly practice, there were 4 fundamental steps to the process. The first was to use the restriction enzymes to digest the component parts, in this case BBa_J04500 and the synthesized subtilisin DNA. Our subtilisin gene and terminator were synthesized by BioBasics, and delivered in a plasmid with compatible BioBrick restriction sites. Second, a gel is run prior to ligation to verify the fragment sizes match the calculated predictions.The third was to ligate the two parts into a plasmid. And the fourth was to transform the E.coli with the plasmid. The engineered bacteria can then be incubated, allowing it to secrete subtilisin, which can then be extracted for potential stain removal.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>As with the 3A assembly practice, there were 4 fundamental steps to the process. The first was to use the restriction enzymes to digest the component parts, in this case BBa_J04500 and the synthesized subtilisin DNA. Our subtilisin gene and terminator were synthesized by BioBasics, and delivered in a plasmid with compatible BioBrick restriction sites. Second, a gel is run prior to ligation to verify the fragment sizes match the calculated predictions.The third was to ligate the two parts into a plasmid. And the fourth was to transform the E.coli with the plasmid. The engineered bacteria can then be incubated, allowing it to secrete subtilisin, which can then be extracted for potential stain removal.</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>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>2: Running The Gel</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>2: Running The Gel</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:proc3.jpg|left|wrap|]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:proc3.jpg|left|wrap|]]</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>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>If digestion worked properly, the parts would be combined in solution and ligated. Although some variations of ligation would be arranged incorrectly, there should be sufficient correct vectors that can could be inserted into ''E. coli''. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>If digestion worked properly, the parts would be combined in solution and ligated. Although some variations of ligation would be arranged incorrectly, there should be sufficient correct vectors that can could be inserted into ''E. coli''. </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>4: Prokaryotic Transformation</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>4: Prokaryotic Transformation</div></td></tr>
</table>Maddoxmhttp://2014hs.igem.org/wiki/index.php?title=Team:WalthamHS_BioHawks/Project/Procedure&diff=25731&oldid=prevMaddoxm: /* Procedure */2014-06-21T00:30:41Z<p><span class="autocomment">Procedure</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[https://2014hs.igem.org/Team:WalthamHS_BioHawks/Project/Results1 View Our Results]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[https://2014hs.igem.org/Team:WalthamHS_BioHawks/Project/Results1 View Our Results]</div></td></tr>
<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>Because the DNA ladder was present and its fragments were correctly distributed, the lack of data from the samples infered a digestion-based error of some kind. Without the vector, the experimental E. coli could not be grown, preventing the biological production of subtilisin. Due to the ultimately insufficient resources of DNA, we were unable to continue our transformation. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Because the DNA ladder was present and its fragments were correctly distributed, the lack of data from the samples infered a digestion-based error of some kind. Without the vector, the experimental <ins class="diffchange diffchange-inline">''</ins>E. coli<ins class="diffchange diffchange-inline">'' </ins>could not be grown, preventing the biological production of subtilisin. Due to the ultimately insufficient resources of DNA, we were unable to continue our transformation. </div></td></tr>
<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: #eee; color:black; font-size: smaller;"><div>We hypothesized that the restriction digest error may have been caused by nonfunctional restriction enzymes. To test this, we digested DNA from the DNA distribution kit with restriction enzymes. Four samples of DNA was incubated each with one type of restiction enzyme. We ran the results on a gel, and the DNA did not appear to be digested. We concluded that the restriction enzymes were nonfunctional. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We hypothesized that the restriction digest error may have been caused by nonfunctional restriction enzymes. To test this, we digested DNA from the DNA distribution kit with restriction enzymes. Four samples of DNA was incubated each with one type of restiction enzyme. We ran the results on a gel, and the DNA did not appear to be digested. We concluded that the restriction enzymes were nonfunctional. </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>3: Ligation of Plasmid</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>3: Ligation of Plasmid</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>If digestion worked properly, the parts would be combined in solution and ligated. Although some variations of ligation would be arranged incorrectly, there should be sufficient correct vectors that can could be inserted into E. coli. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>If digestion worked properly, the parts would be combined in solution and ligated. Although some variations of ligation would be arranged incorrectly, there should be sufficient correct vectors that can could be inserted into <ins class="diffchange diffchange-inline">''</ins>E. coli<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>4: Prokaryotic Transformation</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>4: Prokaryotic Transformation</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>2: Complete biological control test</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>2: Complete biological control test</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>We then wanted to confirm that subtilisin had protein-degrading capabilities, and not E. coli. We also wanted to eliminate liquid dispersion as a source of error. So, we repeated the experiment with three plates, each with 2ml of skim milk applied on top. The first control plate had 10 drops of 10ul of water, the second with 10 drops of 10ul of subtilisin, and the third with 10 drops of 10ul of E. coli, which was grown in LB overnight. Similar to the previous experiment with only milk, the control with water was unchanged and the water diffused into the milk without modifying it. When E. coli was applied to the milk, there were no significant changes. Similar to the last experiment, milk treated with subtilisin was degraded. This proved that subtilisin was solely responsible for protein degradation. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>We then wanted to confirm that subtilisin had protein-degrading capabilities, and not <ins class="diffchange diffchange-inline">''</ins>E. coli<ins class="diffchange diffchange-inline">''</ins>. We also wanted to eliminate liquid dispersion as a source of error. So, we repeated the experiment with three plates, each with 2ml of skim milk applied on top. The first control plate had 10 drops of 10ul of water, the second with 10 drops of 10ul of subtilisin, and the third with 10 drops of 10ul of <ins class="diffchange diffchange-inline">''</ins>E. coli<ins class="diffchange diffchange-inline">''</ins>, which was grown in LB overnight. Similar to the previous experiment with only milk, the control with water was unchanged and the water diffused into the milk without modifying it. When <ins class="diffchange diffchange-inline">''</ins>E. coli<ins class="diffchange diffchange-inline">'' </ins>was applied to the milk, there were no significant changes. Similar to the last experiment, milk treated with subtilisin was degraded. This proved that subtilisin was solely responsible for protein degradation. </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>[https://2014hs.igem.org/Team:WalthamHS_BioHawks/Project/Results4 View Our Results]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[https://2014hs.igem.org/Team:WalthamHS_BioHawks/Project/Results4 View Our Results]</div></td></tr>
</table>Maddoxm