http://2012.igem.org/wiki/index.php?title=Team:TU_Darmstadt/Project/Degradation&feed=atom&action=historyTeam:TU Darmstadt/Project/Degradation - Revision history2024-03-28T12:00:48ZRevision history for this page on the wikiMediaWiki 1.16.0http://2012.igem.org/wiki/index.php?title=Team:TU_Darmstadt/Project/Degradation&diff=235745&oldid=prevArne: /* Degradation */2012-09-27T03:15:35Z<p><span class="autocomment">Degradation</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For further characterisation the enzymes were overexpressed in ''E. coli'' strains [http://ecoliwiki.net/colipedia/index.php/TOP10 Top10], [http://ecoliwiki.net/colipedia/index.php/DH5_alpha DH5&alpha;], [http://ecoliwiki.net/colipedia/index.php/MG1655 Mg1655], screened on tributyrin agar and detected via flow cytometry after performing an antibody staining. The [https://2012.igem.org/Team:TU_Darmstadt/Project/Material_Science material science group] went even further and tried to examine them using AFM.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For further characterisation the enzymes were overexpressed in ''E. coli'' strains [http://ecoliwiki.net/colipedia/index.php/TOP10 Top10], [http://ecoliwiki.net/colipedia/index.php/DH5_alpha DH5&alpha;], [http://ecoliwiki.net/colipedia/index.php/MG1655 Mg1655], screened on tributyrin agar and detected via flow cytometry after performing an antibody staining. The [https://2012.igem.org/Team:TU_Darmstadt/Project/Material_Science material science group] went even further and tried to examine them using AFM.</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>We <del class="diffchange diffchange-inline">are </del>able to control and reproduce expression of <del class="diffchange diffchange-inline">the </del>chimeric protein on the bacterial surface. Furthermore we <del class="diffchange diffchange-inline">achieved </del>the <del class="diffchange diffchange-inline">quantification of </del>activity on selected substrates <del class="diffchange diffchange-inline">and generate reproducible datas</del>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>We <ins class="diffchange diffchange-inline">were </ins>able to control and reproduce <ins class="diffchange diffchange-inline">the </ins>expression of <ins class="diffchange diffchange-inline">our </ins>chimeric protein on the bacterial surface <ins class="diffchange diffchange-inline">in three different ''E.coli'' strains, including the ethylene glycol metabolizing strain [http://ecoliwiki.net/colipedia/index.php/MG1655 Mg1655]</ins>. Furthermore we <ins class="diffchange diffchange-inline">quantified </ins>the <ins class="diffchange diffchange-inline">enzmyatic </ins>activity <ins class="diffchange diffchange-inline">of our membrane bound fusion protein in vivo and the two PET-cleaving enzymes FsC/pNB-Est13 </ins>on selected substrates. </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The corresponding data is available in our [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Degradation labjournal]. If you want to know what happens with the PET after it is degradated to its TPA monomers continue with [https://2012.igem.org/Team:TU_Darmstadt/Project/Transport 2.Transport].</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The corresponding data is available in our [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Degradation labjournal]. If you want to know what happens with the PET after it is degradated to its TPA monomers continue with [https://2012.igem.org/Team:TU_Darmstadt/Project/Transport 2.Transport].</div></td></tr>
</table>Arnehttp://2012.igem.org/wiki/index.php?title=Team:TU_Darmstadt/Project/Degradation&diff=230056&oldid=prevRene: /* Degradation */2012-09-27T01:27:29Z<p><span class="autocomment">Degradation</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For further characterisation the enzymes were overexpressed in ''E. coli'' strains [http://ecoliwiki.net/colipedia/index.php/TOP10 Top10], [http://ecoliwiki.net/colipedia/index.php/DH5_alpha DH5&alpha;], [http://ecoliwiki.net/colipedia/index.php/MG1655 Mg1655], screened on tributyrin agar and detected via flow cytometry after performing an antibody staining. The [https://2012.igem.org/Team:TU_Darmstadt/Project/Material_Science material science group] went even further and tried to examine them using AFM.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For further characterisation the enzymes were overexpressed in ''E. coli'' strains [http://ecoliwiki.net/colipedia/index.php/TOP10 Top10], [http://ecoliwiki.net/colipedia/index.php/DH5_alpha DH5&alpha;], [http://ecoliwiki.net/colipedia/index.php/MG1655 Mg1655], screened on tributyrin agar and detected via flow cytometry after performing an antibody staining. The [https://2012.igem.org/Team:TU_Darmstadt/Project/Material_Science material science group] went even further and tried to examine them using AFM.</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;">We are able to control and reproduce expression of the chimeric protein on the bacterial surface. Furthermore we achieved the quantification of activity on selected substrates and generate reproducible datas.</ins></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The corresponding data is available in our [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Degradation labjournal]. If you want to know what happens with the PET after it is degradated to its TPA monomers continue with [https://2012.igem.org/Team:TU_Darmstadt/Project/Transport 2.Transport].</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The corresponding data is available in our [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Degradation labjournal]. If you want to know what happens with the PET after it is degradated to its TPA monomers continue with [https://2012.igem.org/Team:TU_Darmstadt/Project/Transport 2.Transport].</div></td></tr>
</table>Renehttp://2012.igem.org/wiki/index.php?title=Team:TU_Darmstadt/Project/Degradation&diff=226993&oldid=prevA.Schmidt: /* Degradation */2012-09-27T00:28:16Z<p><span class="autocomment">Degradation</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The signal sequence (PhoA), the catalytic domain (FsC/Est13) and EstA are assembled inline from their respective vectors. This is due to the fact, that by combining multiple parts in the standardized BioBrick vectors, scars with stop codons are generated that would effectively prevent the fusionproteins expression.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The signal sequence (PhoA), the catalytic domain (FsC/Est13) and EstA are assembled inline from their respective vectors. This is due to the fact, that by combining multiple parts in the standardized BioBrick vectors, scars with stop codons are generated that would effectively prevent the fusionproteins expression.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>In consideration of the DNA constructs length we pursued two PCR based synthesis strategies. One being the SKV (standard cloning procedure) the other being the SOE (standard overlap procedure). The first using primers and restriction sites for assembly the latter using overlapping primers. During both assembly procedures restriction sites of PstI, <del class="diffchange diffchange-inline">EcorI</del>, SpeI or XbaI were eliminated from the coding sequence by mutagene PCR. In the end we completely changed our assembly strategy, using synthesis products and BsaI sites to put our parts together.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>In consideration of the DNA constructs length we pursued two PCR based synthesis strategies. One being the SKV (standard cloning procedure) the other being the SOE (standard overlap procedure). The first using primers and restriction sites for assembly the latter using overlapping primers. During both assembly procedures restriction sites of PstI, <ins class="diffchange diffchange-inline">EcoRI</ins>, SpeI or XbaI were eliminated from the coding sequence by mutagene PCR. In the end we completely changed our assembly strategy, using synthesis products and BsaI sites to put our parts together.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>After completion the fusionprotein and its subunits were transfered to the BioBrick standard and sent to the registry. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>After completion the fusionprotein and its subunits were transfered to the BioBrick standard and sent to the registry. </div></td></tr>
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</table>A.Schmidthttp://2012.igem.org/wiki/index.php?title=Team:TU_Darmstadt/Project/Degradation&diff=190377&oldid=prevAdrian E at 09:33, 26 September 20122012-09-26T09:33:42Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href="/Team:TU_Darmstadt/Safety" title="Safety">Safety</a></li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href="/Team:TU_Darmstadt/Safety" title="Safety">Safety</a></li></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href="/Team:TU_Darmstadt/Downloads" title="Downloads">Downloads</a></li></ul></li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href="/Team:TU_Darmstadt/Downloads" title="Downloads">Downloads</a></li></ul></li></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <li><a href="/Team:TU_Darmstadt/Human_Practice" title="Human Practice">Human Practice</a<del class="diffchange diffchange-inline">><ul></del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <li><a href="/Team:TU_Darmstadt/Human_Practice" title="Human Practice">Human Practice</a></li> </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"> <li><a href="/Team:TU_Darmstadt/Human_Practice/Classes" title="Classes">Classes</a></li></ul</del>></li> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href="/Team:TU_Darmstadt/Sponsors" title="Sponsors">Sponsors</a><ul></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href="/Team:TU_Darmstadt/Sponsors" title="Sponsors">Sponsors</a><ul></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href="/Team:TU_Darmstadt/Sponsors" title="Sponsors">Overview</a></li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href="/Team:TU_Darmstadt/Sponsors" title="Sponsors">Overview</a></li></div></td></tr>
</table>Adrian Ehttp://2012.igem.org/wiki/index.php?title=Team:TU_Darmstadt/Project/Degradation&diff=178904&oldid=prevPboba at 19:19, 25 September 20122012-09-25T19:19:29Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Degradation ==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Degradation ==</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>The [https://2012.igem.org/Team:TU_Darmstadt/Team#Degradation degradation group] consists of six undergraduates and two PhD student advisors. Our objective is the expression of a fusion <del class="diffchange diffchange-inline">proteins </del>on the surface of ''E. coli'' to enable a microbial polyethylenterephtalate (PET) degradation. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The [https://2012.igem.org/Team:TU_Darmstadt/Team#Degradation degradation group] consists of six undergraduates and two PhD student advisors. Our objective is the expression of a fusion <ins class="diffchange diffchange-inline">protein </ins>on the surface of ''E. coli'' to enable a microbial polyethylenterephtalate (PET) 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: #ffa; color:black; font-size: smaller;"><div>We identified three potential PET degradation enzymes from literature. Two of them are cutinases HiC (''Humicola insolens'' cutinase) and FsC (''Fusarium solani'' cutinase) the other namely pNB-Est13 beeing an esterase. After <del class="diffchange diffchange-inline">thoroughly </del>examination we dropped the HiC due to a temperature optimum of 80+°C. Shortly after the FsC was dropped as well, due to its toxicity for ''E.coli''.[[File:Project_overview_degradation.png|450px|thumb|right|Enzymatical degradation of polyethylen terephtalate (PET) to terephtalic acid (TPA)]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>We identified three potential PET degradation enzymes from literature. Two of them are cutinases HiC (''Humicola insolens'' cutinase) and FsC (''Fusarium solani'' cutinase)<ins class="diffchange diffchange-inline">, </ins>the other namely pNB-Est13 beeing an esterase. After <ins class="diffchange diffchange-inline">a short </ins>examination we dropped the HiC due to a temperature optimum of 80+°C. Shortly after the FsC was dropped as well, due to its toxicity for ''E.coli''.[[File:Project_overview_degradation.png|450px|thumb|right|Enzymatical degradation of polyethylen terephtalate (PET) to terephtalic acid (TPA)]]</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In order to maximise the activity we decided to anchor and display the cutinase/esterase directly on the surface of the producing bacterial cell. Surface-exposed enzymes are directly accessible to the respective substrates which no longer have to traverse the cellular membrane barriers. Furthermore, the enzyme reaction occurs in a chemically more defined environment as compared to the interior of a microbial cell. We use the outer membrane proteins of ''Pseudomonas aeruginosa'' (EstA) as a membrane anchor and the signaling sequence of PhoA translocators to display the enzyme on the outer surface of ''E. coli'' cells. In addition the fusion protein contains a his-tag and a myc-tag for detection via flow cytometry after antibody staining or Western blot.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In order to maximise the activity we decided to anchor and display the cutinase/esterase directly on the surface of the producing bacterial cell. Surface-exposed enzymes are directly accessible to the respective substrates which no longer have to traverse the cellular membrane barriers. Furthermore, the enzyme reaction occurs in a chemically more defined environment as compared to the interior of a microbial cell. We use the outer membrane proteins of ''Pseudomonas aeruginosa'' (EstA) as a membrane anchor and the signaling sequence of PhoA translocators to display the enzyme on the outer surface of ''E. coli'' cells. In addition the fusion protein contains a his-tag and a myc-tag for detection via flow cytometry after antibody staining or Western blot.</div></td></tr>
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</table>Pbobahttp://2012.igem.org/wiki/index.php?title=Team:TU_Darmstadt/Project/Degradation&diff=157174&oldid=prevAdrian E: /* Degradation */2012-09-24T14:25:15Z<p><span class="autocomment">Degradation</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>After completion the fusionprotein and its subunits were transfered to the BioBrick standard and sent to the registry. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>After completion the fusionprotein and its subunits were transfered to the BioBrick standard and sent to the registry. </div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>For further characterisation the enzymes were overexpressed in ''E. coli'' strains [http://ecoliwiki.net/colipedia/index.php/TOP10 Top10], [http://ecoliwiki.net/colipedia/index.php/DH5_alpha <del class="diffchange diffchange-inline">DH5alpha</del>], [http://ecoliwiki.net/colipedia/index.php/MG1655 Mg1655], screened on tributyrin agar and detected via flow cytometry after performing an antibody staining. The [https://2012.igem.org/Team:TU_Darmstadt/Project/Material_Science material science group] went even further and tried to examine them using AFM.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>For further characterisation the enzymes were overexpressed in ''E. coli'' strains [http://ecoliwiki.net/colipedia/index.php/TOP10 Top10], [http://ecoliwiki.net/colipedia/index.php/DH5_alpha <ins class="diffchange diffchange-inline">DH5&alpha;</ins>], [http://ecoliwiki.net/colipedia/index.php/MG1655 Mg1655], screened on tributyrin agar and detected via flow cytometry after performing an antibody staining. The [https://2012.igem.org/Team:TU_Darmstadt/Project/Material_Science material science group] went even further and tried to examine them using AFM.</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 corresponding data is available in our [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Degradation labjournal]. If you want to know what happens with the PET after it is degradated to its TPA monomers continue with [https://2012.igem.org/Team:TU_Darmstadt/Project/Transport 2.Transport].</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The corresponding data is available in our [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Degradation labjournal]. If you want to know what happens with the PET after it is degradated to its TPA monomers continue with [https://2012.igem.org/Team:TU_Darmstadt/Project/Transport 2.Transport].</div></td></tr>
</table>Adrian Ehttp://2012.igem.org/wiki/index.php?title=Team:TU_Darmstadt/Project/Degradation&diff=154373&oldid=prevArne: /* Degradation */2012-09-24T09:06:16Z<p><span class="autocomment">Degradation</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>After completion the fusionprotein and its subunits were transfered to the BioBrick standard and sent to the registry. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>After completion the fusionprotein and its subunits were transfered to the BioBrick standard and sent to the registry. </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>For further characterisation the enzymes were overexpressed in ''E. coli'' strains [http://ecoliwiki.net/colipedia/index.php/TOP10 Top10] [http://ecoliwiki.net/colipedia/index.php/DH5_alpha DH5alpha] [http://ecoliwiki.net/colipedia/index.php/MG1655 Mg1655], screened on tributyrin agar and detected via flow cytometry after performing an antibody staining. The [https://2012.igem.org/Team:TU_Darmstadt/Project/Material_Science material science group] went even further and tried to examine them using AFM.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>For further characterisation the enzymes were overexpressed in ''E. coli'' strains [http://ecoliwiki.net/colipedia/index.php/TOP10 Top10]<ins class="diffchange diffchange-inline">, </ins>[http://ecoliwiki.net/colipedia/index.php/DH5_alpha DH5alpha]<ins class="diffchange diffchange-inline">, </ins>[http://ecoliwiki.net/colipedia/index.php/MG1655 Mg1655], screened on tributyrin agar and detected via flow cytometry after performing an antibody staining. The [https://2012.igem.org/Team:TU_Darmstadt/Project/Material_Science material science group] went even further and tried to examine them using AFM.</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 corresponding data is available in our [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Degradation labjournal]. If you want to know what happens with the PET after it is degradated to its TPA monomers continue with [https://2012.igem.org/Team:TU_Darmstadt/Project/Transport 2.Transport].</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The corresponding data is available in our [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Degradation labjournal]. If you want to know what happens with the PET after it is degradated to its TPA monomers continue with [https://2012.igem.org/Team:TU_Darmstadt/Project/Transport 2.Transport].</div></td></tr>
</table>Arnehttp://2012.igem.org/wiki/index.php?title=Team:TU_Darmstadt/Project/Degradation&diff=154366&oldid=prevArne: /* Degradation */2012-09-24T09:05:38Z<p><span class="autocomment">Degradation</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>After completion the fusionprotein and its subunits were transfered to the BioBrick standard and sent to the registry. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>After completion the fusionprotein and its subunits were transfered to the BioBrick standard and sent to the registry. </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>For further characterisation the enzymes were overexpressed in ''E. coli'', screened on tributyrin agar and <del class="diffchange diffchange-inline">FACS</del>. The [https://2012.igem.org/Team:TU_Darmstadt/Project/Material_Science material science group] went even further and tried to examine them using AFM.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>For further characterisation the enzymes were overexpressed in ''E. coli'' <ins class="diffchange diffchange-inline">strains [http://ecoliwiki.net/colipedia/index.php/TOP10 Top10] [http://ecoliwiki.net/colipedia/index.php/DH5_alpha DH5alpha] [http://ecoliwiki.net/colipedia/index.php/MG1655 Mg1655]</ins>, screened on tributyrin agar and <ins class="diffchange diffchange-inline">detected via flow cytometry after performing an antibody staining</ins>. The [https://2012.igem.org/Team:TU_Darmstadt/Project/Material_Science material science group] went even further and tried to examine them using AFM.</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 corresponding data is available in our [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Degradation labjournal]. If you want to know what happens with the PET after it is degradated to its TPA monomers continue with [https://2012.igem.org/Team:TU_Darmstadt/Project/Transport 2.Transport].</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The corresponding data is available in our [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Degradation labjournal]. If you want to know what happens with the PET after it is degradated to its TPA monomers continue with [https://2012.igem.org/Team:TU_Darmstadt/Project/Transport 2.Transport].</div></td></tr>
</table>Arnehttp://2012.igem.org/wiki/index.php?title=Team:TU_Darmstadt/Project/Degradation&diff=149807&oldid=prevArne: /* Degradation */2012-09-23T18:40:32Z<p><span class="autocomment">Degradation</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Degradation ==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Degradation ==</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>The [https://2012.igem.org/Team:TU_Darmstadt/Team#Degradation degradation group] consists of six undergraduates and <del class="diffchange diffchange-inline">one </del>PhD <del class="diffchange diffchange-inline">advisor</del>. Our objective is the expression of a fusion proteins on the surface of ''E. coli<del class="diffchange diffchange-inline">'' or as alternative ''S. cerevisiae</del>'' to enable a microbial polyethylenterephtalate (PET) degradation. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The [https://2012.igem.org/Team:TU_Darmstadt/Team#Degradation degradation group] consists of six undergraduates and <ins class="diffchange diffchange-inline">two </ins>PhD <ins class="diffchange diffchange-inline">student advisors</ins>. Our objective is the expression of a fusion proteins on the surface of ''E. coli'' to enable a microbial polyethylenterephtalate (PET) 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: #ffa; color:black; font-size: smaller;"><div>We identified three potential PET degradation enzymes from literature. Two of them are cutinases HiC (''Humicola insolens'' cutinase) and FsC (''Fusarium solani'' cutinase) the other namely pNB-Est13 beeing an esterase. After thoroughly examination we dropped the HiC due to a temperature optimum of 80+°C.[[File:Project_overview_degradation.png|450px|thumb|right|Enzymatical degradation of polyethylen terephtalate (PET) to terephtalic acid (TPA)]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>We identified three potential PET degradation enzymes from literature. Two of them are cutinases HiC (''Humicola insolens'' cutinase) and FsC (''Fusarium solani'' cutinase) the other namely pNB-Est13 beeing an esterase. After thoroughly examination we dropped the HiC due to a temperature optimum of 80+°C<ins class="diffchange diffchange-inline">. Shortly after the FsC was dropped as well, due to its toxicity for ''E.coli''</ins>.[[File:Project_overview_degradation.png|450px|thumb|right|Enzymatical degradation of polyethylen terephtalate (PET) to terephtalic acid (TPA)]]</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>In order to maximise the activity we decided to anchor and display the cutinase/esterase directly on the surface of the producing bacterial cell. Surface-exposed enzymes are directly accessible to the respective substrates which no longer have to traverse the cellular membrane barriers. Furthermore, the enzyme reaction occurs in a chemically more defined environment as compared to the interior of a microbial cell. We use the outer membrane proteins of ''Pseudomonas aeruginosa'' (EstA) as a membrane anchor and the signaling sequence of PhoA translocators to display the enzyme on the outer surface of ''E. coli'' cells. In addition the fusion protein contains a his-tag for <del class="diffchange diffchange-inline">FACS </del>detection <del class="diffchange diffchange-inline">and purification</del>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>In order to maximise the activity we decided to anchor and display the cutinase/esterase directly on the surface of the producing bacterial cell. Surface-exposed enzymes are directly accessible to the respective substrates which no longer have to traverse the cellular membrane barriers. Furthermore, the enzyme reaction occurs in a chemically more defined environment as compared to the interior of a microbial cell. We use the outer membrane proteins of ''Pseudomonas aeruginosa'' (EstA) as a membrane anchor and the signaling sequence of PhoA translocators to display the enzyme on the outer surface of ''E. coli'' cells. In addition the fusion protein contains a his<ins class="diffchange diffchange-inline">-tag and a myc</ins>-tag for <ins class="diffchange diffchange-inline"> </ins>detection <ins class="diffchange diffchange-inline">via flow cytometry after antibody staining or Western blot</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>The signal sequence (PhoA), the catalytic domain (FsC/Est13) and EstA are assembled inline from their respective vectors. This is due to the fact, that by combining multiple parts in the standardized BioBrick vectors, scars with stop codons are generated that would effectively prevent the fusionproteins expression.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The signal sequence (PhoA), the catalytic domain (FsC/Est13) and EstA are assembled inline from their respective vectors. This is due to the fact, that by combining multiple parts in the standardized BioBrick vectors, scars with stop codons are generated that would effectively prevent the fusionproteins expression.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>In consideration of the DNA constructs length we pursued two PCR based synthesis strategies. One being the SKV (standard cloning procedure) the other being the SOE (standard overlap procedure). The first using primers and restriction sites for assembly the latter using overlapping primers. During both assembly procedures restriction sites of PstI, EcorI, SpeI or XbaI were eliminated from the coding sequence by mutagene PCR.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>In consideration of the DNA constructs length we pursued two PCR based synthesis strategies. One being the SKV (standard cloning procedure) the other being the SOE (standard overlap procedure). The first using primers and restriction sites for assembly the latter using overlapping primers. During both assembly procedures restriction sites of PstI, EcorI, SpeI or XbaI were eliminated from the coding sequence by mutagene PCR<ins class="diffchange diffchange-inline">. In the end we completely changed our assembly strategy, using synthesis products and BsaI sites to put our parts together</ins>.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>After completion the <del class="diffchange diffchange-inline">fusionproteins </del>and <del class="diffchange diffchange-inline">their </del>subunits were transfered to the BioBrick standard and sent to the registry. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>After completion the <ins class="diffchange diffchange-inline">fusionprotein </ins>and <ins class="diffchange diffchange-inline">its </ins>subunits were transfered to the BioBrick standard and sent to the registry. </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>For further characterisation the enzymes were overexpressed in ''E. coli'', screened on tributyrin agar and FACS. The [https://2012.igem.org/Team:TU_Darmstadt/Project/Material_Science material science group] went even further and tried to examine them using AFM.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For further characterisation the enzymes were overexpressed in ''E. coli'', screened on tributyrin agar and FACS. The [https://2012.igem.org/Team:TU_Darmstadt/Project/Material_Science material science group] went even further and tried to examine them using AFM.</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 corresponding data is available in our [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Degradation labjournal]. If you want to know what happens with the PET after it is degradated to its TPA monomers continue with [https://2012.igem.org/Team:TU_Darmstadt/Project/Transport 2.Transport].</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The corresponding data is available in our [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Degradation labjournal]. If you want to know what happens with the PET after it is degradated to its TPA monomers continue with [https://2012.igem.org/Team:TU_Darmstadt/Project/Transport 2.Transport].</div></td></tr>
</table>Arnehttp://2012.igem.org/wiki/index.php?title=Team:TU_Darmstadt/Project/Degradation&diff=144196&oldid=prevAdrian E: /* Degradation */2012-09-23T03:00:04Z<p><span class="autocomment">Degradation</span></p>
<table style="background-color: white; color:black;">
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Degradation ==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Degradation ==</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 [https://2012.igem.org/Team:TU_Darmstadt/Team#Degradation degradation group] consists of six undergraduates and one PhD advisor. Our objective is the expression of a fusion proteins on the surface of ''E. coli'' or as alternative ''S. cerevisiae'' to enable a microbial polyethylenterephtalate (PET) degradation. </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: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">Die Gruppe Degradierung des TU Darmstadt iGEM Teams 2012 besteht aus 6 Studenten und 2 Doktoranden</del>. <del class="diffchange diffchange-inline">Unserer Ziel ist es Fusionsproteine auf der Zelloberfläche von E</del>.<del class="diffchange diffchange-inline">Coli und S</del>.<del class="diffchange diffchange-inline">cerevisiae zu bringen um dort das Polymer Polyethylenterephthalat </del>(PET) <del class="diffchange diffchange-inline">zu degradieren und in seine Monomere Terephthalsäure </del>(TPA) <del class="diffchange diffchange-inline">und Ehtylenglyco zul zerlegen</del>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">We identified three potential PET degradation enzymes from literature</ins>. <ins class="diffchange diffchange-inline">Two of them are cutinases HiC (''Humicola insolens'' cutinase) and FsC (''Fusarium solani'' cutinase) the other namely pNB-Est13 beeing an esterase</ins>. <ins class="diffchange diffchange-inline">After thoroughly examination we dropped the HiC due to a temperature optimum of 80+°C</ins>.<ins class="diffchange diffchange-inline">[[File:Project_overview_degradation.png|450px|thumb|right|Enzymatical degradation of polyethylen terephtalate </ins>(PET) <ins class="diffchange diffchange-inline">to terephtalic acid </ins>(TPA)<ins class="diffchange diffchange-inline">]]</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 class="diffchange diffchange-inline">In order to maximise the activity we decided to anchor and display the cutinase/esterase directly on the surface of the producing bacterial cell. Surface-exposed enzymes are directly accessible to the respective substrates which no longer have to traverse the cellular membrane barriers. Furthermore, the enzyme reaction occurs in a chemically more defined environment as compared to the interior of a microbial cell. We use the outer membrane proteins of ''Pseudomonas aeruginosa'' (EstA) as a membrane anchor and the signaling sequence of PhoA translocators to display the enzyme on the outer surface of ''E. coli'' cells. In addition the fusion protein contains a his-tag for FACS detection and purification</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: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">Die Fusionsproteine bestehen aus einer Signalsequenz </del>(PhoA) <del class="diffchange diffchange-inline">zur Sekretion ins Periplasma</del>, <del class="diffchange diffchange-inline">einem His-Tag zur Detektion via FACS</del>, <del class="diffchange diffchange-inline">verschiedenen katalytischen Domänen zur Hydrolyse des PET's und einem C-terminalen Membrananker</del>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">The signal sequence </ins>(PhoA), <ins class="diffchange diffchange-inline">the catalytic domain (FsC/Est13) and EstA are assembled inline from their respective vectors. This is due to the fact, that by combining multiple parts in the standardized BioBrick vectors, scars with stop codons are generated that would effectively prevent the fusionproteins expression.</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 class="diffchange diffchange-inline">In consideration of the DNA constructs length we pursued two PCR based synthesis strategies. One being the SKV (standard cloning procedure) the other being the SOE (standard overlap procedure). The first using primers and restriction sites for assembly the latter using overlapping primers. During both assembly procedures restriction sites of PstI, EcorI</ins>, <ins class="diffchange diffchange-inline">SpeI or XbaI were eliminated from the coding sequence by mutagene PCR.</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 class="diffchange diffchange-inline">After completion the fusionproteins and their subunits were transfered to the BioBrick standard and sent to the registry</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: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">Die Signal Sequenz (PhoA Signal Peptid)</del>, <del class="diffchange diffchange-inline">die katalytische Domänen und der Membrananker werden aus verschiedenen Spender Vektoren entnommen, da das Standard BioBrick System des iGEM-Wettbewerbs nach jeder Insertion Stoppcodons vermittelt und somit die Synthese von Fusionsproteinen nicht erlaubt</del>. <del class="diffchange diffchange-inline">Danach werden störende Schnittstellen (PstI, EcorI, SpeI oder XbaI) in der codierenden Sequenz entfernt</del>. <del class="diffchange diffchange-inline">Wir bedienen uns daher beim Zusammenfügen der einzelnen Funktionselementen der herkömmlichen Klonierungsverfahren</del>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">For further characterisation the enzymes were overexpressed in ''E. coli''</ins>, <ins class="diffchange diffchange-inline">screened on tributyrin agar and FACS</ins>. <ins class="diffchange diffchange-inline">The [https://2012.igem</ins>.<ins class="diffchange diffchange-inline">org/Team:TU_Darmstadt/Project/Material_Science material science group] went even further and tried to examine them using AFM</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: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">Nachdem die Fusionsproteine erstellt und </del>in <del class="diffchange diffchange-inline">das BioBrick System überführt wurden und anschließend in E</del>.<del class="diffchange diffchange-inline">coli Produktionsstämmen inseriert um hohe Mengen an Protein für die Charakterisierung zu erzeugen</del>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">The corresponding data is available </ins>in <ins class="diffchange diffchange-inline">our [https://2012</ins>.<ins class="diffchange diffchange-inline">igem</ins>.<ins class="diffchange diffchange-inline">org/Team</ins>:<ins class="diffchange diffchange-inline">TU_Darmstadt/Labjournal/Degradation labjournal]</ins>. <ins class="diffchange diffchange-inline">If you want to know what happens with the </ins>PET <ins class="diffchange diffchange-inline">after it is degradated to its TPA monomers </ins>continue with [https://2012.igem.org/Team:TU_Darmstadt/Project/Transport 2.Transport]<ins class="diffchange diffchange-inline">.</ins></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">Die Aktivität der auf der Zelloberfläche präsentierten Fusionsproteine wird auf Tributyrin-Agarplatten untersucht. Danach in einen Chassis Stamm eingeführt, der alle Funktionen aus den Arbeitsgurppen Degradierung, Transport und Metabolismus beinhaltet.</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">Die Aktivität unserer Fusionsproteine wird erst qualitativ über die Ausplattierug auf Tributyrin-Agarplatten untersucht. Parallel dazu wird durch einen fluoreszenzmarkierten Antikörper eine quantitative Aussage über die Zelloberflächenexpression via FACS gemacht.</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">Die Charackterisiertung der Enzymaktivität (Quantitative Parameter</del>: <del class="diffchange diffchange-inline">Vmax, Km etc</del>.<del class="diffchange diffchange-inline">) werden photometrisch über ein pNP-Assay mit dem Standardsubstrat (4-nitrophenyl Butyrat) und 2 weiteren von der Metarailwissenschafs Gruppe synthetisierten Substraten gemacht. Die Hydrolyse auf PET-Monomeren wird mittels Säure-Base Titration und HPLC gemessen. Im Anschluss dieser Untersuchungen wird die </del>PET<del class="diffchange diffchange-inline">-spezifische Hydrolyseaktivität auf nativen PET-Mikropartikel mit verschiedenen Körnungen analysiert.</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>continue with [https://2012.igem.org/Team:TU_Darmstadt/Project/Transport 2.Transport]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
</table>Adrian E