http://2014.igem.org/wiki/index.php?title=Team:Heidelberg/pages/Toolbox&feed=atom&action=historyTeam:Heidelberg/pages/Toolbox - Revision history2024-03-29T14:58:10ZRevision history for this page on the wikiMediaWiki 1.16.5http://2014.igem.org/wiki/index.php?title=Team:Heidelberg/pages/Toolbox&diff=400919&oldid=prevMaxW: /* Circularization */2014-10-18T03:53:29Z<p><span class="autocomment">Circularization</span></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Enzymes represent a major tool for many branches of the chemical industry, including food, brewing, paper, detergent or biofuel. Millions of years of evolution have allowed these proteins to performed extremely specific chemical modifications that are not only essential for living organisms but can also be of great benefit to produce useful molecules for our life, efficiently and at low cost. A major limitation of the use of enzyme for industrial application and in general out of their natural environment is their stability. They can be destroyed by other enzymes and they can unfold and take non-functional conformation when exposed to non-physiological temperature and pH. Such limitations has motivated research in species that can grow at extreme temperatures <del class="diffchange diffchange-inline">[[#References|[1]]]</del>. Another major area of chemical research is the design of strategies to stabilize enzymes, and more generally proteins and peptides <del class="diffchange diffchange-inline">[[#References|[2][3]]]</del>. Protein circularization, meaning ligation of the N- and C-terminal ends of a protein, represents a promising way to achieve this stabilization. While conserving the functionality of their linear counterpart, circular proteins can be superior in terms of thermostability <del class="diffchange diffchange-inline">[[#References|[4][5][6]]]</del>, resistance against chemical denaturation <del class="diffchange diffchange-inline">[[#References|[7]]] </del>and protection from exopeptidases <del class="diffchange diffchange-inline">[[#References|[5][7]]]</del>. Moreover, a circular backbone can improve ''in vivo'' stability of therapeutical proteins and peptides <del class="diffchange diffchange-inline">[[#References|[8]]]</del>. All these remarkable properties motivated us to develop new tools to circularize any protein of interest. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Enzymes represent a major tool for many branches of the chemical industry, including food, brewing, paper, detergent or biofuel. Millions of years of evolution have allowed these proteins to performed extremely specific chemical modifications that are not only essential for living organisms but can also be of great benefit to produce useful molecules for our life, efficiently and at low cost. A major limitation of the use of enzyme for industrial application and in general out of their natural environment is their stability. They can be destroyed by other enzymes and they can unfold and take non-functional conformation when exposed to non-physiological temperature and pH. Such limitations has motivated research in species that can grow at extreme temperatures . Another major area of chemical research is the design of strategies to stabilize enzymes, and more generally proteins and peptides. Protein circularization, meaning ligation of the N- and C-terminal ends of a protein, represents a promising way to achieve this stabilization. While conserving the functionality of their linear counterpart, circular proteins can be superior in terms of thermostability, resistance against chemical denaturation and protection from exopeptidases . Moreover, a circular backbone can improve ''in vivo'' stability of therapeutical proteins and peptides. All these remarkable properties motivated us to develop new tools to circularize any protein of interest. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Our [[Team:Heidelberg/Toolbox_Guide | Toolbox Guide]] provides a step-by-step strategy to clone a circularization linker and express it in ''E. coli''. Moreover, in case of complex structures where the protein extremities are far from each other, we have developed the software tool [[Team:Heidelberg/Software/Linker_Software | CRAUT]] that will design the appropriate rigid linkers.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Our [[Team:Heidelberg/Toolbox_Guide | Toolbox Guide]] provides a step-by-step strategy to clone a circularization linker and express it in ''E. coli''. Moreover, in case of complex structures where the protein extremities are far from each other, we have developed the software tool [[Team:Heidelberg/Software/Linker_Software | CRAUT]] that will design the appropriate rigid linkers.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Circularization Constructs==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Circularization Constructs==</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>The most promising approaches to [[Team:Heidelberg/Toolbox/Circularization | circularize]] proteins are protein trans-splicing using [[Team:Heidelberg/Project/Background | split inteins<del class="diffchange diffchange-inline">]] [[#References|[1]</del>]] and Sortase A-catalyzed cyclization <del class="diffchange diffchange-inline">[[#References|[2]]]</del>. Both methods require the addition of specific proteins domains or peptides to the protein to be circularized. Consequently, on DNA level, creating circular proteins is equivalent to creating fusion proteins. However, existing protein fusion standards like [http://parts.igem.org/Help:Standards/Assembly/RFC23 RFC[23]] cause scars. Those scars on protein level may affect protein function and further complicate 3D-structure modeling. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The most promising approaches to [[Team:Heidelberg/Toolbox/Circularization | circularize]] proteins are protein trans-splicing using [[Team:Heidelberg/Project/Background | split inteins]] and Sortase A-catalyzed cyclization. Both methods require the addition of specific proteins domains or peptides to the protein to be circularized. Consequently, on DNA level, creating circular proteins is equivalent to creating fusion proteins. However, existing protein fusion standards like [http://parts.igem.org/Help:Standards/Assembly/RFC23 RFC[23]] cause scars. Those scars on protein level may affect protein function and further complicate 3D-structure modeling. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Therefore, we decided to create the new RFC[i] that allows scarless cloning of inteins. Our intein circularization constructs apply to this standard, while our sortase constructs are closely related and can be used similarly. Detailed instructions on how to use our constructs are provided in our [[Team:Heidelberg/Toolbox_Guide | Toolbox Guide]]. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Therefore, we decided to create the new RFC[i] that allows scarless cloning of inteins. Our intein circularization constructs apply to this standard, while our sortase constructs are closely related and can be used similarly. Detailed instructions on how to use our constructs are provided in our [[Team:Heidelberg/Toolbox_Guide | Toolbox Guide]]. </div></td></tr>
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</table>MaxWhttp://2014.igem.org/wiki/index.php?title=Team:Heidelberg/pages/Toolbox&diff=400731&oldid=prevMaxW: /* Purification */2014-10-18T03:52:12Z<p><span class="autocomment">Purification</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Therapeutic proteins have changed modern medicine. For instance, the monoclonal antibody Trastuzumab, is used to treat breast cancers that are positive for the epidermal growth factor HER2/neu (Ref. Clinical outcome in women with HER2-positive de novo or recurring stage IV breast cancer receiving trastuzumab-based therapy). </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Therapeutic proteins have changed modern medicine. For instance, the monoclonal antibody Trastuzumab, is used to treat breast cancers that are positive for the epidermal growth factor HER2/neu (Ref. Clinical outcome in women with HER2-positive de novo or recurring stage IV breast cancer receiving trastuzumab-based therapy). </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We standardized this split-intein purification system, so it is easy applicable to all kinds of proteins. Visit our [https://2014.igem.org/Team:Heidelberg/Toolbox_Guide toolbox guide] to purify your protein!</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We standardized this split-intein purification system, so it is easy applicable to all kinds of proteins. Visit our [https://2014.igem.org/Team:Heidelberg/Toolbox_Guide toolbox guide] to purify your protein!</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>====Introduction====</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>====Introduction====</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In an extensive assay we proved the principle behind split protein assembly by showing that GFP can be artificially split into two halves and thereafter be reassembled so the fluorescence is restored. Visit the [https://2014.igem.org/Team:Heidelberg/Project/Reconstitution split Fluorescent Protein].</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In an extensive assay we proved the principle behind split protein assembly by showing that GFP can be artificially split into two halves and thereafter be reassembled so the fluorescence is restored. Visit the [https://2014.igem.org/Team:Heidelberg/Project/Reconstitution split Fluorescent Protein].</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>file=BBa_K1362000.png}}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>file=BBa_K1362000.png}}</div></td></tr>
</table>MaxWhttp://2014.igem.org/wiki/index.php?title=Team:Heidelberg/pages/Toolbox&diff=400088&oldid=prevMaxW: /* Split Intein Circularization */2014-10-18T03:47:25Z<p><span class="autocomment">Split Intein Circularization</span></p>
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</table>MaxWhttp://2014.igem.org/wiki/index.php?title=Team:Heidelberg/pages/Toolbox&diff=400001&oldid=prevMaxW: /* Split Intein Circularization */2014-10-18T03:46:51Z<p><span class="autocomment">Split Intein Circularization</span></p>
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