http://2014.igem.org/wiki/index.php?title=Team:USyd-Australia/Project/Design&feed=atom&action=historyTeam:USyd-Australia/Project/Design - Revision history2024-03-29T07:18:44ZRevision history for this page on the wikiMediaWiki 1.16.5http://2014.igem.org/wiki/index.php?title=Team:USyd-Australia/Project/Design&diff=367961&oldid=prevAsis8454 at 23:26, 17 October 20142014-10-17T23:26:54Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br> <p>As gene cassettes are non-replicative it is important that they are incorporated into an AttI site before the cell divides and the cassettes face the possibility of ejection. Therefore it is vital to have a controllable Integrase gene which can be turned on prior to the addition of gene cassettes to ensure that the cassettes are integrated before they are lost. Part <a href="https://2014.igem.org/Team:USyd-Australia/pUS203">pUS203</a> is our submission BioBrick which encodes the controllable Integrase system. We are using the araC-pBAD system from <a href="http://parts.igem.org/Part:BBa_K731201">K731201</a> which allows us to turn on transcription of the Integrase under the activation of arabinose.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br> <p>As gene cassettes are non-replicative it is important that they are incorporated into an AttI site before the cell divides and the cassettes face the possibility of ejection. Therefore it is vital to have a controllable Integrase gene which can be turned on prior to the addition of gene cassettes to ensure that the cassettes are integrated before they are lost. Part <a href="https://2014.igem.org/Team:USyd-Australia/pUS203">pUS203</a> is our submission BioBrick which encodes the controllable Integrase system. We are using the araC-pBAD system from <a href="http://parts.igem.org/Part:BBa_K731201">K731201</a> which allows us to turn on transcription of the Integrase under the activation of arabinose.</p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p> By controlling integrase expression with arabinose, we are also able to turn off integrase, which would stop inappropriate excision of cassettes from the array from occurring. </p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p> By controlling integrase expression with arabinose, we are also able to turn off integrase, which would stop inappropriate excision of cassettes from the array from occurring <ins class="diffchange diffchange-inline">after the array has been assembled</ins>. </p></div></td></tr>
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</table>Asis8454http://2014.igem.org/wiki/index.php?title=Team:USyd-Australia/Project/Design&diff=367845&oldid=prevAsis8454 at 23:25, 17 October 20142014-10-17T23:25:44Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Step 4: Express the array</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Step 4: Express the array</h3></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><br> <p>Once the cassette is integrated, we would induce expression of the genes using an inducible promoter which is placed upstream to the AttI site and cassette array. A single promoter can read through multiple cassettes if a terminator is not present, allowing multiple genes to be expressed <del class="diffchange diffchange-inline">from a single signal</del></p><br><h2> </h2></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><br> <p>Once the cassette is integrated, we would induce expression of the genes using an inducible promoter which is placed upstream to the AttI site and cassette array. A single promoter can read through multiple cassettes if a terminator is not present, allowing multiple genes to be expressed <ins class="diffchange diffchange-inline">in one go.</ins></p<ins class="diffchange diffchange-inline">><br><br><br</ins>><br><h2> </h2></div></td></tr>
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</table>Asis8454http://2014.igem.org/wiki/index.php?title=Team:USyd-Australia/Project/Design&diff=367708&oldid=prevAsis8454 at 23:24, 17 October 20142014-10-17T23:24:33Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Step 1: Produce Cassettes</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Step 1: Produce Cassettes</h3></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><img src="https://static.igem.org/mediawiki/2014/c/c4/USyd-Australia_Integron_Design-Produce_Cassettes.png" width="340px" align="right" hspace="10px"><br> <p>Gene Cassettes are the carriers of genetic information in our design. They are small, modular chunks of DNA containing a gene flanked by AttC sites. <del class="diffchange diffchange-inline">Cassettes</del>, unlike plasmids, are non-replicative and so do not require extra genes to modulate or initiate replication. To create a cassette simply requires the use of primers to create half AttC sites at either end and circularizing the product by using <a href="https://2014.igem.org/Team:USyd-Australia/Project/Protocols#ELAN">Enzymatic Ligation Assisted by Nucleases (ELAN)</a>. This is a reaction in which ligation and enzymatic digestion of compatible but non-identical ends, </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><img src="https://static.igem.org/mediawiki/2014/c/c4/USyd-Australia_Integron_Design-Produce_Cassettes.png" width="340px" align="right" hspace="10px"><br> <p>Gene Cassettes are the carriers of genetic information in our design. They are small, modular chunks of DNA containing a gene flanked by AttC sites. <ins class="diffchange diffchange-inline">In their mobile circular form, cassettes</ins>, unlike plasmids, are non-replicative and so do not require extra genes to modulate or initiate replication. To create a cassette simply requires the use of primers to create half AttC sites at either end and circularizing the product by using <a href="https://2014.igem.org/Team:USyd-Australia/Project/Protocols#ELAN">Enzymatic Ligation Assisted by Nucleases (ELAN)</a>. This is a reaction in which ligation and enzymatic digestion of compatible but non-identical ends, </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>like XbaI and SpeI, occurs simultaneously. This should result only in stable single-cassette circles or concatemers of several cassettes. For systems that already contain an intact AttC site, XbaI and SpeI sites can be introduced by PCR for ELAN, or overlapping ends can be introduced by PCR for <a href="https://2014.igem.org/Team:USyd-Australia/Project/Protocols#gibson">Gibson Assembly</a>.</p><br><h2> </h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>like XbaI and SpeI, occurs simultaneously. This should result only in stable single-cassette circles or concatemers of several cassettes. For systems that already contain an intact AttC site, XbaI and SpeI sites can be introduced by PCR for ELAN, or overlapping ends can be introduced by PCR for <a href="https://2014.igem.org/Team:USyd-Australia/Project/Protocols#gibson">Gibson Assembly</a>.</p><br><h2> </h2></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Step 3: Add Cassettes</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Step 3: Add Cassettes</h3></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><img src="https://static.igem.org/mediawiki/2014/5/55/USyd-Australia_Integron_design-add_cassettes.png" width="300px" align="right" hspace="10px"></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><img src="https://static.igem.org/mediawiki/2014/5/55/USyd-Australia_Integron_design-add_cassettes.png" width="300px" align="right" hspace="10px"></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><br> <p>As gene cassettes enter the cell site the integrase acts to cause specific recombination between the AttC site on the cassette and an AttI site which may be present on the plasmid or bacterial chromosome. Integrase facilitates the formation of a holiday junction between the AttI and AttC regions at the recombination site, resulting in insertion of the cassette into the middle of the AttI site. </p<del class="diffchange diffchange-inline">><br><br><br><br</del>><br><br><h2> </h2></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><br> <p>As gene cassettes enter the cell site the integrase acts to cause specific recombination between the AttC site on the cassette and an AttI site which may be present on the plasmid or bacterial chromosome. Integrase facilitates the formation of a holiday junction between the AttI and AttC regions at the recombination site, resulting in insertion of the cassette into the middle of the AttI site. </p><br><br><h2> </h2></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><img src="https://static.igem.org/mediawiki/2014/3/34/USyd-Australia_Integron_design-express_the_array.png" width="<del class="diffchange diffchange-inline">60%</del>"></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><h3>Step 4: Express the array</h3></ins></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><br> <p><del class="diffchange diffchange-inline">Now that </del>the <del class="diffchange diffchange-inline">required </del>cassette is <del class="diffchange diffchange-inline">present in the array </del>we <del class="diffchange diffchange-inline">can </del>induce expression of the genes <del class="diffchange diffchange-inline">from </del>an <del class="diffchange diffchange-inline">upstream </del>promoter to the cassette array. A single promoter can read through multiple cassettes if a terminator is not present, allowing multiple genes to be expressed from a single signal</p><br><h2> </h2></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><img src="https://static.igem.org/mediawiki/2014/3/34/USyd-Australia_Integron_design-express_the_array.png" width="<ins class="diffchange diffchange-inline">300px" align="left" hspace="10px</ins>"></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><br> <p><ins class="diffchange diffchange-inline">Once </ins>the cassette is <ins class="diffchange diffchange-inline">integrated, </ins>we <ins class="diffchange diffchange-inline">would </ins>induce expression of the genes <ins class="diffchange diffchange-inline">using </ins>an <ins class="diffchange diffchange-inline">inducible </ins>promoter <ins class="diffchange diffchange-inline">which is placed upstream </ins>to the <ins class="diffchange diffchange-inline">AttI site and </ins>cassette array. A single promoter can read through multiple cassettes if a terminator is not present, allowing multiple genes to be expressed from a single signal</p><br><h2> </h2></div></td></tr>
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</table>Asis8454http://2014.igem.org/wiki/index.php?title=Team:USyd-Australia/Project/Design&diff=366909&oldid=prevAsis8454 at 23:17, 17 October 20142014-10-17T23:17:32Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Step 1: Produce Cassettes</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Step 1: Produce Cassettes</h3></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><img src="https://static.igem.org/mediawiki/2014/c/c4/USyd-Australia_Integron_Design-Produce_Cassettes.png" width="340px" align="right"><br> <p>Gene Cassettes are the carriers of genetic information in our design. They are small, modular chunks of DNA containing a gene flanked by AttC sites. Cassettes, unlike plasmids, are non-replicative and so do not require extra genes to modulate or initiate replication. To create a cassette simply requires the use of primers to create half AttC sites at either end and circularizing the product by using <a href="https://2014.igem.org/Team:USyd-Australia/Project/Protocols#ELAN">Enzymatic Ligation Assisted by Nucleases (ELAN)</a>. This is a reaction in which ligation and enzymatic digestion of compatible but non-identical ends, </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><img src="https://static.igem.org/mediawiki/2014/c/c4/USyd-Australia_Integron_Design-Produce_Cassettes.png" width="340px" align="right<ins class="diffchange diffchange-inline">" hspace="10px</ins>"><br> <p>Gene Cassettes are the carriers of genetic information in our design. They are small, modular chunks of DNA containing a gene flanked by AttC sites. Cassettes, unlike plasmids, are non-replicative and so do not require extra genes to modulate or initiate replication. To create a cassette simply requires the use of primers to create half AttC sites at either end and circularizing the product by using <a href="https://2014.igem.org/Team:USyd-Australia/Project/Protocols#ELAN">Enzymatic Ligation Assisted by Nucleases (ELAN)</a>. This is a reaction in which ligation and enzymatic digestion of compatible but non-identical ends, </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>like XbaI and SpeI, occurs simultaneously. This should result only in stable single-cassette circles or concatemers of several cassettes. For systems that already contain an intact AttC site, XbaI and SpeI sites can be introduced by PCR for ELAN, or overlapping ends can be introduced by PCR for <a href="https://2014.igem.org/Team:USyd-Australia/Project/Protocols#gibson">Gibson Assembly</a>.</p><br><h2> </h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>like XbaI and SpeI, occurs simultaneously. This should result only in stable single-cassette circles or concatemers of several cassettes. For systems that already contain an intact AttC site, XbaI and SpeI sites can be introduced by PCR for ELAN, or overlapping ends can be introduced by PCR for <a href="https://2014.igem.org/Team:USyd-Australia/Project/Protocols#gibson">Gibson Assembly</a>.</p><br><h2> </h2></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Step2: Express Integrase</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Step2: Express Integrase</h3></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><img src="https://static.igem.org/mediawiki/2014/1/1f/USyd-Australia_Integron_Design-Express_Integrase.png" width="315px" align="left"></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><img src="https://static.igem.org/mediawiki/2014/1/1f/USyd-Australia_Integron_Design-Express_Integrase.png" width="315px" align="left<ins class="diffchange diffchange-inline">" hspace="10px</ins>"></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br> <p>As gene cassettes are non-replicative it is important that they are incorporated into an AttI site before the cell divides and the cassettes face the possibility of ejection. Therefore it is vital to have a controllable Integrase gene which can be turned on prior to the addition of gene cassettes to ensure that the cassettes are integrated before they are lost. Part <a href="https://2014.igem.org/Team:USyd-Australia/pUS203">pUS203</a> is our submission BioBrick which encodes the controllable Integrase system. We are using the araC-pBAD system from <a href="http://parts.igem.org/Part:BBa_K731201">K731201</a> which allows us to turn on transcription of the Integrase under the activation of arabinose.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br> <p>As gene cassettes are non-replicative it is important that they are incorporated into an AttI site before the cell divides and the cassettes face the possibility of ejection. Therefore it is vital to have a controllable Integrase gene which can be turned on prior to the addition of gene cassettes to ensure that the cassettes are integrated before they are lost. Part <a href="https://2014.igem.org/Team:USyd-Australia/pUS203">pUS203</a> is our submission BioBrick which encodes the controllable Integrase system. We are using the araC-pBAD system from <a href="http://parts.igem.org/Part:BBa_K731201">K731201</a> which allows us to turn on transcription of the Integrase under the activation of arabinose.</p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p> By controlling integrase expression with arabinose, we are also able to turn off integrase, which would stop inappropriate excision of cassettes from the array from occurring. </p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p> By controlling integrase expression with arabinose, we are also able to turn off integrase, which would stop inappropriate excision of cassettes from the array from occurring. </p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><h2> </h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><h2> </h2></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><img src="https://static.igem.org/mediawiki/2014/5/55/USyd-Australia_Integron_design-add_cassettes.png" width="<del class="diffchange diffchange-inline">60%</del>"></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><h3>Step 3: Add Cassettes</h3></ins></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><br> <p>As gene cassettes enter the cell site the integrase acts to cause specific recombination between the AttC site on the cassette and an AttI site which may be present on <del class="diffchange diffchange-inline">a </del>plasmid or <del class="diffchange diffchange-inline">the </del>bacterial chromosome. <del class="diffchange diffchange-inline">The cassette forms </del>a holiday junction at the recombination site. </p><br><h2> </h2></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><img src="https://static.igem.org/mediawiki/2014/5/55/USyd-Australia_Integron_design-add_cassettes.png" width="<ins class="diffchange diffchange-inline">300px" align="right" hspace="10px</ins>"></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><br> <p>As gene cassettes enter the cell site the integrase acts to cause specific recombination between the AttC site on the cassette and an AttI site which may be present on <ins class="diffchange diffchange-inline">the </ins>plasmid or bacterial chromosome. <ins class="diffchange diffchange-inline">Integrase facilitates the formation of </ins>a holiday junction <ins class="diffchange diffchange-inline">between the AttI and AttC regions </ins>at the recombination <ins class="diffchange diffchange-inline">site, resulting in insertion of the cassette into the middle of the AttI </ins>site. </p<ins class="diffchange diffchange-inline">><br><br><br><br><br</ins>><br><h2> </h2></div></td></tr>
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</table>Asis8454http://2014.igem.org/wiki/index.php?title=Team:USyd-Australia/Project/Design&diff=365353&oldid=prevAsis8454 at 23:03, 17 October 20142014-10-17T23:03:01Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Step2: Express Integrase</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Step2: Express Integrase</h3></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><img src="https://static.igem.org/mediawiki/2014/1/1f/USyd-Australia_Integron_Design-Express_Integrase.png" width="<del class="diffchange diffchange-inline">340px</del>" align="left"></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><img src="https://static.igem.org/mediawiki/2014/1/1f/USyd-Australia_Integron_Design-Express_Integrase.png" width="<ins class="diffchange diffchange-inline">315px</ins>" align="left"></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br> <p>As gene cassettes are non-replicative it is important that they are incorporated into an AttI site before the cell divides and the cassettes face the possibility of ejection. Therefore it is vital to have a controllable Integrase gene which can be turned on prior to the addition of gene cassettes to ensure that the cassettes are integrated before they are lost. Part <a href="https://2014.igem.org/Team:USyd-Australia/pUS203">pUS203</a> is our submission BioBrick which encodes the controllable Integrase system. We are using the araC-pBAD system from <a href="http://parts.igem.org/Part:BBa_K731201">K731201</a> which allows us to turn on transcription of the Integrase under the activation of arabinose.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br> <p>As gene cassettes are non-replicative it is important that they are incorporated into an AttI site before the cell divides and the cassettes face the possibility of ejection. Therefore it is vital to have a controllable Integrase gene which can be turned on prior to the addition of gene cassettes to ensure that the cassettes are integrated before they are lost. Part <a href="https://2014.igem.org/Team:USyd-Australia/pUS203">pUS203</a> is our submission BioBrick which encodes the controllable Integrase system. We are using the araC-pBAD system from <a href="http://parts.igem.org/Part:BBa_K731201">K731201</a> which allows us to turn on transcription of the Integrase under the activation of arabinose.</p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p> By controlling integrase expression with arabinose, we are also able to turn off integrase, which would stop excision of cassettes from the array from occurring. </p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p> By controlling integrase expression with arabinose, we are also able to turn off integrase, which would stop <ins class="diffchange diffchange-inline">inappropriate </ins>excision of cassettes from the array from occurring. </p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><h2> </h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><h2> </h2></div></td></tr>
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</table>Asis8454http://2014.igem.org/wiki/index.php?title=Team:USyd-Australia/Project/Design&diff=365238&oldid=prevAsis8454 at 23:01, 17 October 20142014-10-17T23:01:57Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>like XbaI and SpeI, occurs simultaneously. This should result only in stable single-cassette circles or concatemers of several cassettes. For systems that already contain an intact AttC site, XbaI and SpeI sites can be introduced by PCR for ELAN, or overlapping ends can be introduced by PCR for <a href="https://2014.igem.org/Team:USyd-Australia/Project/Protocols#gibson">Gibson Assembly</a>.</p><br><h2> </h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>like XbaI and SpeI, occurs simultaneously. This should result only in stable single-cassette circles or concatemers of several cassettes. For systems that already contain an intact AttC site, XbaI and SpeI sites can be introduced by PCR for ELAN, or overlapping ends can be introduced by PCR for <a href="https://2014.igem.org/Team:USyd-Australia/Project/Protocols#gibson">Gibson Assembly</a>.</p><br><h2> </h2></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><img src="https://static.igem.org/mediawiki/2014/1/1f/USyd-Australia_Integron_Design-Express_Integrase.png" width="<del class="diffchange diffchange-inline">60%</del>"></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><h3>Step2: Express Integrase</h3></ins></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><br> <p>As gene cassettes are non-replicative it is important that they are incorporated into an AttI site before the cell divides and the cassettes face the possibility of ejection. Therefore it is vital to have a controllable Integrase gene which can be turned on prior to the addition of gene cassettes to ensure that the cassettes are integrated before they are lost. Part <a href="https://2014.igem.org/Team:USyd-Australia/pUS203">pUS203</a> is our submission BioBrick which encodes the controllable Integrase system. We are using the araC-pBAD system from <a href="http://parts.igem.org/Part:BBa_K731201">K731201</a> which allows us to turn on transcription of the Integrase under the activation of arabinose.</p><br><h2> </h2></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><img src="https://static.igem.org/mediawiki/2014/1/1f/USyd-Australia_Integron_Design-Express_Integrase.png" width="<ins class="diffchange diffchange-inline">340px" align="left</ins>"></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><br> <p>As gene cassettes are non-replicative it is important that they are incorporated into an AttI site before the cell divides and the cassettes face the possibility of ejection. Therefore it is vital to have a controllable Integrase gene which can be turned on prior to the addition of gene cassettes to ensure that the cassettes are integrated before they are lost. Part <a href="https://2014.igem.org/Team:USyd-Australia/pUS203">pUS203</a> is our submission BioBrick which encodes the controllable Integrase system. We are using the araC-pBAD system from <a href="http://parts.igem.org/Part:BBa_K731201">K731201</a> which allows us to turn on transcription of the Integrase under the activation of arabinose.</p></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"><p> By controlling integrase expression with arabinose, we are also able to turn off integrase, which would stop excision of cassettes from the array from occurring. </p></ins></div></td></tr>
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</table>Asis8454http://2014.igem.org/wiki/index.php?title=Team:USyd-Australia/Project/Design&diff=362169&oldid=prevAsis8454 at 22:30, 17 October 20142014-10-17T22:30:17Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2>Project Design</h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2>Project Design</h2></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> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><h3>Step 1: Produce Cassettes</h3></ins></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><img src="https://static.igem.org/mediawiki/2014/c/c4/USyd-Australia_Integron_Design-Produce_Cassettes.png" width="<del class="diffchange diffchange-inline">60%</del>" align="<del class="diffchange diffchange-inline">center</del>"><br> <p>Gene Cassettes are the carriers of genetic information in our design. They are small, modular chunks of DNA containing a gene flanked by AttC sites. Cassettes, unlike plasmids, are non-replicative and so do not require extra genes to modulate or initiate replication. To create a cassette simply requires the use of primers to create half AttC sites at either end and circularizing the product <del class="diffchange diffchange-inline">through </del><a href="https://2014.igem.org/Team:USyd-Australia/Project/Protocols#gibson">Gibson Assembly</a> <del class="diffchange diffchange-inline">or in other ways</del>.</p><br><h2> </h2></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><img src="https://static.igem.org/mediawiki/2014/c/c4/USyd-Australia_Integron_Design-Produce_Cassettes.png" width="<ins class="diffchange diffchange-inline">340px</ins>" align="<ins class="diffchange diffchange-inline">right</ins>"><br> <p>Gene Cassettes are the carriers of genetic information in our design. They are small, modular chunks of DNA containing a gene flanked by AttC sites. Cassettes, unlike plasmids, are non-replicative and so do not require extra genes to modulate or initiate replication. To create a cassette simply requires the use of primers to create half AttC sites at either end and circularizing the product <ins class="diffchange diffchange-inline">by using <a href="https://2014.igem.org/Team:USyd-Australia/Project/Protocols#ELAN">Enzymatic Ligation Assisted by Nucleases (ELAN)</a>. This is a reaction in which ligation and enzymatic digestion of compatible but non-identical ends, </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">like XbaI and SpeI, occurs simultaneously. This should result only in stable single-cassette circles or concatemers of several cassettes. For systems that already contain an intact AttC site, XbaI and SpeI sites can be introduced by PCR for ELAN, or overlapping ends can be introduced by PCR for </ins><a href="https://2014.igem.org/Team:USyd-Australia/Project/Protocols#gibson">Gibson Assembly</a>.</p><br><h2> </h2></div></td></tr>
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</table>Asis8454http://2014.igem.org/wiki/index.php?title=Team:USyd-Australia/Project/Design&diff=339460&oldid=prevAndy.Bachler at 18:11, 17 October 20142014-10-17T18:11:50Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><img src="https://static.igem.org/mediawiki/2014/5/55/USyd-Australia_Integron_design-add_cassettes.png" width="60%"></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><img src="https://static.igem.org/mediawiki/2014/5/55/USyd-Australia_Integron_design-add_cassettes.png" width="60%"></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><br> <p></p><br><h2> </h2></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><br> <p><ins class="diffchange diffchange-inline">As gene cassettes enter the cell site the integrase acts to cause specific recombination between the AttC site on the cassette and an AttI site which may be present on a plasmid or the bacterial chromosome. The cassette forms a holiday junction at the recombination site. </ins></p><br><h2> </h2></div></td></tr>
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<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;"><br> <p>Now that the required cassette is present in the array we can induce expression of the genes from an upstream promoter to the cassette array. A single promoter can read through multiple cassettes if a terminator is not present, allowing multiple genes to be expressed from a single signal</p><br><h2> </h2></ins></div></td></tr>
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</table>Andy.Bachlerhttp://2014.igem.org/wiki/index.php?title=Team:USyd-Australia/Project/Design&diff=338278&oldid=prevAndy.Bachler at 17:56, 17 October 20142014-10-17T17:56:12Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><img src="https://static.igem.org/mediawiki/2014/c/c4/USyd-Australia_Integron_Design-Produce_Cassettes.png" width="60%" align="center"><br> <p>Gene Cassettes are the carriers of genetic information in our design. They are small, modular chunks of DNA containing a gene flanked by AttC sites. Cassettes, unlike plasmids, are non-replicative and so do not require extra genes to modulate or initiate replication. To create a cassette simply requires the use of primers to create half AttC sites at either end and circularizing the product through <a href="https://2014.igem.org/Team:USyd-Australia/Project/Protocols#gibson">Gibson Assembly</a> or in other ways.</p><br><h2> </h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><img src="https://static.igem.org/mediawiki/2014/c/c4/USyd-Australia_Integron_Design-Produce_Cassettes.png" width="60%" align="center"><br> <p>Gene Cassettes are the carriers of genetic information in our design. They are small, modular chunks of DNA containing a gene flanked by AttC sites. Cassettes, unlike plasmids, are non-replicative and so do not require extra genes to modulate or initiate replication. To create a cassette simply requires the use of primers to create half AttC sites at either end and circularizing the product through <a href="https://2014.igem.org/Team:USyd-Australia/Project/Protocols#gibson">Gibson Assembly</a> or in other ways.</p><br><h2> </h2></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br> <p>As gene cassettes are non-replicative it is important that they are incorporated into an AttI site before the cell divides and the cassettes face the possibility of ejection. Therefore it is vital to have a controllable Integrase gene which can be turned on prior to the addition of gene cassettes to ensure that the cassettes are integrated before they are lost. Part <a href="https://2014.igem.org/Team:USyd-Australia/pUS203">pUS203</a> is our submission BioBrick which encodes the controllable Integrase system. We are using the araC-pBAD system from <a href="http://parts.igem.org/Part:BBa_K731201">K731201</a> which allows us to turn on transcription of the Integrase under the activation of arabinose.</p><br><h2> </h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br> <p>As gene cassettes are non-replicative it is important that they are incorporated into an AttI site before the cell divides and the cassettes face the possibility of ejection. Therefore it is vital to have a controllable Integrase gene which can be turned on prior to the addition of gene cassettes to ensure that the cassettes are integrated before they are lost. Part <a href="https://2014.igem.org/Team:USyd-Australia/pUS203">pUS203</a> is our submission BioBrick which encodes the controllable Integrase system. We are using the araC-pBAD system from <a href="http://parts.igem.org/Part:BBa_K731201">K731201</a> which allows us to turn on transcription of the Integrase under the activation of arabinose.</p><br><h2> </h2></div></td></tr>
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</table>Andy.Bachlerhttp://2014.igem.org/wiki/index.php?title=Team:USyd-Australia/Project/Design&diff=335708&oldid=prevAndy.Bachler at 17:27, 17 October 20142014-10-17T17:27:00Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><img src="https://static.igem.org/mediawiki/2014/c/c4/USyd-Australia_Integron_Design-Produce_Cassettes.png" width="60%" align="center"><br> <p>Gene Cassettes are the carriers of genetic information in our design. They are small, modular chunks of DNA containing a gene flanked by AttC sites. Cassettes, unlike plasmids, are non-replicative and so do not require extra genes to modulate or initiate replication. To create a cassette simply requires the use of primers to create half AttC sites at either end and circularizing the product through <a href="https://2014.igem.org/Team:USyd-Australia/Project/Protocols#gibson">Gibson Assembly</a> or in other ways.</p><br><h2> </h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><img src="https://static.igem.org/mediawiki/2014/c/c4/USyd-Australia_Integron_Design-Produce_Cassettes.png" width="60%" align="center"><br> <p>Gene Cassettes are the carriers of genetic information in our design. They are small, modular chunks of DNA containing a gene flanked by AttC sites. Cassettes, unlike plasmids, are non-replicative and so do not require extra genes to modulate or initiate replication. To create a cassette simply requires the use of primers to create half AttC sites at either end and circularizing the product through <a href="https://2014.igem.org/Team:USyd-Australia/Project/Protocols#gibson">Gibson Assembly</a> or in other ways.</p><br><h2> </h2></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><img src="https://static.igem.org/mediawiki/2014/1/1f/USyd-Australia_Integron_Design-Express_Integrase.png" width="60%"></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><img src="https://static.igem.org/mediawiki/2014/1/1f/USyd-Australia_Integron_Design-Express_Integrase.png" width="60%"></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><br> <p>As gene cassettes are non-replicative it is important that they are incorporated into an AttI site before the cell divides and the cassettes face the possibility of ejection. Therefore it is <del class="diffchange diffchange-inline">important </del>to have a controllable Integrase gene which can be turned on prior to the addition of gene cassettes to ensure that the cassettes are integrated before they are lost. Part <a href="https://2014.igem.org/Team:USyd-Australia/pUS203">pUS203</a> is our submission BioBrick which encodes the controllable Integrase system. We are using the araC-pBAD system from <a href="http://parts.igem.org/Part:BBa_K731201">K731201</a> which allows us to turn on transcription of the Integrase under the activation of arabinose.</p><br><h2> </h2></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><br> <p>As gene cassettes are non-replicative it is important that they are incorporated into an AttI site before the cell divides and the cassettes face the possibility of ejection. Therefore it is <ins class="diffchange diffchange-inline">vital </ins>to have a controllable Integrase gene which can be turned on prior to the addition of gene cassettes to ensure that the cassettes are integrated before they are lost. Part <a href="https://2014.igem.org/Team:USyd-Australia/pUS203">pUS203</a> is our submission BioBrick which encodes the controllable Integrase system. We are using the araC-pBAD system from <a href="http://parts.igem.org/Part:BBa_K731201">K731201</a> which allows us to turn on transcription of the Integrase under the activation of arabinose.</p><br><h2> </h2></div></td></tr>
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</table>Andy.Bachler