http://2014.igem.org/wiki/index.php?title=Team:Toronto/Parts&feed=atom&action=historyTeam:Toronto/Parts - Revision history2024-03-28T20:17:15ZRevision history for this page on the wikiMediaWiki 1.16.5http://2014.igem.org/wiki/index.php?title=Team:Toronto/Parts&diff=403759&oldid=prevC4Rex at 18:40, 9 December 20142014-12-09T18:40:49Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Assembly Strategy</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Assembly Strategy</h3></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We planned on using Gibson assembly to assemble our complete plasmids, including the rearrangement of pCas9 described above, all in one reaction. The four fragments of our assembly are as follows:</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We planned on using Gibson assembly to assemble our complete plasmids, including the rearrangement of pCas9 described above, all in one reaction. The four fragments of our assembly are as follows:</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li>the low-copy origin of replication and ampicillin resistance marker from pTRC99A (PCR from <a href="http://www.addgene.org/vector-database/4402/">pTRC99A</a>)</li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li>the low-copy origin of replication and ampicillin resistance marker from pTRC99A (PCR from <a href="http://www.addgene.org/vector-database/4402/">pTRC99A</a>)</li></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>CRISPR activity assay</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>CRISPR activity assay</h3></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In our case, we chose to use <a href="http://parts.igem.org/Part:BBa_J04450">RFP</a> in pSB1C3 as our target plasmid because it has an easily recognizable phenotype (red colonies), as well as a different antibiotic resistance marker (Chloramphenicol resistance) than our CRISPR plasmids (which are Ampicillin resistant). To make our CRISPR plasmids target the RFP plasmid, we found a <a href="http://parts.igem.org/Part:BBa_K1559010">suitable protospacer</a> within the sequence of RFP to insert into the spacer insertion site of our CRISPR plasmids.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In our case, we chose to use <a href="http://parts.igem.org/Part:BBa_J04450">RFP</a> in pSB1C3 as our target plasmid because it has an easily recognizable phenotype (red colonies), as well as a different antibiotic resistance marker (Chloramphenicol resistance) than our CRISPR plasmids (which are Ampicillin resistant). To make our CRISPR plasmids target the RFP plasmid, we found a <a href="http://parts.igem.org/Part:BBa_K1559010">suitable protospacer</a> within the sequence of RFP to insert into the spacer insertion site of our CRISPR plasmids.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Summary of the two plasmids:</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Summary of the two plasmids:</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><i>Target plasmid:</i> constitutively active RFP (red colonies), Chloramphenicol resistance</li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><i>Target plasmid:</i> constitutively active RFP (red colonies), Chloramphenicol resistance</li></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><i>CRISPR plasmid:</i> Cas9 (controlled by various promoters), crRNA array containing spacer targeting a sequence in RFP, tracrRNA, Ampicillin resistance.</li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><i>CRISPR plasmid:</i> Cas9 (controlled by various promoters), crRNA array containing spacer targeting a sequence in RFP, tracrRNA, Ampicillin resistance.</li></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Before starting our assay, it is necessary to transform the target plasmid (RFP) into the wildtype (MG1655) strain of E. coli. This allows us to obtain a strain of cells known to carry the target plasmid. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Before starting our assay, it is necessary to transform the target plasmid (RFP) into the wildtype (MG1655) strain of E. coli. This allows us to obtain a strain of cells known to carry the target plasmid. </div></td></tr>
</table>C4Rexhttp://2014.igem.org/wiki/index.php?title=Team:Toronto/Parts&diff=403758&oldid=prevC4Rex at 18:39, 9 December 20142014-12-09T18:39:17Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Assembly Strategy</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Assembly Strategy</h3></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;"><p></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We planned on using Gibson assembly to assemble our complete plasmids, including the rearrangement of pCas9 described above, all in one reaction. The four fragments of our assembly are as follows:</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We planned on using Gibson assembly to assemble our complete plasmids, including the rearrangement of pCas9 described above, all in one reaction. The four fragments of our assembly are as follows:</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li>the low-copy origin of replication and ampicillin resistance marker from pTRC99A (PCR from <a href="http://www.addgene.org/vector-database/4402/">pTRC99A</a>)</li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li>the low-copy origin of replication and ampicillin resistance marker from pTRC99A (PCR from <a href="http://www.addgene.org/vector-database/4402/">pTRC99A</a>)</li></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>CRISPR activity assay</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>CRISPR activity assay</h3></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><i>CRISPR plasmid:</i> Cas9 (controlled by various promoters), crRNA array containing spacer targeting a sequence in RFP, tracrRNA, Ampicillin resistance.</li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><i>CRISPR plasmid:</i> Cas9 (controlled by various promoters), crRNA array containing spacer targeting a sequence in RFP, tracrRNA, Ampicillin resistance.</li></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Before starting our assay, it is necessary to transform the target plasmid (RFP) into the wildtype (MG1655) strain of E. coli. This allows us to obtain a strain of cells known to carry the target plasmid. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Before starting our assay, it is necessary to transform the target plasmid (RFP) into the wildtype (MG1655) strain of E. coli. This allows us to obtain a strain of cells known to carry the target plasmid. </div></td></tr>
</table>C4Rexhttp://2014.igem.org/wiki/index.php?title=Team:Toronto/Parts&diff=403757&oldid=prevC4Rex: replaced the question mark characters2014-12-09T18:33:46Z<p>replaced the question mark characters</p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>What is CRISPR?</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>What is CRISPR?</h3></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>CRISPR <del class="diffchange diffchange-inline">� </del>Clustered Regularly Interspaced Short Palindromic Repeats <del class="diffchange diffchange-inline">� </del>is a long name for a characteristic DNA locus that is responsible for the adaptive immunity in most bacteria and in many species of archaea. CRISPRs have been divided into three types (I-III), of which type II is the best understood. A CRISPR type II locus consists of three components: tracrRNA, the Cas genes, and the <del class="diffchange diffchange-inline">�array�</del>. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>CRISPR <ins class="diffchange diffchange-inline">(</ins>Clustered Regularly Interspaced Short Palindromic Repeats<ins class="diffchange diffchange-inline">) </ins>is a long name for a characteristic DNA locus that is responsible for the adaptive immunity in most bacteria and in many species of archaea. CRISPRs have been divided into three types (I-III), of which type II is the best understood. A CRISPR type II locus consists of three components: tracrRNA, the Cas genes, and the <ins class="diffchange diffchange-inline">"array"</ins>. </div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><i>Cas gene(s):</i> The <del class="diffchange diffchange-inline">�signature gene� </del>of a type II CRISPR system is Cas9, an endonuclease. Its original purpose in bacteria is to degrade foreign DNA.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><i>Cas gene(s):</i> The <ins class="diffchange diffchange-inline">core </ins>of a type II CRISPR system is Cas9, an endonuclease. Its original purpose in bacteria is to degrade foreign DNA.</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><i>Array:</i> By incorporating bits of foreign DNA into this array, the bacteria gains immunity to it in the future. Those bits, which are called spacers, are the instructions that tell Cas9 where to cut. They are complementary to part of the DNA of a previous invader; the exact sequence it matches is called the protospacer. So, the array follows a pattern of <del class="diffchange diffchange-inline">�spacer � </del>repeat <del class="diffchange diffchange-inline">� </del>spacer <del class="diffchange diffchange-inline">� repeat�</del>, where the repeats are palindromic sequences that aid in identification of the individual spacers. When foreign DNA enters the bacterium, if the bacterium possesses a complementary spacer to it, that spacer is transcribed separately from its surrounding parts as what is called a crRNA.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><i>Array:</i> By incorporating bits of foreign DNA into this array, the bacteria gains immunity to it in the future. Those bits, which are called spacers, are the instructions that tell Cas9 where to cut. They are complementary to part of the DNA of a previous invader; the exact sequence it matches is called the protospacer. So, the array follows a pattern of <ins class="diffchange diffchange-inline">spacer - </ins>repeat <ins class="diffchange diffchange-inline">- </ins>spacer <ins class="diffchange diffchange-inline">- repeat</ins>, where the repeats are palindromic sequences that aid in identification of the individual spacers. When foreign DNA enters the bacterium, if the bacterium possesses a complementary spacer to it, that spacer is transcribed separately from its surrounding parts as what is called a crRNA.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>The viability of CRISPR tech in biosafety systems</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>The viability of CRISPR tech in biosafety systems</h3></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>There are have been dozens of solutions proposed to solve the biocontainment problem. The 2012 Paris-Bettencourt iGEM team created <a href="http://parts.igem.org/Biosafety?title=Biosafety">a very useful page</a> that lists, amongst other things, all parts that have been created so far tagged in the <del class="diffchange diffchange-inline">�biosafety� </del>category and divides them into 3 types: <b>Semantic Containment</b>, <b>Kill Switch</b>, and <b>XNase</b>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>There are have been dozens of solutions proposed to solve the biocontainment problem. The 2012 Paris-Bettencourt iGEM team created <a href="http://parts.igem.org/Biosafety?title=Biosafety">a very useful page</a> that lists, amongst other things, all parts that have been created so far tagged in the <ins class="diffchange diffchange-inline">biosafety </ins>category and divides them into 3 types: <b>Semantic Containment</b>, <b>Kill Switch</b>, and <b>XNase</b>.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h4>Semantic Containment</h4></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h4>Semantic Containment</h4></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>The genetic construct can only be <del class="diffchange diffchange-inline">�understood� </del>in the synthetic cell and <del class="diffchange diffchange-inline">can�t </del>confer its advantages to wild-type population partners (if the construct were to be shared by horizontal gene transfer) because the cell expresses a <del class="diffchange diffchange-inline">�nonsense suppressor� </del>i.e. a mutated form of tRNA. Paris-Bettencourt 2012 is the only team to have a created a part of this type.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The genetic construct can only be <ins class="diffchange diffchange-inline">"understood" </ins>in the synthetic cell and <ins class="diffchange diffchange-inline">can't </ins>confer its advantages to wild-type population partners (if the construct were to be shared by horizontal gene transfer) because the cell expresses a <ins class="diffchange diffchange-inline">"nonsense suppressor" </ins>i.e. a mutated form of tRNA. Paris-Bettencourt 2012 is the only team to have a created a part of this type.</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>This assay involves two plasmids, a <del class="diffchange diffchange-inline">�target� </del>plasmid, containing a protospacer and PAM, as well as the CRISPR plasmid itself (promoter, cas9, crRNA array, tracrRNA), with the corresponding spacer sequence inserted into the spacer insertion site of the crRNA array. The two plasmids must have different antibiotic selectable markers.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>This assay involves two plasmids, a <ins class="diffchange diffchange-inline">"target" </ins>plasmid, containing a protospacer and PAM, as well as the CRISPR plasmid itself (promoter, cas9, crRNA array, tracrRNA), with the corresponding spacer sequence inserted into the spacer insertion site of the crRNA array. The two plasmids must have different antibiotic selectable markers.</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>In the case of inducible promoters, the assay can be repeated with and without the inducer present in the medium, to determine the <del class="diffchange diffchange-inline">�on� </del>and <del class="diffchange diffchange-inline">�off� </del>rates of CRISPR activity.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>In the case of inducible promoters, the assay can be repeated with and without the inducer present in the medium, to determine the <ins class="diffchange diffchange-inline">"on" </ins>and <ins class="diffchange diffchange-inline">"off" </ins>rates of CRISPR activity.</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> Jiang, W., Bikard, D., Cox, D., Zhang, F., & Marraffini, L. A. (2013). RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature Biotechnology, 31(3), 233 <del class="diffchange diffchange-inline">� </del>239. doi:10.1038/nbt.2508</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> Jiang, W., Bikard, D., Cox, D., Zhang, F., & Marraffini, L. A. (2013). RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature Biotechnology, 31(3), 233 <ins class="diffchange diffchange-inline">- </ins>239. doi:10.1038/nbt.2508</div></td></tr>
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</table>C4Rexhttp://2014.igem.org/wiki/index.php?title=Team:Toronto/Parts&diff=403752&oldid=prevC4Rex: added design section to parts page2014-12-09T18:09:49Z<p>added design section to parts page</p>
<a href="http://2014.igem.org/wiki/index.php?title=Team:Toronto/Parts&diff=403752&oldid=386957">Show changes</a>C4Rexhttp://2014.igem.org/wiki/index.php?title=Team:Toronto/Parts&diff=386957&oldid=prevC4Rex at 02:10, 18 October 20142014-10-18T02:10:34Z<p></p>
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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">In addition, we </del>have documented and added the sequences of the parts we designed to the parts registry in the hopes that future iGEM teams may find them useful. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">We </ins>have <ins class="diffchange diffchange-inline">also </ins>documented and added the sequences <ins class="diffchange diffchange-inline">of the rest </ins>of the parts we designed to the parts registry in the hopes that future iGEM teams may find them useful. </div></td></tr>
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</table>C4Rexhttp://2014.igem.org/wiki/index.php?title=Team:Toronto/Parts&diff=386878&oldid=prevSamantha.chow at 02:09, 18 October 20142014-10-18T02:09:50Z<p></p>
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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;"> <h1><span>Parts</span></h1></ins></div></td></tr>
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</table>Samantha.chowhttp://2014.igem.org/wiki/index.php?title=Team:Toronto/Parts&diff=386623&oldid=prevSamantha.chow at 02:07, 18 October 20142014-10-18T02:07:34Z<p></p>
<a href="http://2014.igem.org/wiki/index.php?title=Team:Toronto/Parts&diff=386623&oldid=345842">Show changes</a>Samantha.chowhttp://2014.igem.org/wiki/index.php?title=Team:Toronto/Parts&diff=345842&oldid=prevSamantha.chow at 19:38, 17 October 20142014-10-17T19:38:48Z<p></p>
<a href="http://2014.igem.org/wiki/index.php?title=Team:Toronto/Parts&diff=345842&oldid=8869">Show changes</a>Samantha.chowhttp://2014.igem.org/wiki/index.php?title=Team:Toronto/Parts&diff=8869&oldid=prevIGEM HQ: Prototype team page2014-05-27T17:55:55Z<p>Prototype team page</p>
<p><b>New page</b></p><div>{{CSS/Main}}<br />
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<h1 >WELCOME TO iGEM 2014! </h1><br />
<p>Your team has been approved and you are ready to start the iGEM season!<br />
<br>On this page you can document your project, introduce your team members, document your progress <br> and share your iGEM experience with the rest of the world! </p><br />
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<p style="color:#E7E7E7"> <a href="https://2014.igem.org/wiki/index.php?title=Team:Toronto/Parts&action=edit"style="color:#FFFFFF"> Click here to edit this page!</a> </p><br />
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<a href="https://2014.igem.org/Team:Toronto"style="color:#000000">Home </a> </td><br />
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<a href="https://2014.igem.org/Team:Toronto/Team"style="color:#000000"> Team </a> </td><br />
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<a href="https://igem.org/Team.cgi?year=2014&team_name=Toronto"style="color:#000000"> Official Team Profile </a></td><br />
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<a href="https://2014.igem.org/Team:Toronto/Attributions"style="color:#000000"> Attributions </a></td><br />
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<td > <h3>What information do I need to start putting my parts on the Registry? </h3></td><br />
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<p><br />
An important aspect of the iGEM competition is the use and creation of standard biological parts. Each team will make new parts during iGEM and will submit them to the <a href="http://partsregistry.org"> Registry of Standard Biological Parts</a>. The iGEM software provides an easy way to present the parts your team has created. The "groupparts" tag will generate a table with all of the parts that your team adds to your team sandbox. <br />
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<strong>Note that if you want to document a part you need to document it on the <a href="http://partsregistry.org Registry"> Registry</a>, not on your team wiki.</strong> Future teams and other users and are much more likely to find parts on the Registry than on your team wiki. <br />
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Remember that the goal of proper part documentation is to describe and define a part, so that it can be used without a need to refer to the primary literature. Registry users in future years should be able to read your documentation and be able to use the part successfully. Also, you should provide proper references to acknowledge previous authors and to provide for users who wish to know more.<br />
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<h3>When should you put parts into the Registry?</h3><br />
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As soon as possible! We encourage teams to start completing documentation for their parts on the Registry as soon as you have it available. The sooner you put up your parts, the better recall you will have of all details surrounding your parts. Remember you don't need to send us the DNA to create an entry for a part on the Registry. However, you must send us the sample/DNA before the Jamboree. Only parts for which you have sent us samples/DNA are eligible for awards and medal requirements. <br />
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The information needed to initially create a part on the Registry is:<br />
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<li>Part Name</li><br />
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<li>Sequence</li><br />
<li>Short Description (60 characters on what the DNA does)</li><br />
<li>Long Description (Longer description of what the DNA does)</li><br />
<li>Design considerations</li><br />
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We encourage you to put up <em>much more</em> information as you gather it over the summer. If you have images, plots, characterization data and other information, please also put it up on the part page. Check out part <a href="http://parts.igem.org/Part:BBa_K404003">BBa_K404003</a> for an excellent example of a highly characterized part. <br />
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You can add parts to the Registry at our <a href="http://parts.igem.org/Add_a_Part_to_the_Registry"> Add a Part to the Registry</a> link.<br />
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<tr><td colspan="3" > <h3> Parts Table</h3></td></tr><br />
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Any parts your team has created will appear in this table below:</td></tr><br />
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