http://2011.igem.org/wiki/index.php?title=Team:Washington/Magnetosomes/GibsonVectors&feed=atom&action=historyTeam:Washington/Magnetosomes/GibsonVectors - Revision history2024-03-28T12:44:46ZRevision history for this page on the wikiMediaWiki 1.16.0http://2011.igem.org/wiki/index.php?title=Team:Washington/Magnetosomes/GibsonVectors&diff=197446&oldid=prevSaushun at 00:24, 29 September 20112011-09-29T00:24:23Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=== What happened last year?===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=== What happened last year?===</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The Gibson Cloning method is definitely not a new method to be introduced to the iGEM community. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The Gibson Cloning method is definitely not a new method to be introduced to the iGEM community. </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>In 2010, the Cambridge iGEM team created the [http://www.cambridgeigem.org/RFC57.pdf BBF RFC 57] document which outlines a protocol for Gibson Assembly using standard BioBricks (BBF RFC 10) that would allow many fragment inserts during a single cloning step. However, while creating the Magnetosome Toolkit, we found that this BioBrick standard was incapable of producing high yields of desired Gibson products even for two-fragment assemblies.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>In 2010, the Cambridge iGEM team created the [http://www.cambridgeigem.org/RFC57.pdf BBF RFC 57] document which outlines a protocol for Gibson Assembly using standard BioBricks (<ins class="diffchange diffchange-inline">[http://dspace.mit.edu/bitstream/handle/1721.1/45138/BBFRFC10.txt?sequence=1 </ins>BBF RFC 10<ins class="diffchange diffchange-inline">]</ins>) that would allow many fragment inserts during a single cloning step. However, while creating the Magnetosome Toolkit, we found that this BioBrick standard was incapable of producing high yields of desired Gibson products even for two-fragment assemblies.</div></td></tr>
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</table>Saushunhttp://2011.igem.org/wiki/index.php?title=Team:Washington/Magnetosomes/GibsonVectors&diff=196681&oldid=prevRobere: /* What about this year? */2011-09-28T23:46:34Z<p><span class="autocomment">What about this year?</span></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>=== What <del class="diffchange diffchange-inline">about </del>this year?===</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>=== What <ins class="diffchange diffchange-inline">did we do </ins>this year?===</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Seeing that this is a very efficient method to do cloning, we continued to make improvements to the methods and created a '''Gibson Assembly Toolkit'''! <br> <br/></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Seeing that this is a very efficient method to do cloning, we continued to make improvements to the methods and created a '''Gibson Assembly Toolkit'''! <br> <br/></div></td></tr>
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</table>Roberehttp://2011.igem.org/wiki/index.php?title=Team:Washington/Magnetosomes/GibsonVectors&diff=196676&oldid=prevRobere: /* What happened last year? */2011-09-28T23:46:13Z<p><span class="autocomment">What happened last year?</span></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>To overcome this problem, the [https://2010.igem.org/Team:Washington/Tools_Used/Next-Gen_Cloning 2010 UW iGEM] team developed new prefix and suffix regions that are based on BglBrick (BBF [http://dspace.mit.edu/handle/1721.1/46747 RFC 21]) standard and designed to eliminate self-complementarity from the prefix and suffix of the plasmid. These vectors dramatically increase the Gibson assembly efficiency of large-scale gene assemblies and are also compatible with iGEM standard BioBrick parts. <br/></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>To overcome this problem, the [https://2010.igem.org/Team:Washington/Tools_Used/Next-Gen_Cloning 2010 UW iGEM] team developed new prefix and suffix regions that are based on BglBrick (BBF [http://dspace.mit.edu/handle/1721.1/46747 RFC 21]) standard and designed to eliminate self-complementarity from the prefix and suffix of the plasmid. These vectors <ins class="diffchange diffchange-inline">[https://2011.igem.org/Team:Washington/Magnetosomes/GibsonResults </ins>dramatically<ins class="diffchange diffchange-inline">] </ins>increase the Gibson assembly efficiency of large-scale gene assemblies and are also compatible with iGEM standard BioBrick parts. <br/></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:Igem2011_gibsonbrick.png|600px|center]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:Igem2011_gibsonbrick.png|600px|center]]</div></td></tr>
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</table>Roberehttp://2011.igem.org/wiki/index.php?title=Team:Washington/Magnetosomes/GibsonVectors&diff=196652&oldid=prevRobere: /* What happened last year? */2011-09-28T23:45:01Z<p><span class="autocomment">What happened last year?</span></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>To overcome this problem, the [https://2010.igem.org/Team:Washington/Tools_Used/Next-Gen_Cloning 2010 UW iGEM] team developed new prefix and suffix regions that are based on BglBrick (BBF [http://dspace.mit.edu/handle/1721.1/46747 RFC 21]) <del class="diffchange diffchange-inline">standards </del>and designed to eliminate self-complementarity of the plasmid. These vectors <del class="diffchange diffchange-inline">drastically </del>increase the Gibson assembly efficiency of large-scale gene assemblies and are also compatible with iGEM standard BioBrick parts. <br/></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>To overcome this problem, the [https://2010.igem.org/Team:Washington/Tools_Used/Next-Gen_Cloning 2010 UW iGEM] team developed new prefix and suffix regions that are based on BglBrick (BBF [http://dspace.mit.edu/handle/1721.1/46747 RFC 21]) <ins class="diffchange diffchange-inline">standard </ins>and designed to eliminate self-complementarity <ins class="diffchange diffchange-inline">from the prefix and suffix </ins>of the plasmid. These vectors <ins class="diffchange diffchange-inline">dramatically </ins>increase the Gibson assembly efficiency of large-scale gene assemblies and are also compatible with iGEM standard BioBrick parts. <br/></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:Igem2011_gibsonbrick.png|600px|center]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:Igem2011_gibsonbrick.png|600px|center]]</div></td></tr>
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</table>Roberehttp://2011.igem.org/wiki/index.php?title=Team:Washington/Magnetosomes/GibsonVectors&diff=196633&oldid=prevRobere: /* What happened last year? */2011-09-28T23:44:02Z<p><span class="autocomment">What happened last year?</span></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>To overcome this problem, the [https://2010.igem.org/Team:Washington/Tools_Used/Next-Gen_Cloning 2010 UW iGEM] team developed new prefix and suffix regions that are based on BglBrick standards and designed to eliminate self-complementarity of the plasmid. These vectors drastically increase the Gibson assembly efficiency of large-scale gene assemblies and are also compatible with iGEM standard BioBrick parts. <br/></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>To overcome this problem, the [https://2010.igem.org/Team:Washington/Tools_Used/Next-Gen_Cloning 2010 UW iGEM] team developed new prefix and suffix regions that are based on BglBrick <ins class="diffchange diffchange-inline">(BBF [http://dspace.mit.edu/handle/1721.1/46747 RFC 21]) </ins>standards and designed to eliminate self-complementarity of the plasmid. These vectors drastically increase the Gibson assembly efficiency of large-scale gene assemblies and are also compatible with iGEM standard BioBrick parts. <br/></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:Igem2011_gibsonbrick.png|600px|center]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:Igem2011_gibsonbrick.png|600px|center]]</div></td></tr>
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</table>Roberehttp://2011.igem.org/wiki/index.php?title=Team:Washington/Magnetosomes/GibsonVectors&diff=196610&oldid=prevRobere at 23:42, 28 September 20112011-09-28T23:42:50Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>To overcome this problem, the [2010 UW iGEM] team developed new prefix and suffix regions that are based on BglBrick standards and designed to eliminate self-complementarity of the plasmid. These vectors drastically increase the Gibson assembly efficiency of large-scale gene assemblies and are also compatible with iGEM standard BioBrick parts. <br/></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>To overcome this problem, the [<ins class="diffchange diffchange-inline">https://2010.igem.org/Team:Washington/Tools_Used/Next-Gen_Cloning </ins>2010 UW iGEM] team developed new prefix and suffix regions that are based on BglBrick standards and designed to eliminate self-complementarity of the plasmid. These vectors drastically increase the Gibson assembly efficiency of large-scale gene assemblies and are also compatible with iGEM standard BioBrick parts. <br/></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:Igem2011_gibsonbrick.png|600px|center]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:Igem2011_gibsonbrick.png|600px|center]]</div></td></tr>
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</table>Roberehttp://2011.igem.org/wiki/index.php?title=Team:Washington/Magnetosomes/GibsonVectors&diff=196601&oldid=prevRobere at 23:42, 28 September 20112011-09-28T23:42:14Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><big><big><big><big>'''iGEM Toolkits: Gibson Assembly Vectors'''</big></big></big></big></center><br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><big><big><big><big>'''iGEM Toolkits: Gibson Assembly Vectors'''</big></big></big></big></center><br><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>=== About Gibson <del class="diffchange diffchange-inline">Cloning/</del>Assembly===</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>=== About Gibson Assembly===</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Gibson <del class="diffchange diffchange-inline">Cloning/Assembly </del>is a <del class="diffchange diffchange-inline">novel </del>synthetic biology tool that allows multiple gene-inserts <del class="diffchange diffchange-inline">during a single </del>isothermal reaction <del class="diffchange diffchange-inline">that is used for assembling overlapping </del>DNA fragments. This method is gaining popularity as it tends be more efficient, <del class="diffchange diffchange-inline">saving </del>a great amount of time <del class="diffchange diffchange-inline">during </del>the cloning process. Overall, Gibson <del class="diffchange diffchange-inline">cloning </del>allows teams to built large gene constructs with ease. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Gibson <ins class="diffchange diffchange-inline">assembly </ins>is a <ins class="diffchange diffchange-inline">new </ins>synthetic biology tool that allows <ins class="diffchange diffchange-inline">scar-free assembly of </ins>multiple gene-inserts <ins class="diffchange diffchange-inline">in one </ins>isothermal reaction <ins class="diffchange diffchange-inline">by using an exonuclease, polymerase, and heat-stable ligase to chew back, anneal, and repair gaps from homologous </ins>DNA fragments. This method is gaining popularity as it tends <ins class="diffchange diffchange-inline">to </ins>be more efficient <ins class="diffchange diffchange-inline">than standard restriction/ligation assembly</ins>, <ins class="diffchange diffchange-inline">which can save </ins>a great amount of time <ins class="diffchange diffchange-inline">in </ins>the cloning process. Overall, Gibson <ins class="diffchange diffchange-inline">assembly </ins>allows teams to built large gene constructs with ease. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br/> [[File:Igem2011_GibsonReacion.png|center|400px]] </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br/> [[File:Igem2011_GibsonReacion.png|center|400px]] </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>-------------</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>-------------</div></td></tr>
</table>Roberehttp://2011.igem.org/wiki/index.php?title=Team:Washington/Magnetosomes/GibsonVectors&diff=195974&oldid=prevRobere: /* Creation of 5 plasmid vectors */2011-09-28T23:06:25Z<p><span class="autocomment">Creation of 5 plasmid vectors</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>As listed above, each of our vectors have varying copy numbers, antibiotic resistances, and purposes within the magnetosome gene assembly. However, they all appear to be very efficient will multiple gene inserts.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>As listed above, each of our vectors have varying copy numbers, antibiotic resistances, and purposes within the magnetosome gene assembly. However, they all appear to be very efficient will multiple gene inserts.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br/><br/><br/><br/><br/></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br/><br/><br/><br/><br/></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>For experimental details comparing the efficiencies of pSB1A3 and pGA1A3, see [https://2011.igem.org/Team:Washington/Magnetosomes/GibsonResults our assay results]. In addition to the characterization of assembly efficiency, we used the pGA vectors for the [https://2011.igem.org/Team:Washington/Magnetosomes/Magnet_Toolkit Magnetosome Toolkit] project. First, we extracted all the 18 <i>mamAB</i> magnetosome genes <del class="diffchange diffchange-inline">and put them in BioBricks</del>. We also made super-assemblies from these BioBricks to make 10 kilobase plasmids from 2 kilobase pieces by assembling up to five fragments all in one cloning step! </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>For experimental details comparing the efficiencies of pSB1A3 and pGA1A3, see [https://2011.igem.org/Team:Washington/Magnetosomes/GibsonResults our assay results]. In addition to the characterization of assembly efficiency, we used the pGA vectors for the [https://2011.igem.org/Team:Washington/Magnetosomes/Magnet_Toolkit Magnetosome Toolkit] project. First, we <ins class="diffchange diffchange-inline">efficiently </ins>extracted <ins class="diffchange diffchange-inline">and BioBricked </ins>all the 18 <i>mamAB</i> magnetosome genes. We also made super-assemblies from these BioBricks to make 10 kilobase plasmids <ins class="diffchange diffchange-inline">(see [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590017 mamHIEJKL] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590019 mamQRBSTUV]) </ins>from 2 kilobase pieces by assembling up to five fragments all in one cloning step! </div></td></tr>
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</table>Roberehttp://2011.igem.org/wiki/index.php?title=Team:Washington/Magnetosomes/GibsonVectors&diff=195908&oldid=prevRobere: /* Creation of 5 plasmid vectors */2011-09-28T23:02:02Z<p><span class="autocomment">Creation of 5 plasmid vectors</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>As listed above, each of our vectors have varying copy numbers, antibiotic resistances, and purposes within the magnetosome gene assembly. However, they all appear to be very efficient will multiple gene inserts.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>As listed above, each of our vectors have varying copy numbers, antibiotic resistances, and purposes within the magnetosome gene assembly. However, they all appear to be very efficient will multiple gene inserts.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><br/><br/><br/></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><br/><br/></ins><br/><br/><br/></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For experimental details comparing the efficiencies of pSB1A3 and pGA1A3, see [https://2011.igem.org/Team:Washington/Magnetosomes/GibsonResults our assay results]. In addition to the characterization of assembly efficiency, we used the pGA vectors for the [https://2011.igem.org/Team:Washington/Magnetosomes/Magnet_Toolkit Magnetosome Toolkit] project. First, we extracted all the 18 <i>mamAB</i> magnetosome genes and put them in BioBricks. We also made super-assemblies from these BioBricks to make 10 kilobase plasmids from 2 kilobase pieces by assembling up to five fragments all in one cloning step! </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For experimental details comparing the efficiencies of pSB1A3 and pGA1A3, see [https://2011.igem.org/Team:Washington/Magnetosomes/GibsonResults our assay results]. In addition to the characterization of assembly efficiency, we used the pGA vectors for the [https://2011.igem.org/Team:Washington/Magnetosomes/Magnet_Toolkit Magnetosome Toolkit] project. First, we extracted all the 18 <i>mamAB</i> magnetosome genes and put them in BioBricks. We also made super-assemblies from these BioBricks to make 10 kilobase plasmids from 2 kilobase pieces by assembling up to five fragments all in one cloning step! </div></td></tr>
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</table>Roberehttp://2011.igem.org/wiki/index.php?title=Team:Washington/Magnetosomes/GibsonVectors&diff=195899&oldid=prevRobere: /* Creation of 5 plasmid vectors */2011-09-28T23:01:49Z<p><span class="autocomment">Creation of 5 plasmid vectors</span></p>
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<td colspan='2' style="background-color: white; color:black;">Revision as of 23:01, 28 September 2011</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>As listed above, each of our vectors have varying copy numbers, antibiotic resistances, and purposes within the magnetosome gene assembly. However, they all appear to be very efficient will multiple gene inserts.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>As listed above, each of our vectors have varying copy numbers, antibiotic resistances, and purposes within the magnetosome gene assembly. However, they all appear to be very efficient will multiple gene inserts.</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><ins class="diffchange diffchange-inline"><br/><br/><br/></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For experimental details comparing the efficiencies of pSB1A3 and pGA1A3, see [https://2011.igem.org/Team:Washington/Magnetosomes/GibsonResults our assay results]. In addition to the characterization of assembly efficiency, we used the pGA vectors for the [https://2011.igem.org/Team:Washington/Magnetosomes/Magnet_Toolkit Magnetosome Toolkit] project. First, we extracted all the 18 <i>mamAB</i> magnetosome genes and put them in BioBricks. We also made super-assemblies from these BioBricks to make 10 kilobase plasmids from 2 kilobase pieces by assembling up to five fragments all in one cloning step! </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For experimental details comparing the efficiencies of pSB1A3 and pGA1A3, see [https://2011.igem.org/Team:Washington/Magnetosomes/GibsonResults our assay results]. In addition to the characterization of assembly efficiency, we used the pGA vectors for the [https://2011.igem.org/Team:Washington/Magnetosomes/Magnet_Toolkit Magnetosome Toolkit] project. First, we extracted all the 18 <i>mamAB</i> magnetosome genes and put them in BioBricks. We also made super-assemblies from these BioBricks to make 10 kilobase plasmids from 2 kilobase pieces by assembling up to five fragments all in one cloning step! </div></td></tr>
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</table>Robere