http://2012.igem.org/wiki/index.php?title=Team:Macquarie_Australia/Protocols/GibsonTips&feed=atom&action=historyTeam:Macquarie Australia/Protocols/GibsonTips - Revision history2024-03-28T10:25:02ZRevision history for this page on the wikiMediaWiki 1.16.0http://2012.igem.org/wiki/index.php?title=Team:Macquarie_Australia/Protocols/GibsonTips&diff=232890&oldid=prevElleworgan at 02:22, 27 September 20122012-09-27T02:22:03Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>If the company you are ordering your gBlocks from is not in your time zone the development of the gBlocks may be slow. It is important that you develop good communication with one of their sales representatives. Communication is essential for this process, and poor communication can make a 4-day process significantly longer.</ol></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>If the company you are ordering your gBlocks from is not in your time zone the development of the gBlocks may be slow. It is important that you develop good communication with one of their sales representatives. Communication is essential for this process, and poor communication can make a 4-day process significantly longer.</ol></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>Our practical experience with Gibson assembly reassured us that this technique provides a successful method of gene cloning. Its high levels of efficiency convinced us that this procedure will quickly become the most widely used and accepted method of gene cloning <del class="diffchange diffchange-inline">in research and industry and will allow significant advances to be made in the field of synthetic biology</del>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Our practical experience with Gibson assembly reassured us that this technique provides a successful method of gene cloning. Its high levels of efficiency <ins class="diffchange diffchange-inline">and <b>high success rate</b> (with all five constructs successful) </ins>convinced us that this procedure will quickly become the most widely used and accepted method of gene cloning.</div></td></tr>
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</table>Elleworganhttp://2012.igem.org/wiki/index.php?title=Team:Macquarie_Australia/Protocols/GibsonTips&diff=232819&oldid=prevElleworgan at 02:20, 27 September 20122012-09-27T02:20:50Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li><b>gBlock Provider</b></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li><b>gBlock Provider</b></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>If the company you are ordering your gBlocks from is not in your time zone the development of the gBlocks may be slow. It is important that you develop good communication with one of their sales representatives. Communication is essential for this process, and poor communication can make a 4-day process significantly longer.</ol></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>If the company you are ordering your gBlocks from is not in your time zone the development of the gBlocks may be slow. It is important that you develop good communication with one of their sales representatives. Communication is essential for this process, and poor communication can make a 4-day process significantly longer.</ol></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 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;">Our practical experience with Gibson assembly reassured us that this technique provides a successful method of gene cloning. Its high levels of efficiency convinced us that this procedure will quickly become the most widely used and accepted method of gene cloning in research and industry and will allow significant advances to be made in the field of synthetic biology.</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></p></ins></div></td></tr>
</table>Elleworganhttp://2012.igem.org/wiki/index.php?title=Team:Macquarie_Australia/Protocols/GibsonTips&diff=231733&oldid=prevElleworgan at 01:58, 27 September 20122012-09-27T01:58:35Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li><b>Be Meticulous</b><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li><b>Be Meticulous</b><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>Like all molecular techniques, Gibson is very sensitive and so take care during the assembly. Ensure complete mixing and keep the enzymes being used on ice- otherwise they will denature and the reaction will not occur</p></li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>Like all molecular techniques, Gibson is very sensitive and so take care during the assembly. Ensure complete mixing and keep the enzymes being used on ice- otherwise they will denature and the reaction will not occur</p></li></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><li><b>Use Electroporation for the transformation</b><del class="diffchange diffchange-inline"><br></del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><li><b>Use Electroporation for the transformation</b><p>You want to have the most efficient transformation or you will be doing several rounds of Gibson Assembly. In our project we found that electroporation was significantly more successful than heat shock. </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"><li><b>Use High Competency Cells</b><br></del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"><p>Gibson Assembly is a sensitive technique but only a small amount of complete plasmid is produced. To maximise to product obtained, highly efficient competent cells need to be used, or transformation procedures that provide higher efficiency need to be used.</p></li></del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>You want to have the most efficient transformation or you will be doing several rounds of Gibson Assembly. In our project we found that electroporation was significantly more successful than heat shock. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></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;"><li><b>Use High Competency Cells</b><br></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"><p>Gibson Assembly is a sensitive technique but only a small amount of complete plasmid is produced. To maximise to product obtained, highly efficient competent cells need to be used, or transformation procedures that provide higher efficiency need to be used.</p></li><br></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><img src="https://static.igem.org/mediawiki/2012/7/7a/TRANS_GRAPH.png" width=480 height=300></p></li></center></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><img src="https://static.igem.org/mediawiki/2012/7/7a/TRANS_GRAPH.png" width=480 height=300></p></li></center></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><center>Figure 1: the blue bars represent the electroporated transformation counts and the red represent the heat shock transformation counts.</p></center></li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><center>Figure 1: the blue bars represent the electroporated transformation counts and the red represent the heat shock transformation counts.</p></center></li></div></td></tr>
</table>Elleworganhttp://2012.igem.org/wiki/index.php?title=Team:Macquarie_Australia/Protocols/GibsonTips&diff=231663&oldid=prevElleworgan at 01:57, 27 September 20122012-09-27T01:57:31Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li><b>Check for Restriction Sites</b><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li><b>Check for Restriction Sites</b><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>With changes in the sequence following codon optimisation there is the risk that new restriction sites have been introduced. BioBricks require there to be no internalised EcoR1, Spe1, Xba1, or Pst1 sites. Therefore, the finals G Block sequence needs to be proofread for these sites or else the BioBrick is non-functional and so the Gibson Assembly becomes irrelevant.</p></li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>With changes in the sequence following codon optimisation there is the risk that new restriction sites have been introduced. BioBricks require there to be no internalised EcoR1, Spe1, Xba1, or Pst1 sites. Therefore, the finals G Block sequence needs to be proofread for these sites or else the BioBrick is non-functional and so the Gibson Assembly becomes irrelevant.</p></li></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"><li><b>Use High Competency Cells</b><br></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"><p>Gibson Assembly is a sensitive technique but only a small amount of complete plasmid is produced. To maximise to product obtained, highly efficient competent cells need to be used, or transformation procedures that provide higher efficiency need to be used.</p></li></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li><b>Be Meticulous</b><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li><b>Be Meticulous</b><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>Like all molecular techniques, Gibson is very sensitive and so take care during the assembly. Ensure complete mixing and keep the enzymes being used on ice- otherwise they will denature and the reaction will not occur</p></li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>Like all molecular techniques, Gibson is very sensitive and so take care during the assembly. Ensure complete mixing and keep the enzymes being used on ice- otherwise they will denature and the reaction will not occur</p></li></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li><b>Use Electroporation for the transformation</b><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li><b>Use Electroporation for the transformation</b><br></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;"><li><b>Use High Competency Cells</b><br></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"><p>Gibson Assembly is a sensitive technique but only a small amount of complete plasmid is produced. To maximise to product obtained, highly efficient competent cells need to be used, or transformation procedures that provide higher efficiency need to be used.</p></li></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>You want to have the most efficient transformation or you will be doing several rounds of Gibson Assembly. In our project we found that electroporation was significantly more successful than heat shock. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>You want to have the most efficient transformation or you will be doing several rounds of Gibson Assembly. In our project we found that electroporation was significantly more successful than heat shock. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td></tr>
</table>Elleworganhttp://2012.igem.org/wiki/index.php?title=Team:Macquarie_Australia/Protocols/GibsonTips&diff=231548&oldid=prevElleworgan at 01:55, 27 September 20122012-09-27T01:55:20Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><p>Optimising codons is one of the great things about Gibson Assembly, it allows us to increase the yield of our protein. Unfortunately, in our experience, optimising for <i>E. Coli</i> tends to increase the GC content considerably. As such a lot of the optimisation may need to be reversed to allow for the G Blocks to be synthesised.</li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><p>Optimising codons is one of the great things about Gibson Assembly, it allows us to increase the yield of our protein. Unfortunately, in our experience, optimising for <i>E. Coli</i> tends to increase the GC content considerably. As such a lot of the optimisation may need to be reversed to allow for the G Blocks to be synthesised.</li></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li><b>Be Wary of GC Rich Regions</p></b></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li><b>Be Wary of GC Rich Regions</p></b></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>As touched on in the previous point, GC rich regions make synthesis <del class="diffchange diffchange-inline">impossible</del>. Hairpin loops are formed which prevent elongation. As such, when developing the G Blocks be wary of GC rich regions. If possible, using translation software, change bases in the wobble position to an A or T to keep the integrity of the protein sequence. We aimed to produce our gBlocks with less then 60% GC content.</p></li></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>As touched on in the previous point, GC rich regions make synthesis <ins class="diffchange diffchange-inline">problematic</ins>. Hairpin loops are formed which prevent elongation. As such, when developing the G Blocks be wary of GC rich regions. If possible, using translation software, change bases in the wobble position to an A or T to keep the integrity of the protein sequence. We aimed to produce our gBlocks with less then 60% GC content.</p></li></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li><b>Check for Restriction Sites</b><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li><b>Check for Restriction Sites</b><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>With changes in the sequence following codon optimisation there is the risk that new restriction sites have been introduced. BioBricks require there to be no internalised EcoR1, Spe1, Xba1, or Pst1 sites. Therefore, the finals G Block sequence needs to be proofread for these sites or else the BioBrick is non-functional and so the Gibson Assembly becomes irrelevant.</p></li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>With changes in the sequence following codon optimisation there is the risk that new restriction sites have been introduced. BioBricks require there to be no internalised EcoR1, Spe1, Xba1, or Pst1 sites. Therefore, the finals G Block sequence needs to be proofread for these sites or else the BioBrick is non-functional and so the Gibson Assembly becomes irrelevant.</p></li></div></td></tr>
</table>Elleworganhttp://2012.igem.org/wiki/index.php?title=Team:Macquarie_Australia/Protocols/GibsonTips&diff=231251&oldid=prevElleworgan at 01:49, 27 September 20122012-09-27T01:49:35Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h1><center>Top Ten Tips for Gibson Assembly</center></h1></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h1><center>Top Ten Tips for Gibson Assembly</center></h1></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>Gibson assembly is a powerful and useful tool for synthetic biologists. In our project, it was particularly useful for producing <i>E. coli</i> codon optimised BioBricks. In order to most effectively employ the technique of Gibson assembly, we designed DNA fragments known as G-blocks, which we assembled (as below) <del class="diffchange diffchange-inline">in order </del>to <del class="diffchange diffchange-inline">construct </del>our BioBricks. We found this process to be efficient and successful. We would recommend using the technique, but before you do- consider our top ten tips for designing and assembling G-blocks using Gibson assembly.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>Gibson assembly is a powerful and useful tool for synthetic biologists. In our project, it was particularly useful for producing <i>E. coli</i> codon optimised BioBricks. In order to most effectively employ the technique of Gibson assembly, we designed DNA fragments known as G-blocks, which we assembled (as below) <ins class="diffchange diffchange-inline">before combining and ligating them </ins>to <ins class="diffchange diffchange-inline">produce </ins>our BioBricks. </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 class="diffchange diffchange-inline"><center><img src="http://24.media.tumblr.com/tumblr_mazjjagDVw1rg4kjpo1_500.jpg"></center></ins></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>We found this process to be efficient and successful. We would recommend using the technique, but before you do- consider our top ten tips for designing and assembling G-blocks using Gibson assembly.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><blockquote><ol></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><blockquote><ol></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li><b>Consider the overlapping sequence</b></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li><b>Consider the overlapping sequence</b></div></td></tr>
</table>Elleworganhttp://2012.igem.org/wiki/index.php?title=Team:Macquarie_Australia/Protocols/GibsonTips&diff=229992&oldid=prevElleworgan at 01:26, 27 September 20122012-09-27T01:26:22Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h1><center>Top Ten Tips for Gibson Assembly</center></h1></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h1><center>Top Ten Tips for Gibson Assembly</center></h1></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>Gibson assembly is a powerful and useful tool for synthetic biologists. In our project, it was particularly useful for producing <i>E. coli</i> codon optimised BioBricks. In order to most effectively employ the technique of Gibson assembly, we designed DNA fragments known as G-blocks, which we assembled in order to construct our BioBricks. We found this process to be efficient and successful. We would recommend using the technique, but before you do- consider our top ten tips for designing and assembling G-blocks using Gibson assembly.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>Gibson assembly is a powerful and useful tool for synthetic biologists. In our project, it was particularly useful for producing <i>E. coli</i> codon optimised BioBricks. In order to most effectively employ the technique of Gibson assembly, we designed DNA fragments known as G-blocks, which we assembled <ins class="diffchange diffchange-inline">(as below) </ins>in order to construct our BioBricks. We found this process to be efficient and successful. We would recommend using the technique, but before you do- consider our top ten tips for designing and assembling G-blocks using Gibson assembly.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><blockquote><ol></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><blockquote><ol></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li><b>Consider the overlapping sequence</b></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li><b>Consider the overlapping sequence</b></div></td></tr>
</table>Elleworganhttp://2012.igem.org/wiki/index.php?title=Team:Macquarie_Australia/Protocols/GibsonTips&diff=229625&oldid=prevElleworgan at 01:19, 27 September 20122012-09-27T01:19:25Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h1><center>Top Ten Tips for Gibson Assembly</center></h1></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h1><center>Top Ten Tips for Gibson Assembly</center></h1></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>Gibson assembly is a powerful and useful tool for synthetic biologists. In our project, it was particularly useful for producing <i>E. coli</i> codon optimised BioBricks. We would recommend using the technique, but before you do- consider our top ten tips.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>Gibson assembly is a powerful and useful tool for synthetic biologists. In our project, it was particularly useful for producing <i>E. coli</i> codon optimised BioBricks<ins class="diffchange diffchange-inline">. In order to most effectively employ the technique of Gibson assembly, we designed DNA fragments known as G-blocks, which we assembled in order to construct our BioBricks. We found this process to be efficient and successful</ins>. We would recommend using the technique, but before you do- consider our top ten tips <ins class="diffchange diffchange-inline">for designing and assembling G-blocks using Gibson assembly</ins>.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><blockquote><ol></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><blockquote><ol></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li><b>Consider the overlapping sequence</b></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li><b>Consider the overlapping sequence</b></div></td></tr>
</table>Elleworganhttp://2012.igem.org/wiki/index.php?title=Team:Macquarie_Australia/Protocols/GibsonTips&diff=193011&oldid=prevRyankenny at 11:21, 26 September 20122012-09-26T11:21:24Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><p>Optimising codons is one of the great things about Gibson Assembly, it allows us to increase the yield of our protein. Unfortunately, in our experience, optimising for <i>E. Coli</i> tends to increase the GC content considerably. As such a lot of the optimisation may need to be reversed to allow for the G Blocks to be synthesised.</li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><p>Optimising codons is one of the great things about Gibson Assembly, it allows us to increase the yield of our protein. Unfortunately, in our experience, optimising for <i>E. Coli</i> tends to increase the GC content considerably. As such a lot of the optimisation may need to be reversed to allow for the G Blocks to be synthesised.</li></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li><b>Be Wary of GC Rich Regions</p></b></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li><b>Be Wary of GC Rich Regions</p></b></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>As touched on in the previous point, GC rich regions make synthesis impossible. Hairpin loops are formed which prevent elongation. As such, when developing the G Blocks be wary of GC rich regions. If possible, using translation software, change bases in the wobble position to an A or T to keep the integrity of the protein sequence.</p></li></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>As touched on in the previous point, GC rich regions make synthesis impossible. Hairpin loops are formed which prevent elongation. As such, when developing the G Blocks be wary of GC rich regions. If possible, using translation software, change bases in the wobble position to an A or T to keep the integrity of the protein sequence<ins class="diffchange diffchange-inline">. We aimed to produce our gBlocks with less then 60% GC content</ins>.</p></li></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li><b>Check for Restriction Sites</b><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li><b>Check for Restriction Sites</b><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>With changes in the sequence following codon optimisation there is the risk that new restriction sites have been introduced. BioBricks require there to be no internalised EcoR1, Spe1, Xba1, or Pst1 sites. Therefore, the finals G Block sequence needs to be proofread for these sites or else the BioBrick is non-functional and so the Gibson Assembly becomes irrelevant.</p></li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>With changes in the sequence following codon optimisation there is the risk that new restriction sites have been introduced. BioBricks require there to be no internalised EcoR1, Spe1, Xba1, or Pst1 sites. Therefore, the finals G Block sequence needs to be proofread for these sites or else the BioBrick is non-functional and so the Gibson Assembly becomes irrelevant.</p></li></div></td></tr>
</table>Ryankennyhttp://2012.igem.org/wiki/index.php?title=Team:Macquarie_Australia/Protocols/GibsonTips&diff=171164&oldid=prevRyankenny at 13:19, 25 September 20122012-09-25T13:19:41Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p><center>Figure 1: the blue bars represent the electroporated <del class="diffchange diffchange-inline">transformations </del>and the red represent the heat shock <del class="diffchange diffchange-inline">transformations</del>.</p></center></li></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p><center>Figure 1: the blue bars represent the electroporated <ins class="diffchange diffchange-inline">transformation counts </ins>and the red represent the heat shock <ins class="diffchange diffchange-inline">transformation counts</ins>.</p></center></li></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li><b>Utilise the vector</b></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li><b>Utilise the vector</b></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>When developing the gBlocks you can remove the need to perform restriction digests to produce a BioBrick. By taking the destination vector's 5' and 3' ends into account during development entire steps can be removed and the pure BioBrick can be easily produced. This is part of the power of Gibson Assembly and could be used to make complex plasmids.</p></li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>When developing the gBlocks you can remove the need to perform restriction digests to produce a BioBrick. By taking the destination vector's 5' and 3' ends into account during development entire steps can be removed and the pure BioBrick can be easily produced. This is part of the power of Gibson Assembly and could be used to make complex plasmids.</p></li></div></td></tr>
</table>Ryankenny