"
Team:Macquarie Australia/Protocols/GibsonTips
From 2012.igem.org
| Line 5: | Line 5: | ||
<blockquote><ol> | <blockquote><ol> | ||
<li><b>Consider the overlapping sequence</b> | <li><b>Consider the overlapping sequence</b> | ||
| - | <br>The whole technique revolves around the overlapping sequence, <b>everything</b> can be planned but if this is not correct then the blocks won't anneal. Therefore, the overlapping regions need to be identified and conserved between the two adjacent sections G Blocks. Keep an eye on the Melting Temperature (T<sub>M</sub>), if this gets to high then significant problems will arise.</li> | + | <br><p>The whole technique revolves around the overlapping sequence, <b>everything</b> can be planned but if this is not correct then the blocks won't anneal. Therefore, the overlapping regions need to be identified and conserved between the two adjacent sections G Blocks. Keep an eye on the Melting Temperature (T<sub>M</sub>), if this gets to high then significant problems will arise.</p></li> |
<li><b>Codon Usage</b> | <li><b>Codon Usage</b> | ||
| - | <br>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> | + | <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> |
| - | <li><b>Be Wary of GC Rich Regions</b> | + | <li><b>Be Wary of GC Rich Regions</p></b> |
| - | <br>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.</li> | + | <br><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> |
<li><b>Check for Restriction Sites</b><br> | <li><b>Check for Restriction Sites</b><br> | ||
| - | 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.</li> | + | <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> |
<li><b>Use High Competency Cells</b><br> | <li><b>Use High Competency Cells</b><br> | ||
| - | 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.</li> | + | <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> |
<li><b>Be Meticulous</b><br> | <li><b>Be Meticulous</b><br> | ||
| - | 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</li> | + | <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> |
| + | <li><b>Use Electroporation for the transformation</b><br> | ||
| + | <p></p></li> | ||
</ol> | </ol> | ||
Revision as of 04:27, 21 September 2012
Top Ten Tips for Gibson Assembly
- Consider the overlapping sequence
The whole technique revolves around the overlapping sequence, everything can be planned but if this is not correct then the blocks won't anneal. Therefore, the overlapping regions need to be identified and conserved between the two adjacent sections G Blocks. Keep an eye on the Melting Temperature (TM), if this gets to high then significant problems will arise.
- Codon Usage
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 E. Coli 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.
- Be Wary of GC Rich Regions
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.
- Check for Restriction Sites
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.
- Use High Competency Cells
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.
- Be Meticulous
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
- Use Electroporation for the transformation
