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Team:Macquarie Australia/Protocols/GibsonTips
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<li><b>Use Electroporation for the transformation</b><br> | <li><b>Use Electroporation for the transformation</b><br> | ||
<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. | <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. | ||
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| + | <img src="https://static.igem.org/mediawiki/2012/7/7a/TRANS_GRAPH.png" width=320 height=200></p></li> | ||
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Revision as of 05:57, 22 September 2012
Top Ten Tips for Gibson Assembly
Gibson assembly is a powerful and useful tool for synthetic biologists. In our project, it was particularly useful for producing E. coli codon optimised BioBricks. We would recommend using the technique, but before you do- consider our top ten tips.
- 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
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.
