Team:Linkoping Sweden/Gibson assembly
Gibson assembly
Gibson assembly is an exonuclease-based method to assembly multiple DNA fragments in correct order (1). The method was invented in 2009 by Daniel G. Gibson, of the J. Craig Venter Institute. The assembly reaction is carried out in one single reaction-tube, all at once, at 50° Celsius for 15-60 minutes. The process involves three different enzymatic actions. A 5’ exonuclease creates overhangs, enabling matched fragments to anneal. Then a DNA polymerase fills gap between the annealed strands and the 5´ end. Finally, a DNA ligase seals the gaps between the filled in gap and the annealed strands.
Since the method is exonuclease-based, no digestion by restriction enzymes is required for the assembly to take place. For correct assembly, the ends of each DNA fragment must be designed to fit the ends of adjacent fragments. These fragments can be created through PCR or DNA synthesis. The principles of designing fragments for Gibson assembly are very simple. Each DNA-insert should have 15-30 bp of overlapping ends to each other (2). One easy way to do this is to simply add 15-30 bp of the 5´end sequence of the next fragment, to the 3´ end of your fragment.
One obstacle we encountered in the design of our fragments, was the presence of repeatedly occurring sequences, due to the usage of same introns and terminators at various sites. It is very important in a multiple fragment Gibson assembly that all ends are unique for all parts to assemble correctly. Avoiding unspecific annealing was the most time consuming step in design, remodelling of the fragments solved the problem, and the adding of one way overlaps to ends that were not unique.
The Gibson assembly method has been used to successfully assemble up to six fragments in one reaction, this was not achieved with all six fragments. Yet the NEBuilder® HiFi DNA Assembly kit proved to be very efficient when inserting single fragments into a vector, according to our results.
References
1. Gibson DG, Young L, Chuang R-Y, Venter JC, Hutchison CA, Smith HO. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods [Internet]. 2009 May [cited 2016 Sep 30];6(5):343–5. Available from: http://www.ncbi.nlm.nih.gov/pubmed/19363495
2. New England Biolabs. NEBuilder® HiFi DNA Assembly | NEB [Internet]. Available from: https://www.neb.com/applications/cloning-and-synthetic-biology/dna-assembly-and-cloning/nebuilder-hifi-dna-assembly
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