Difference between revisions of "Team:UIUC Illinois/Project"

Gibson Assembly is a revolutionary method for assembling multiple linear DNA fragments (original paper link here) by Dr. Daniel Gibson at the J. Craig Venter Institute in 2009. The multiple overlapping DNA fragments can be joined by a single reaction regardless of the fragment length, which adds to the versatility of the method. By adding the three different enzymes (5’ exonuclease, DNA polymerase, and DNA ligase), a fully ligated double-stranded DNA molecule is acquired. This method is proven to be efficient due to the ease of the reaction – needing only one tube of reaction – and the effectiveness of the reaction: no scars on the ligated DNA, non-selective compatibility of DNA fragments, no specific restriction sites needed.

Preparation before undergoing Gibson Assembly reaction
1. Primer Design In designing the primer, adjacent segments in the plasmid should have identical sequences on the ends; the insert sequence and the vector sequence should be compatible with each other. These identical sequences can be created via PCR with primers containing a 5’ end identical to the adjacent segment and a 3’ that would anneal to the target sequence. Also, an effective amount of 60 bp primers might be more effective due to the more targeted approach by the enzymes. Once a preferred primer design has been achieved, amplify the amount of primer DNA by PCR.
2. Check size and yield from the PCR product. When the product is found to be impure, consider using gel purification protocol to rinse the impurities.

Undergoing the Gibson Assembly Reaction

For our method, we used NEB’s Gibson Assembly Master Mix. The Master Mix consists of three enzymes in a single buffer:

T5 exonuclease: chews back the 5’ end of the DNA to create a 3’ overhang, so the complementary strand would anneal to each other.

Phusion DNA Polymerase: incorporates nucleotides to fill in the gaps in the annealed DNA fragment

Taq DNA ligase: joining the annealed DNA fragments and removing the ‘nicks’ and ‘scars’.