Gibson Assembly, also known as Gibson Cloning, is a method to assemble two or more linear fragments together without the use of restriction enzymes. Instead, the fragments have to be homologous at the sequence end (see image below, part (a)) so that they can ligate when a single strand is created. This principle is also found in various other related cloning methods like SLIC (sequence and ligase independent cloning (Li 2007)), CPEC (circular polymerase extension cloning (Quan 2009) & SLiCE (Seamless Ligation Cloning Extract (Zhang 2012)). The difference between each of the methods is how the single strand is created, either by melting the complete sequence (CPEC), excising with 3' exonuclease activity (SLIC) or 5' exonuclease activity (Gibson Assembly).
Gibson is an isothermal method where a T5 exonuclease is used to chew back the nucleotides from the 5' end at 50°C (b). The T5 exonuclease is not heat stable, so it will get inactivated after a certain time (c). The overlapping homologous ends can then anneal, non-complementary parts get filled up with a polymerase (d) and the nick gets sealed with a ligase (e). The product is a scarless joint of the two initial sequences (f). Furthermore, this can be done with multiple sequences at once in one pot, allowing to create very large products with each cloning step.
This tutorial covers assembly of single and multiple fragments using Gibson cloning in Geneious. The Gibson cloning tool allows you to simulate your Gibson reaction and will produce a list of the PCR primers required to create the homologous ends.
Exercise 1: Basic Gibson Cloning with a single insert
Exercise 2: Advanced Batch-Cloning