CH391L/S14/Gibson Assembly

Gibson assembly is an enzymatic method for assembling fragments of DNA into longer constructs or plasmids. The assembly reaction is a simple isothermal procedure that takes no more than one hour and can join multiple fragments in one step. The method was developed by Daniel Gibson at the J. Craig Venter Institute in 2009 and has since become an increasingly popular method of assembling DNA.

Historical Context
Before Gibson assembly, the predominant method for assembling DNA fragments was by using restriction enzymes to make sticky end cuts at restriction sites and match sticky ends to each other to join the DNA. Being the first method of recombinant DNA, restriction enzyme assembly has produced massively influential research and technology. The method has many disadvantages, the primary one being that restriction enzymes that have sites that appear within the sequence to be assembled cannot be used. As sequences become longer, the probability of additional restriction sites appearing increases dramatically, which limits the use of restriction enzyme assembly to shorter sequences than commonly appear in nature. Newer restriction enzyme assembly standards, such as BioBricks and its associated 3A assembly, have increased the audience of potential researchers, but retain the problems inherent in restriction enzyme cutting.

Background
Daniel Gibson, while working at JCVI, was researching whole genome assembly from smaller parts. Assembling whole genomes is a task that requires a very open method for assembling DNA due to the fact that even the smallest genomes are inevitably going to be cut numerous times in many inopportune locations. The method he and his colleagues developed uses overlaps of homology and an exonuclease to create sticky ends on any fragment of DNA, leaving no scar or trace that the assembled sequence was joined from smaller parts. The reaction uses a high fidelity thermostable DNA polymerase, Phusion sold by New England Biolabs, to polymerize nucleotides at annealing temperature. In addition, a thermostable Taq ligase is necessary to rejoin the nicked phosphodiester backbone that is broken when filling in gaps left by the exonuclease. The T5 exonuclease in question is not thermostable, however, because the method requires that the exonuclease become nonfunctional after creating sticky ends long enough to anneal adjacent fragments. These three enzymes, along with an isothermal reaction buffer containing deoxynucleotide triphosphates, Tris buffer, ions and other stabilizers, are sold as a kit or master mix by New England Biolabs under license from Synthetic Genomics. However, all the necessary ingredients are available individually and a Gibson assembly master mix can be put together by any competent laboratory researcher based on Daniel Gibson's published recipe or alternative variations published elsewhere.

Recipe
5x isothermal reaction buffer: 25% PEG-8000 500 mM Tris-HCl pH 7.5 50 mM MgCl2 50mM DTT 1mM each of the four dNTPs 5mM NAD

40 µl mix: 8 µl 5x isothermal buffer 0.8 µl of 0.2 U µl–1 or 1.0 U µl–1 T5 exonuclease 4 µl of 40 U µl–1 Taq DNA ligase 0.5 µl of 2 U µl–1 Phusion DNA polymerase

T5 exonuclease was diluted 1:50 or 1:10 from 10 U ml–1 in its stored buffer (50% glycerol, 50 mM Tris-HCl pH 7.5, 0.1 mM EDTA, 1 mM DTT, 0.1 M NaCl and 0.1% Triton X-100) depending on the overlap size. For overlaps shorter than 150 bp, 0.2 U ml–1 T5 exonuclease is used. For overlaps larger than 150 bp, 1.0 U ml–1 T5 exonuclease was used.

To constitute a reaction, 5 µl DNA was added to 15 ml of this mixture. Incubations were carried out at 50˙C for 15 to 60 min, with 60 min being optimal.

The Method
Before the assembly reaction, one needs to first create a construct of the DNA fragments they plan to assemble in one of many CAD software programs and use the sequence information to design primers approximately 40 base pairs long with about 20 base pairs of homology to the adjacent sequence, in addition to any other standard length primers necessary to amplify linear DNA constructs. These primers can be ordered easily from a synthetic DNA service, such as IDT. Once the purified DNA fragments to be assembled and the necessary primers are obtained, the fragments must be amplified by PCR to create many copies of new, extended fragments with double stranded sequence regions that overlap with the adjacent fragment to be joined. Once the extended fragments are purified, they are added to the Gibson assembly master mix containing the buffer, exonuclease polymerase and ligase, recipe as outlined above, and held at 50˙C for as few as 15 minutes, but one hour produces the best results. The resulting assembled DNA can then be transformed into the organism of interest if cloning or expression are the goal. If using electroporation as a method of transformation for E. coli, some find that the Gibson mixture is salty and prone to arcing in an electroporator, which would kill the electrocompetent cells. This can be resolved by desalting after the isothermal assembly step using nanopore filters floated over deionized water.

Advantages
Gibson assembly has many advantages for those looking to assemble any kind of DNA constructs. Without the use of restriction enzyme, the presence of restriction enzyme sites in the sequence has no effect on the assembly, allowing the use of wide ranging sequences and fusions of very long proteins from smaller subsequences. Additionally, a large number of fragments--assemblages of over 10 fragments have been reported--make short work of long, complex constructs from varying sources. The design element of the overlapping primers also allows for synthetic DNA sequences to be inserted between conjoined sequences, allowing for new ribosome binding sites or promoters to be inserted. Finally, the process is simple enough that it can be automated.

Disadvantages
There are few disadvantages to the method, and the main focus of problems that some have involve the use of very long oligonucleotide primers, or ultramers. When these longer than normal primers reach a length approaching 60 base pairs, they become difficult to assemble without secondary structure interference and are many times more expensive than shorter oligonucleotide primers, due to their difficulty synthesizing reliably. Additionally, the Phusion polymerase called for by the method for high fidelity polymerization is another expensive component. Taq polymerase will work in many situations, but is not usually recommended because it is more prone to creating errors in the sequence.

iGEM
Since being published in 2009, Gibson assembly has begun to be utilized by many undergraduate iGEM competition teams around the world. One of the first notable teams to explore the use of the method was the Cambridge team from 2010. The team used Gibson assembly to construct bioluminescent E. coli, and in the process created a free web tool using the python programming language called Gibthon. Their tool allows sequence input and construct design in a simplified CAD form, as well as a tool for primer design. Additionally, the team submitted a BioBricks Foundation Request for Comment, RFC 57, outlining the use of Gibson assembly as a standardized assembly method.