You could begin by reading the detailed information on their respective websites and if and when you have a more specific question, ask for assistance. 

Both methods are based on homolog fragment ends and a single-tube enzymatic reaction. The difference is in the composition of the enzyme mix (e.g. Exonuclease, DNA Polymerase and DNA Ligase) which has to be carefully balanced to achieve a high cloning efficiency.

In-Fusion HD Cloning Kits are designed for fast, directional cloning of one or more fragments of DNA into any vector. The cornerstone of In-Fusion cloning technology is Clontech’s proprietary In-Fusion Enzyme, which fuses DNA fragments e.g. PCR-generated sequences and linearized vectors, efficiently and precisely by recognizing a 15 bp overlap at their ends. This 15 bp overlap can be engineered by designing primers for amplification of the desired sequences. In-Fusion HD Kits offer increased cloning efficiency over previous generations of In-Fusion Kits, especially for long fragments, short oligonucleotides, and multiple fragments.

• Clone any insert, into any location, within any vector you choose

• Efficiently clone a broad range of fragment sizes

• Clone multiple DNA fragments simultaneously into any vector in a single reaction

• No restriction digestion, phosphatase treatment, or ligation required

• Final constructs are seamless with no extra or unwanted base pairs

Daniel G. Gibson, of the J. Craig Venter Institute, described a robust exonuclease-based method to assemble DNA seamlessly and in the correct order, eponymously known as Gibson Assembly. The reaction is carried out under isothermal conditions using three enzymatic activities: a 5’ exonuclease generates long overhangs, a polymerase fills in the gaps of the annealed single strand regions, and a DNA ligase seals the nicks of the annealed and filled-in gaps. This method has been widely adopted and is a major workhorse of synthetic biology projects worldwide. Applying this methodology, the 16.3 kb mouse mitochondrial genome was assembled from 600 overlapping 60-mers. In combination with in vivo assembly in yeast, Gibson Assembly was used to synthesize the 1.1 Mbp Mycoplasma mycoides genome. The synthesized genome was transplanted to a M. capricolum recipient cell, creating new self-replicating M. mycoides cells.

Dear Mr Jorge，Thanks for you answer. You had made me successfully get through my puzzle. It that true that In-fusion have both assemble and cloning and Gibson only have assemble without cloning occur? 

Dear Dongchang,

I believe your question has not been answered.

Previous anwers just copy-pasted information about both cloning methods directly from the websites.

Wojtek is the closest to a real answer; "Both methods are based on homolog fragment ends and a single-tube enzymatic reaction. The difference is in the composition of the enzyme mix (e.g. Exonuclease, DNA Polymerase and DNA Ligase)".

Both methods use an enzyme mix, and both methods incubate at 50C. There may not be any difference at all. The composition of the enzyme mix could be the same. I didn't find the InFusion enzyme mix composition yet. But it would be interesting if someone can.

some differences:

1) gibson leaves no nick on the product, while infusion does. this usually doesn't matter, unless you want to use the product as a pcr template or for natural transformation. (https://www.neb.com/applications/cloning-and-synthetic-biology/dna-assembly-and-cloning/nebuilder-hifi-dna-assembly/comparison-of-dna-assembly-reaction-types)

2) gibson is more robust, it tolerate 3´- & 5´-end mismatch, while infusion doesn't. so you can use gibson to do nest assembly but not with infusion. this also means gibson has a higher chance to do unwanted assembly. again this is usually not a big problem. most false positive colonies i got when using gibson were actually caused by primer dimer or unspecific pcr products (they have the same overlap ends as the designed products) (https://www.researchgate.net/file.PostFileLoader.html?id=59ade373217e204f872f3e2c&assetKey=AS%3A535011875344385%401504568179549)

3) there is ligase in gibson. so if you use only one restriction enzyme to linearize your plasmid backbone and use it in the assembly. The ligase may repair the backbone back to empty plasmid.

Hi,

In-Fusion and Gibson assembly works on Homology at the ends.

Infusion cloning uses vaccenia virus DNA polymerase which uses its 3' exonuclease activity to remove 15-20 bp and makes 5" overhangs. 15 bp single standard regions of vector and insert anneals and form double standard DNA, single standard dna binding proteins stabilizes the single standard DNA outside the complementary duplex region .There will be a scare (nick)and reaction mixture directly transformed in to E.coli.

Gibson assembly uses three enzymes (5' Exonuclease , phusion DNA polymerase ,T4 DNA Ligase). 5'Exonuclease makes 3' overhangs which different from infusion. After annealing of vector and insert overhangs, phusion polymerase fills the gap and ligase seals it.There is no scare (nick) left in this reaction .

in Gibson Exonuclease activity at 5' end and polymerase activity at 3' end don't cross-talk

finally,

Both methods are single-tube enzymatic reaction based on homolog fragment ends and both methods incubate at 50C.

both works good

Cheers

@Survi Mahesh

in some protocols single standard dna binding protein was not included. They use only 3' exonuclease of DNA polymerase. how important is the ssb do you think?

@Xinglin Jiang

Yes , infusion works with and without single standard DNA binding proteins but addition of ssb increase the efficiency of cloning by stabilising single standard dna outside the complementary region (15 no homology ). Clontrch infusion mastermix do contain ssb in it. clontech infusion mastermix (patent information on web) composition includes ssb .

There is a similar kits available named wit COLD FUSION from system bioscience and GENEART SEAMlESS CLONING from thermo .

Cheers

Dear He,

There has been a lot of confusion and not a single answer for your question about joining vs cloning. Gibson Assembly creates covalently- linked fragments WITH the vector or whatever part you chose to join. If you use vector part, you get a covalently-linked assembly in the vector. Simply speaking, you get a ready-to-transform plasmid. After joining, you do not have any overhang from the vector or your fragments.

In In-fusion cloning, you get a fragment from the joining due to the reaction design. It is a trick they do not want you to know. Also, you get covalently-linked vector with your fragment which you can use for direct transformation.

In summary:

1. Gibson Assembly produces ready-to-transform plasmid with no scars in the vector.

2. In-fusion cloning will produce ready-to-transform plasmid with scars.

Due to size limitation and the number of fragments, Gibson Assembly works for joining 3-4 max fragments up to 10-15 kb in the commercial version from NEB (better than 2 fragments for the In-fusion cloning). Above that, you are out of luck. A commercial solution for overcoming the problem is from ThermoFisher Scientific:

GeneArt™ Seamless PLUS Cloning and Assembly Kit, Catalog number: A14603.

Bacterial systems will uptake up to 40kb DNA while yeast systems- up to 110 kb. If you fiddle enough, you may get bacteria match the yeast for the maximum size uptake.

In-Fusion technology was the clear winner in terms of background, speed, and overall accuracy—especially when more complicated cloning projects were considered. In providing such a high level of cloning accuracy, In-Fusion technology reveals that the real measure of success is not in sheer numbers of colonies, but instead in the number of correct, error-free colonies. Researchers should be able to expect the right clone every time, and In-Fusion Cloning makes that possible. Detailed comparison as below:

https://www.takarabio.com/learning-centers/cloning/in-fusion-cloning-tech-notes/improving-background-over-gibson-assembly

One note, if you want to do cloning of single-stranded oligos, InFusion does NOT work (they mention it in the FAQs) while Gibson does. For the reason, compare the direction of the exonuclease in InFusion vs Gibson.

So the comment above that reads "Both methods use an enzyme mix, and both methods incubate at 50C. There may not be any difference at all. The composition of the enzyme mix could be the same." is not true, enzyme compositions are actually very different in this and more aspects.

Basic Principal of both techniques in similar, they join the overlapping fragments

Gibson is ( 5' exonuclease+ polymerase+ ligase+ buffer optimization)

While infusion( Vaccinia polymerase+ vaccinia SSB protein + buffer optimization)

Infusion is the parental invention, while Gibsons used a similar concept with two enzymes which were available on Vaccinia polymerase only in the infusion

Weak patent claims of Infusion made Gibson assembly tough competitor