Gibson Assembly® | NEB (2024)

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(1). 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(2).

To help select the best DNA assembly method for your needs, please use our Synthetic Biology/DNA Assembly Selection Chart.

Have you tried NEBuilder HiFi DNA Assembly? NEBuilder HiFi offers several advantages over NEB Gibson Assembly. For more information, visit NEBuilderHiFi.com.

Reference:

1. Gibson, D.G., et al. (2009) Nat. Methods 6, 343-345.
2. Gibson, D.G., et al. (2010) Science 329, 52-56.

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Protocols
Application Notes
Publications
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Protocols for Gibson Assembly®

PCR Protocol Phusion® DNA Polymerase
Gibson Assembly® Master Mix – Assembly (E2611)
Gibson Assembly® Protocol (E5510)
Gibson Assembly® Chemical Transformation Protocol (E5510)
Gibson Assembly® Chemical Transformation Protocol (E2611)
Gibson Assembly® Electrocompetent Transformation Protocol (E2611)
Gibson Assembly® Electrocompetent Transformation Protocol (E5510)
PCR Using Q5® High-Fidelity DNA Polymerase (M0491)
Protocol for T5 Exonuclease (M0363)
Protocol for Taq DNA Ligase (M0208)

Application Notes for Gibson Assembly®

Improved methods for site-directed mutagenesis using Gibson Assembly® Master Mix

Tools & Resources

Feature Articles

Restriction Endonucleases: Molecular Cloning and Beyond

Brochures

Molecular Cloning Technical Guide

Selection Tools

Synthetic Biology/DNA Assembly Selection Chart

Troubleshooting Guides

Troubleshooting Guide for Cloning

Publications related to Gibson Assembly®

Vandergaast R, Hoover LI, Zheng K, Fredericksen BL(2014)Generation of West Nile virus infectious clones containing amino acid insertions between capsid and capsid anchor Viruses;6(4), 1637-53. PubMedID: 24721788,DOI: 10.3390/v6041637
Meinke G, Phelan PJ, Kalekar R, Shin J, Archambault J, Bohm A, Bullock PA(2014)Insights into the initiation of JC virus DNA replication derived from the crystal structure of the T-antigen origin binding domain PLoS Pathog;10(2), e1003966. PubMedID: 24586168,DOI: 10.1371/journal.ppat.1003966
Lipscomb GL, Schut GJ, Thorgersen MP, Nixon WJ, Kelly RM, Adams MW(2014)Engineering hydrogen gas production from formate in a hyperthermophile by heterologous production of an 18-subunit membrane-bound complex J Biol Chem;289(5), 2873-9. PubMedID: 24318960,DOI: 10.1074/jbc.M113.530725
Law SH, Sargent TD(2014)The Serine-Threonine Protein Kinase PAK4 Is Dispensable in Zebrafish: Identification of a Morpholino-Generated Pseudophenotype PLoS One;9(6), e100268. PubMedID: 24945275,DOI: 10.1371/journal.pone.0100268
Li Y, Thompson CM, Lipsitch M(2014)A Modified Janus Cassette (Sweet Janus) to Improve Allelic Replacement Efficiency by High-Stringency Negative Selection in Streptococcus pneumoniae PLoS One;9(6), e100510. PubMedID: 24959661,DOI: 10.1371/journal.pone.0100510
Gai CS, Lu J, Brigham CJ, Bernardi AC, Sinskey AJ(2014)Insights into bacterial CO2 metabolism revealed by the characterization of four carbonic anhydrases in Ralstonia eutropha H16 AMB Express;4(1), 2. PubMedID: 24410804,DOI: 10.1186/2191-0855-4-2
Schöner TA, Fuchs SW, Reinhold-Hurek B, Bode HB(2014)Identification and Biosynthesis of a Novel Xanthom*onadin-Dialkylresorcinol-Hybrid from Azoarcus sp. BH72 PLoS One;9(3), e90922. PubMedID: 24618669,DOI: 10.1371/journal.pone.0090922
Ikmi A, Gaertner B, Seidel C, Srivastava M, Zeitlinger J, Gibson MC(2014)Molecular evolution of the Yap/Yorkie proto-oncogene and elucidation of its core transcriptional program Mol Biol Evol;31(6), 1375-90. PubMedID: 24509725,DOI: 10.1093/molbev/msu071
Ikmi A, Gaertner B, Seidel C, Srivastava M, Zeitlinger J, Gibson MC(2014)Molecular evolution of the Yap/Yorkie proto-oncogene and elucidation of its core transcriptional program Mol Biol Evol;31(6), 1375-90. PubMedID: 24509725,DOI: 10.1093/molbev/msu071
Phelan VV, Moree WJ, Aguilar J, Cornett DS, Koumoutsi A, Noble SM, Pogliano K, Guerrero CA, Dorrestein PC(2014)Impact of a transposon insertion in phzF2 on the specialized metabolite production and interkingdom J Bacteriol;196(9), 1683-93. PubMedID: 24532776,DOI: 10.1128/JB.01258-13
Chinnici JL, Fu C, Caccamise LM, Arnold JW, Free SJ(2014)Neurospora crassa Female Development Requires the PACC and Other Signal Transduction Pathways, Transcription Factors, Chromatin Remodeling, Cell-To-Cell Fusion, and Autophagy PLoS One;9(10), e110603. PubMedID: 25333968,DOI: 10.1371/journal.pone.0110603
Guilinger JP, Thompson DB, Liu DR(2014)Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification Nat Biotechnol;32(6), 577-82. PubMedID: 24770324,DOI: 10.1038/nbt.2909
Gutjahr A, Xu SY(2014)Engineering nicking enzymes that preferentially nick 5-methylcytosine-modified DNA Nucleic Acids Res;42(9), e77. PubMedID: 24609382,DOI: 10.1093/nar/gku192
Royce LA, Boggess E, Fu Y, Liu P, Shanks JV, Dickerson J, Jarboe LR(2014)Transcriptomic analysis of carboxylic acid challenge in Escherichia coli: beyond membrane damage PLoS One;9(2), e89580. PubMedID: 24586888,DOI: 10.1371/journal.pone.0089580
Horii T, Arai Y, Yamazaki M, Morita S, Kimura M, Itoh M, Abe Y, Hatada I(2014)Validation of microinjection methods for generating knockout mice by CRISPR/Cas-mediated genome engineering Sci Rep;4, 4513. PubMedID: 24675426,DOI: 10.1038/srep04513
DiCarlo JE, Norville JE, Mali P, Rios X, Aach J, Church GM(2013)Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems Nucleic Acids Res;41(7), 4336-43. PubMedID: 23460208,DOI: 10.1093/nar/gkt135
Guye P, Li Y, Wroblewska L, Duportet X, Weiss R(2013)Rapid, modular and reliable construction of complex mammalian gene circuits Nucleic Acids Res;41(16), e156. PubMedID: 23847100,DOI: 10.1093/nar/gkt605
Singh R, Low ET, Ooi LC, Ong-Abdullah M, Ting NC, Nagappan J, Nookiah R, Amiruddin MD, Rosli R, Manaf MA, Chan KL, Halim MA, Azizi N, Lakey N, Smith SW, Budiman MA, Hogan M, Bacher B, Van Brunt A, Wang C, Ordway JM, Sambanthamurthi R, Martienssen RA(2013)The oil palm SHELL gene controls oil yield and encodes a hom*ologue of SEEDSTICK Nature;500(7462), 340-4. PubMedID: 23883930,DOI: 10.1038/nature12356.
Ramirez-Peralta A, Gupta S, Butzin XY, Setlow B, Korza G, Leyva-Vazquez MA, Christie G, Setlow P(2013)Identification of new proteins that modulate the germination of spores of bacillus species J Bacteriol;195(13), 3009-21. PubMedID: 23625846,DOI: 10.1128/JB.00257-13
Chen C, Fenk LA, de Bono M(2013)Efficient genome editing in Caenorhabditis elegans by CRISPR-targeted hom*ologous recombination Nucleic Acids Res;41(20), e193. PubMedID: 24013562,DOI: 10.1093/nar/gkt805
Ng S, Ivanova A, Duncan O, Law SR, Van Aken O, De Clercq I, Wang Y, Carrie C, Xu L, Kmiec B, Walker H, Van Breusegem F, Whelan J, Giraud E(2013)A membrane-bound NAC transcription factor, ANAC017, mediates mitochondrial retrograde signaling in Arabidopsis Plant Cell;25(9), 3450-71. PubMedID: 24045017,DOI: 10.1105/tpc.113.113985

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