Gibson Assembly®

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

Gibson Assembly Workflow

GibsonOverview
Gibson Assembly employs three enzymatic activities in a single-tube reaction: 5´ exonuclease, the 3´ extension activity of a DNA polymerase and DNA ligase activity. The 5´ exonuclease activity chews back the 5´ end sequences and exposes the complementary sequence for annealing. The polymerase activity then fills in the gaps on the annealed regions. A DNA ligase then seals the nick and covalently links the DNA fragments together. The overlapping sequence of adjoining fragments is much longer than those used in Golden Gate Assembly, and therefore results in a higher percentage of correct assemblies. The NEB Gibson Assembly Master Mix (NEB #E2611) and Gibson Assembly Cloning Kit (NEB #E5510S) enable rapid assembly at 50˚C.



Reference:

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

Protocols for Gibson Assembly®

    Publications related to Gibson Assembly®

  1. 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), PubMedID: 24959661, DOI: 10.1371/journal.pone.0100510
  2. 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), PubMedID: 25333968, DOI: 10.1371/journal.pone.0110603
  3. 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), PubMedID: 24509725, DOI: 10.1093/molbev/msu071
  4. 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), PubMedID: 24509725, DOI: 10.1093/molbev/msu071
  5. 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), PubMedID: 24586888, DOI: 10.1371/journal.pone.0089580
  6. 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), PubMedID: 24945275, DOI: 10.1371/journal.pone.0100268
  7. 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, PubMedID: 24675426, DOI: 10.1038/srep04513
  8. 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), PubMedID: 24532776, DOI: 10.1128/JB.01258-13
  9. Schöner TA, Fuchs SW, Reinhold-Hurek B, Bode HB 2014. Identification and Biosynthesis of a Novel Xanthomonadin-Dialkylresorcinol-Hybrid from Azoarcus sp. BH72 PLoS One. 9(3), PubMedID: 24618669, DOI: 10.1371/journal.pone.0090922
  10. 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), PubMedID: 24586168, DOI: 10.1371/journal.ppat.1003966
  11. 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), PubMedID: 24410804, DOI: 10.1186/2191-0855-4-2
  12. 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), PubMedID: 24318960, DOI: 10.1074/jbc.M113.530725
  13. Gutjahr A, Xu SY 2014. Engineering nicking enzymes that preferentially nick 5-methylcytosine-modified DNA Nucleic Acids Res. 42(9), PubMedID: 24609382, DOI: 10.1093/nar/gku192
  14. 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), PubMedID: 24770324, DOI: 10.1038/nbt.2909
  15. 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), PubMedID: 24721788, DOI: 10.3390/v6041637
  16. 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), PubMedID: 24045017, DOI: 10.1105/tpc.113.113985
  17. 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), PubMedID: 23847100, DOI: 10.1093/nar/gkt605
  18. 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 homologue of SEEDSTICK Nature. 500(7462), PubMedID: 23883930, DOI: 10.1038/nature12356.
  19. Chen C, Fenk LA, de Bono M 2013. Efficient genome editing in Caenorhabditis elegans by CRISPR-targeted homologous recombination Nucleic Acids Res. 41(20), PubMedID: 24013562, DOI: 10.1093/nar/gkt805
  20. 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), PubMedID: 23625846, DOI: 10.1128/JB.00257-13
  21. 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), PubMedID: 23460208, DOI: 10.1093/nar/gkt135

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  1. GibsonOverviewVideo_thumb

    Introduction to Gibson Assembly® E5510 E2611

    Watch an interactive tutorial that details the process by which Gibson Assembly® joins DNA fragments in a single tube, isothermal reaction.

  2. PrimerDesignFragmentAssembly_720

    Primer Design and Fragment Assembly Using NEBuilder® HiFi DNA Assembly or Gibson Assembly® E5510 E5520

    Watch an interactive tutorial on primer design to see how simple it really is to clone with either NEBuilder HiFi DNA Assembly or the Gibson Assembly Cloning Kit.

  3. GA_JoVEVideo_282x210_thumb

    Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1α Promoter

    See how Petar & Clinton, NEB/JoVE video abstract contest winners, have been using NEB's Gibson Assembly products in their laboratory!