Gibson Assembly®

  • My NEB
  • Print
  • PDF
  • 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). NEB also offers a higher fidelity line of DNA assembly products, NEBuilder HiFi DNA Assembly Master Mix (NEB #E2621) and NEBuilder HiFi DNA Assembly Cloning Kit (NEB #E5520). Learn more at NEBuilderHiFi.com

    1. Introduction to Gibson Assembly

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

    2. Primer Design and Fragment Assembly Using NEBuilder HiFi DNA Assembly™ or Gibson Assembly™

      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. 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!

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

    Gibson Assembly Workflow

    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.

    Try NEBuilder® Assembly Tool

    Reference:

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

    Featured Products

    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), e100510. PubMedID: 24959661, DOI: 10.1371/journal.pone.0100510
    2. 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
    3. 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
    4. 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
    5. 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
    6. 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), e90922. PubMedID: 24618669, DOI: 10.1371/journal.pone.0090922
    7. 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
    8. 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
    9. 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
    10. 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
    11. 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
    12. 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
    13. 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
    14. 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
    15. 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
    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), 3450-71. PubMedID: 24045017, DOI: 10.1105/tpc.113.113985
    17. Chen C, Fenk LA, de Bono M (2013)Efficient genome editing in Caenorhabditis elegans by CRISPR-targeted homologous recombination Nucleic Acids Res 41(20), e193. PubMedID: 24013562, DOI: 10.1093/nar/gkt805
    18. 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
    19. 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
    20. 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
    21. 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), 340-4. PubMedID: 23883930, DOI: 10.1038/nature12356.