Gibson Assembly
Introduction
Gibson Assembly[1] is a straightforward cloning technique that allows the combination of multiple DNA fragments in a single isothermal reaction.[2][3] It can assemble up to 15 different fragments in one reaction, is easy to set up, and requires only a few basic components.[3] Successful assembly requires overlapping sequences of approximately 20 bp between fragments (Fig. 1).
Components necessary for the Gibson Assembly reaction and their Functions[4]:
Exonuclease – digests the 5’ ends of DNA fragments, creating single-stranded 3’ overhangs. These complementary overhangs in the overlapping regions allow the DNA fragments to anneal precisely to each other.
DNA Polymerase – fills in gaps by synthesizing DNA complementary to the annealed strand, completing the sequence.
DNA Ligase – seals the remaining nicks, generating a continuous DNA molecule.
The combined action of these three enzymes produces a single, continuous DNA fragment, which can be circularized to form plasmids.
Schematic Workflow
Applications in the NRPieceS Project
We applied Gibson Assembly to create plasmids for heterologous expression and the acceptor and donor plasmids to build the basis for Golden Gate Assembly to generate our library.
References
[1] Gibson et al., 2009
[2] Gibson, D. G., Young, L., Chuang, R.-Y., Venter, J. C., Hutchison, C. A., & Smith, H. O. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods, 6(5), 343–345. https://doi.org/10.1038/nmeth.1318
[3] Avilan, L. (2023). Assembling Multiple Fragments: The Gibson Assembly. Methods in molecular biology, 2633, 45-53. https://doi.org/10.1007/978-1-0716-3004-4_4
[4] Chiocchini, C. (2020). Evaluation of GeneArt Gibson Assembly EX Cloning technology to build large and complex assemblies. Thermo Fisher Scientific White Paper. https://assets.fishersci.com/TFS-Assets/LSG/manuals/MAN0019063_GeneArtGibsonEX_UG.pdf
