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From 2011.igem.org

• Home
• About us
  • The Team
  • Supervisors
  • Gallery
• The Project
  • Abstract
  • Introduction
  • The Plug 'n' Play
    • Assembly system
    • Customization
  • Other assembly systems
  • Achievements
  • Attributions
• Results
  • Background
  • Characterization
  • Proof of concept
    • Mammalian Cells
    • Fungi
  • Data page
  • Submitted Biobricks
  • Collaboration
• Safety
  • Safety
  • Safety rules
• Notebook
  • Lab notes
  • Protocols
• Our Partners
  • Sponsors
  • Advisors
  • Acknowledgements

	1 Gateway system 2 Gibson Assembly 3 In fusion 4 Sandwich 5 3A	Gibson Assembly Gibson Assembly is an isothermal, single-reaction method for assembling multiple overlapping DNA molecules. The method was developed by Daniel G. Gibson at the J. Craig Venter Institute in 2009. The assembly system employs 5´-T5 exonuclease, Phusion DNA polymerase, and Taq lig. Gibson can be used to assemble both ssDNA and dsDNA fragments. This methode makes it possible to join DNA molecules there are as large as 583kb and clone joined products in ''E. coli'' with a length up to 300kb. Among the advantages is that it takes the same amount of time to ligate n DNA fragments than two. The way it's done The isothermal One-step The 5´-T5 exonuclease removes the bases from the 5'-end of double strained DNA molecule, leaving a recess in the DNA. The ssDNA overhang is used to assemble the DNA fragments. The T5 exonuclease are inactivated during the incubation at 50C. Phusion polymerase and Taq ligase fills the gaps of the annealed complementary ssDNA overhangs and seals the nicks in the end, leaving a joined DNA molecules. Gateway assembly MultiSite Gateway Assembly enables the assembly of multiple DNA fragments by utilizing site-specific recombination. The MultriSite Gateway Assambly can clone up to 4 DNA fragments into one vector with the use of a flanked DOI by ''att'' sites.