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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

The world calls for a better Assembly System

The Standard Assembly of BioBricks make use of restrictionsites for four restriction enzymes, which means that a functional iGEM plasmid should only contain the four given restriction sites. If if other restriction sites are present, they have to be eliminated by alterations like site-directed mutagenesis, which can be both time consuming and cause unwanted alterations. Restriction sites have to be completely avoided in the BioBrick and the BioBrick devices, which can be a problem when making new BioBricks from natural sources. Also when assembling the BioBricks with the Standard Assembly System scars occur, which makes it impossible to create fusion proteins. Additionally, the Standard Assembly of BioBricks is limited in that it is only possible to put two things together at a time.

All in all, the iGEM competition and the fast growing field of synthethic biology calls for a simpler, faster and more efficient assembly system that are easily applied to both bacteria, fungi, and mammalian cells.

USER cloning

In early 1990s, the uracil excision-based (USER) cloning was invented as a ligation-independent cloning technique that could substitute the conventional assembly systems that made use of restriction enzymes and ligase. In 2003 New England Biolabs (NEB) introduced the USER Friendly Cloning Kit. Although NEBs USER kit was simple and efficient, it was not compatible with proofreading polymerases that stalled when encountering a uracil base in the DNA template (2. New England Biolabs, 2004). This made the USER Friendly Kit unattractive, although the concept was brilliant. In recent years, proofreading polymerases have been developed that are compatible with the concept of USER cloning, since they can read through uracil (1. Nour-Eldin HH etc)..

The USER method applies long complementary overhangs on the PCR product(s) as well as on the destination vector. The overhangs on the PCR product are custom made, between 7-15 nucleotides long and deoxy uridine nucleotides substitute selected deoxy thymidine nucleotides. The PCR products containing the customized overhangs are treated with the USER enzyme, which is a mix of DNA glycosidase and DNA glycosylase-lyase endo VIII. This treatment results in release of the DNA sequence upstream the deoxy uridine nucleotide and the resulting exposed overhangs can anneal to each other to form a stable hybridization product. This product can now be transformed directly into E.coli without prior ligation (1,2). In order to avoid template carry-over after PCR, the PCR product is usually treated with the restriction enzyme DpnI. DpnI cleaves only when its recognition site is methylated. Unmethylated PCR-derived DNA will be left intact (1)……

Plug 'n' Play with DNA

DTU-Denmark-2 introduces a customized system called "Plug ‘n’ Play with DNA", where certain categories of biological parts can be gathered. This means that the parts in the form of pre-produced PCR-products are directly mixed with a vector, which makes assembly of an expression vector possible within a very short time. All the parts in the form of PCR-products will be distributed in microtiter plates directly ready for cloning.Furthermore, the “Plug’n’Play” kit will contain a back-up where all parts are contained on a plasmid to ensure amplification of a mutation free template if needed. The simple and easy use of the system will be demonstrated by developing a reporter targeting system for the fungus Aspergillus niger. Furthermore, will we demonstrate its application in mammalian cells.

References

(1) Hussam H. Nour-Eldin, Fernando Geu-Flores, and Barbara A. Halkier. USER Cloning and USER Fusion: The Ideal Cloning Techniques for Small and Big Laboratories. Methods in Molecular Biology 643.

(2) Nørholm, M. H. H. A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering. BMC Biotechnol. 10, 21 (2010).