DNA Assembly

Workpackage D: Genome Engineering

Hierarchical DNA assembly methods are a necessary part of genome construction and modification. Large-scale assemblies require specialized cloning vehicles like artificial chromosomes for construction and transfer of genetic circuits to target organisms. Recent developments are producing efficient tools for modifying chromosomal sequences in situ.

 OpenPlant is setting standards for plant synthetic biology and establishing registries for sharing of plant­-specific DNA parts. We are also creating standard tools for the engineering of plant genomes. Development of Phytobrick and UNS standards for efficient hierarchical assembly of DNA circuits extends DNA Assembly using Gibson assembly and higher order Mo­Clo and Golden Braid techniques.

Type IIS restriction endonuclease mediated cloning

An introduction to Type IIS restriction enzymes and how they differ from classical Type II restriction enzymes.

  • Type IIS-mediated assembly methods are based on the original description of "Golden Gate" cloning and allows efficient simultaneous assembly of multiple DNA parts, without requiring the isolation of individual fragments.

Nicola Patron has assembled a short online course on Type IIS that covers:

  • How type IIS restriction enzymes enable a one-pot reaction
  • How to easily and reliably assemble multiple standard parts in a single reaction, and
  • How to make new parts

You can order the Universal Acceptor Plasmid, pUAP1 and other plasmids via Addgene.

Image credit: DNA by MIKI Yoshihito on Flickr, licensed under CC-BY 2.0