In-Fusion BioBrick assembly and re-engineering

Nucleic Acids Res. 2010 May;38(8):2624-36. doi: 10.1093/nar/gkq179. Epub 2010 Apr 12.

Abstract

Genetic circuits can be assembled from standardized biological parts called BioBricks. Examples of BioBricks include promoters, ribosome-binding sites, coding sequences and transcriptional terminators. Standard BioBrick assembly normally involves restriction enzyme digestion and ligation of two BioBricks at a time. The method described here is an alternative assembly strategy that allows for two or more PCR-amplified BioBricks to be quickly assembled and re-engineered using the Clontech In-Fusion PCR Cloning Kit. This method allows for a large number of parallel assemblies to be performed and is a flexible way to mix and match BioBricks. In-Fusion assembly can be semi-standardized by the use of simple primer design rules that minimize the time involved in planning assembly reactions. We describe the success rate and mutation rate of In-Fusion assembled genetic circuits using various homology and primer lengths. We also demonstrate the success and flexibility of this method with six specific examples of BioBrick assembly and re-engineering. These examples include assembly of two basic parts, part swapping, a deletion, an insertion, and three-way In-Fusion assemblies.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • DNA / chemistry
  • DNA Primers
  • Drug Resistance, Microbial / genetics
  • Genetic Engineering / methods*
  • Mutation
  • Polymerase Chain Reaction
  • Promoter Regions, Genetic
  • Recombinant Fusion Proteins / genetics
  • Ribosomes / metabolism
  • Sequence Homology, Nucleic Acid
  • Terminator Regions, Genetic

Substances

  • DNA Primers
  • Recombinant Fusion Proteins
  • DNA