title: In-Fusion BioBrick assembly and re-engineering.
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.
authors.affiliation: Department of Bioengineering, University of Washington, Seattle, WA 98195, USA. sleight@u.washington.edu
authors.forename: Sean C
authors.initials: SC
authors.lastname: Sleight
authors.forename: Bryan A
authors.initials: BA
authors.lastname: Bartley
authors.forename: Jane A
authors.initials: JA
authors.lastname: Lieviant
authors.forename: Herbert M
authors.initials: HM
authors.lastname: Sauro
mesh_terms: D004247:DNA / Q000737:chemistry; D017931:DNA Primers; D004352:Drug Resistance, Microbial / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D009154:Mutation; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012270:Ribosomes / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013728:Terminator Regions, Genetic
chemical_list: D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D004247:DNA
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