In-Fusion BioBrick assembly and re-engineering
Open Access
- 10 April 2010
- journal article
- research article
- Published by Oxford University Press (OUP) in Nucleic Acids Research
- Vol. 38 (8), 2624-2636
- https://doi.org/10.1093/nar/gkq179
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.This publication has 17 references indexed in Scilit:
- BglBricks: A flexible standard for biological part assemblyJournal of Biological Engineering, 2010
- USER friendly DNA recombination (USERec): a simple and flexible near homology-independent method for gene library construction"Protein Engineering, Design and Selection", 2009
- Synthesis of DNA fragments in yeast by one-step assembly of overlapping oligonucleotidesNucleic Acids Research, 2009
- Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction EnzymesPLOS ONE, 2009
- Measuring the activity of BioBrick promoters using an in vivo reference standardJournal of Biological Engineering, 2009
- Refinement and standardization of synthetic biological parts and devicesNature Biotechnology, 2008
- Engineering BioBrick vectors from BioBrick partsJournal of Biological Engineering, 2008
- In-Fusion™ assembly: seamless engineering of multidomain fusion proteins, modular vectors, and mutationsBioTechniques, 2007
- USER fusion: a rapid and efficient method for simultaneous fusion and cloning of multiple PCR productsNucleic Acids Research, 2007
- Harnessing homologous recombination in vitro to generate recombinant DNA via SLICNature Methods, 2007