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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.

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