2624–2636 Nucleic Acids Research, 2010, Vol. 38, No. 8 doi:10.1093/nar/gkq179 Published online 12 April 2010 In-Fusion BioBrick assembly and re-engineering Sean C. Sleight*, Bryan A. Bartley, Jane A. Lieviant and Herbert M. Sauro Department of Bioengineering, University of Washington, Seattle, WA 98195, USA Received November 30, 2009; Revised March 1, 2010; Accepted March 2, 2010 ABSTRACT Genetic circuits can be assembled from standardized biological parts called BioBricks. Examples of BioBricks include promoters, ribosome-binding sites, coding sequences and tran- scriptional 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 inser- tion, and three-way In-Fusion assemblies. INTRODUCTION Synthetic biology is an emerging discipline that aims to design and construct novel biological organisms programmed by genetic circuits. Many synthetic biologists assemble genetic circuits from standardized biological parts called BioBricks. Examples of BioBricks include promoters, ribosome-binding sites (RBS), protein or RNA-coding sequences, and transcriptional terminators. Currently every BioBrick is a physical DNA sequence on a circular plasmid that is stored and distributed by the Registry of Biological Parts (http://www.partsregistry .org) as lyophilized DNA in 384-well plate format. Standardized sequences on BioBricks enable Standard Assembly of two BioBricks via restriction enzyme diges- tion and ligation in an idempotent fashion (Figure 1a) (1–5). Standard Assembly involves digestion of two plasmids with diﬀerent restriction enzymes that leave com- patible sticky ends which can be ligated together into a new plasmid. This eﬀectively replaces the restriction sites between the assembled parts with a ‘scar’ sequence, allowing for the new BioBrick to be later combined with other BioBricks. This standardized procedure takes much of the planning out of DNA fragment assembly since the same restriction enzymes can be used for every assembly reaction. Currently several BioBrick assembly standards (http://openwetware.org/wiki/The_BioBricks_Foundation :RFC) have been proposed to improve upon the original BioBrick standard, largely due to the fact that this original standard produces an 8-bp scar between assembled BioBricks and hence does not allow for the creation of fusion proteins. Nearly all of these current assembly standards involve assembly by restriction enzyme diges- tion and ligation. There are also several PCR-based methods currently being used for DNA assembly that have the potential for standardization. These methods include In-Fusion (6,7), SLIC (8), T5 exonuclease recom- bination (9), USER (10), oligonucleotide assembly (11) and SOEing (12). The former four methods generally involve converting overlapping, blunt-end PCR products into fragments with sticky overhangs that can anneal to form circular plasmids, but the method for generating the overhangs diﬀers. For example, the SLIC method (8) uses T4 DNA polymerase while the USER method (10) uses a uracil exonuclease. Unlike restriction digestion, the site at which the overhangs are created is generally not con- strained by a speciﬁc sequence. The latter two methods involve overlapping oligonucleotides with a PCR- ampliﬁed vector (11) or extending overlapping PCR products (12) and do not use subsequent enzymatic treatment. Here, we describe an alternative BioBrick assembly method that allows for BioBricks to be quickly assembled and re-engineered using the Clontech In-Fusion PCR Cloning Kit (6,7). The proprietary In-Fusion enzyme with exonuclease activity fuses together any PCR *To whom correspondence should be addressed. Tel: +1 206 616 1928; Fax: +1 206 685 3300; Email: sleight@u.washington.edu The Author(s) 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.