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 2624���2636 Nucleic Acids Research, 2010, Vol. 38, No. 8 Published online 12 April 2010 doi:10.1093/nar/gkq179 �� 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.

Figure 1. Standard assembly versus In-Fusion assembly. (a) Standard Assembly of two BioBricks (Parts A and B) involves restriction digestion and ligation. Both parts are on pSB1A2 vectors encoding ampicillin resistance. The Part A plasmid is digested with EcoRI (E) and SpeI (S), while the second plasmid is digested with EcoRI (E) and XbaI (X). SpeI and XbaI restricted fragments have compatible sticky ends for ligation. The desired digested fragments are gel purified and ligated together to create the assembled plasmid with both parts. A scar sequence is left between both parts that does not have the original restriction site and the restriction sites flanking the parts are maintained. (b) In-Fusion assembly of two BioBricks involves PCR, purification, and a subsequent In-Fusion reaction. Parts A and B are PCR-amplified (in this example the vector is amplified with Part B) and purified without gel extraction. Each assembly requires four primers, where two are specific to the junction (parts to assemble) and two are general vector primers. BioBrick Part A (blue) and Part B (red) are on pSB1A2 plasmids encoding ampicillin resistance. Primers described in ���Materials and Methods��� section are color-coded to show their homology. The thick black line indicates BioBrick prefix or su���x homology on the pSB1A2 vector. The yellow sequence is the scar that is normally between parts after standard BioBrick assembly, if this is desired, or can be a linker sequence for a fusion protein. The purified PCR products are fused together in the In-Fusion reaction to create a circular plasmid. Restriction sites flanking the parts maintain the standard BioBrick format. Nucleic Acids Research, 2010, Vol. 38, No. 8 2625