Use of In-Fusion Cloning for Simple and Efficient Assembly of Gene Constructs No restriction enzymes or ligation reactions necessary

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1 No restriction enzymes or ligation reactions necessary Background The creation of genetic circuits and artificial biological systems typically involves the use of modular genetic components biological parts that can be arranged to form more complex systems. Several groups have attempted to create standardized formats to streamline the assembly of these biological parts. The BioBrick assembly standard was the first attempt to standardize biological parts to allow a single assembly method; it has become the basis for many similar formats (1 4). Basic BioBrick standard biological parts consist of a DNA sequence encoding a particular genetic component, such as a promoter or transcription termination signal. Each part is flanked by specific unique restriction sites and stored in a plasmid that is distributed by the Registry of Biological Parts. Most BioBrick-based assembly formats require repeated restriction enzyme digestion and ligation reactions to create increasingly complex parts (Figure 1, Panel A); the ligation of two parts generates a scar sequence between the parts that contains neither of the original restriction sites. The result is a composite part that is flanked by the same unique restriction sites as the original parts, so assembly of increasingly larger, more complex parts is possible. Challenges Each of the BioBrick-based assembly methods has advantages and disadvantages. The most common assembly standards are relatively time-consuming and labor intensive, requiring the plasmids containing each BioBrick part to be amplified by transformation into bacteria, growth of an overnight bacterial culture, and plasmid purification. Standard assembly also requires the performance of tedious restriction enzyme digestions, gel electrophoresis and purification of the digested DNA fragments, and ligation reactions. In addition, each of these methods leaves a scar sequence that is not always benign. For example, the scar sequence produced by the original BioBrick assembly standard encodes a stop codon that prevents the creation of fusion proteins. In-Fusion BioBrick Assembly Clontech s In-Fusion Cloning Kit provides a simple and efficient method for assembling and re-engineering BioBricks; this fast, flexible assembly method works without the use of restriction enzymes or ligation reactions, and greatly simplifies the creation of fusion proteins. In-Fusion cloning lets you simultaneously combine one or more products with a linearized vector when the DNA to be joined shares at least 15 bp of homology at each end. The required homology can be easily generated by adding complementary sequence to the ends of the primers. In-Fusion BioBrick assembly is faster, more flexible, and has a higher success rate than standard assembly. Once the primers are made, BioBrick parts can be immediately -amplified from the samples on the BioBrick Parts Distribution plate (Figure 2). In-Fusion Assembly allows several parts to be combined in a variety of different ways because it doesn t require the individual parts to exist as separate BioBricks, or previously combined parts to exist in the correct order. This lets you -amplify any sequence from any part, then assemble the products together in one step. It also allows you to easily engineer mutations using mutagenic primers (5). Once you have the products, the In-Fusion reaction takes only 30 minutes to complete. The In- Fusion reaction is then transformed into bacterial cells and plated. Nearly every assembly reaction yields the desired construct (6). Primer Design Two simple primer design rules allow successful, semi-standardized BioBrick assembly: (i) the reverse primer for () should contain the reverse complement of the following: the last 20 bases of, a scar sequence (if desired), and the first 20 bases (Figure 2); and (ii) the forward primer for () should contain the reverse complement of the primer. This gives a total of 40 bp of homology at the junction of Parts A and B. In contrast, the forward primer of () and the reverse primer of () do not need to be customized, as they are homologous to the standard BioBrick sequence immediately upstream () or downstream () of each part, and may be re-used when assembling different constructs (6). Figures 2 4 illustrate a variety of ways In-Fusion can be used in BioBrick Assembly. In-Fusion assembly is ideal when engineering a circuit with specific parts distributed among several BioBricks, or re-engineering an existing BioBrick with many parts. In- Fusion BioBrick assembly is an especially useful method for small synthetic biology labs that don t perform high throughput restriction enzyme digestions and ligations. In addition, In- Fusion assembly works regardless of the format used to create the BioBrick-based parts (6). We would like to thank Dr. Sean Sleight for allowing us to share his In-Fusion BioBrick assembly method with our customers. For more information, please see: Sleight, S. C. et al. (2010) In-Fusion BioBrick assembly and re-engineering. Nucleic Acids Res. 38(8): References 1. Knight, T. F. (2003) DSpace: 2. Shetty, R. P. et al. (2008) J Biol Eng. 2: Phillips, I. E. and Silver, P. A. (2006) DSpace: 4. Anderson, J. C. et al. (2010) J. Biol. Eng. 4(1): Zhu, B. et al. (2007) Biotechniques 43(3): Sleight, S. C. et al. (2010) Nucleic Acids Res. 38(8): Notice to Purchaser The use of these products and technologies may be governed by one or more licensing agreements. To view specific and current licensing information for any Clontech product, please consult the product s webpage on our website at BioBrick is a trademark of the BioBricks Foundation. Clontech Laboratories, Inc. A Takara Bio Company United States/Canada: Asia Pacific: Europe: +33.(0) Japan: +81.(0) For Research Use Only. Not for use in diagnostic or therapeutic procedures. Not for resale. Clontech, the Clontech logo, and all other trademarks are the property of Clontech unless noted otherwise Clontech Laboratories, Inc. AN US (633220)

2 EX EX Digest with EcoRI & SpeI Digest with EcoRI & XbaI E X S E X Ligate E X Scar Sequence Figure 1. Standard BioBrick assembly. Standard assembly of two BioBricks (Parts A and B) requires restriction digestion and ligation. In this schematic, the plasmid containing is digested with EcoRI (E) and SpeI (S), and the plasmid containing is digested with EcoRI (E) and XbaI (X). The digested fragments are gel purified and ligated together to create the new circuit. Although SpeI and XbaI have different recognition sites, they leave compatible sticky ends that allow ligation of the digested fragments. The resulting scar sequence is not recognized by either enzyme; however, the restriction sites flanking new construct are maintained. 2 Clontech Laboratories, Inc.

3 Figure 2. In-Fusion BioBrick assembly. In-Fusion assembly of two BioBricks requires, a brief purification step, and a subsequent In-Fusion reaction. Parts A and B are -amplified and purified. Each assembly requires four primers: two of these primers are specific for the junction between the assembled parts, and the other two ( and ) are general vector primers. The junction-specific primers are color-coded to indicate regions of homology. The orange portion indicates either (i) a scar sequence like the one that results between parts after standard BioBrick assembly, if desired; (ii) a fusion protein linker sequence; or (iii) regular vector sequence. The all black arrows ( and ) indicate homology with the BioBrick vectors just upstream or downstream of a particular part. The purified products are fused together in the In-Fusion reaction to create a circular plasmid. Restriction sites flanking the parts maintain the standard BioBrick format. Clontech Laboratories, Inc. 3

4 Figure 3. Part-Swapping with In-Fusion. One strategy for using In-Fusion to swap the coding and transcription termination sequences in with the corresponding components in : Use primers and to amplify DNA containing the promoter (green arrow) and vector from. At the same time, use primers and to amplify the insert containing a protein coding sequence (blue arrow) and a transcription terminator (red hexagon) from. During the In-Fusion reaction, the products undergo homologous recombination to form a new circuit. The use of different primers allows different circuits to be formed from the same parts. The primers are described in the caption to Figure 2. 4 Clontech Laboratories, Inc.

5 CF CR Part C Part C Figure 4. Multiple ssembly with In-Fusion. One strategy for combining three parts located in three different plasmids: The inserts (promoter; green arrow) and (protein coding sequence; blue arrow) are amplified using primers and, and and, respectively. The vector, including Part C (transcription termination signal; red hexagon), is amplified using primers CF and CR. All three products are combined in a single In-Fusion reaction: a new circuit forms when the homologous sequences at the ends of the products undergo recombination. Different circuits can be formed from the same parts simply by using different primers. Clontech Laboratories, Inc. 5