2634 Nucleic Acids Research, 2010, Vol. 38, No. 8 Colony PCR results showed that 4 out of 12 (33%) colonies had the correct construct and three were veriﬁed by sequencing (Figure 5c). Three-way assembly: creation of a fusion protein In this assembly, a luxR-GFP fusion protein is incorporated in the middle of the I731014 transcriptional cascade (Figure 6a and b). As in the previous example, an otherwise complex construction using Standard Assembly is eﬃciently streamlined using the In-Fusion cloning method. The I731014 circuit comprises the backbone assembly component. The GFP-coding sequence and a terminator comprise one of the insert components. The ﬁnal component consists of a linker sequence ampliﬁed from a non-Biobrick vector (pBAD/HisA, Invitrogen). To determine the eﬀect of the vector:inserts ratio for a three-way assembly, we tested 1:2:2, 1:5:5 and 1:10:10 ratios. A 1:2:2 ratio resulted in 0/24 correct constructs, a 1:5:5 ratio resulted in 4 out of 24 (16.7%) successes, and 1:10:10 ratio resulted in 9 out of 24 (37.5%) correct con- structs (Figure 6c). While three-way assemblies generally exhibit a lower success rate compared with two-way assemblies, optimizing the vector:inserts ratio improves the odds of success. Furthermore, this example demon- strates that assembly components representing a wide range of sizes (3.8 kb, 0.8 kb and 0.2 kb) may be simultan- eously assembled in a single assembly step. In this example, the minimum 15 bp of homology was used. It is likely that the success rate of three-way assemblies could be further improved by increasing the length of homology at the junctions. DISCUSSION There are currently several assembly methods used to con- struct plasmids for synthetic biology research or other ap- plications. In the synthetic biology community, Standard Assembly is the most widely used method despite competing standards. Custom DNA synthesis is still too expensive for most synthetic biology labs to perform routine constructs, but is ideal when constructing DNA from scratch when there is no template DNA available for PCR or when there are many assembly reactions to perform (14). Overlap extension PCR methods (11,12) are also useful to construct novel DNA sequences without a template, but can be somewhat expensive de- pending on the length of the construct. For many synthet- ic biologists, the enormous number of parts available in the Registry allows for diverse circuits to be constructed without the use of DNA synthesis or overlap extension PCR methods. In our experience, there are three major advantages to In-Fusion assembly over Standard Assembly. First, In-Fusion provides a ﬂexible method to perform large-scale assemblies by mixing and matching parts from the Registry. Second, this method is faster since gel extraction is unnecessary, there are fewer experimental steps, and fewer reactions to optimize, allowing for many reactions to be performed in parallel. Third, we ﬁnd the success rate for In-Fusion to be high and consistently are able to engineer the desired construct. Our results indicate that the In-Fusion success rate in- creases with the homology length without the cost of frequent mutations in assembled plasmids which we found to be rare. More homology between assembly com- ponents requires longer primer lengths, increasing the primer cost slightly, but the corresponding increase in success rate may justify the extra cost when performing diﬃcult assemblies. We found that in general two-way In-Fusion assemblies have >60% success rate, so in this case using the minimal homology of 15 bp may be suﬃ- cient. However, we found that three-way assemblies have lower and more variable success rates (lower than 40% in the examples shown here) depending on the construct. For these more diﬃcult assembly reactions, having more homology will maximize the chances for success and outweigh the extra primer costs. We also did not ﬁnd it necessary to purchase expensive puriﬁed primers because the mutation rate is low enough for it to not be an issue. Although we didn’t perform a systematic analysis, there’s no obvious relationship between success rate and PCR product size, consistent with (6), but in some special cases optimizing the insert:vector ratio may be necessary. In regard to assembly standards, ideally an assembly standard will use the same laboratory components (e.g. restriction enzymes and ligase) so that the same compo- nents can be used to assemble any two (or more) BioBricks together. This In-Fusion BioBrick assembly method in its current state cannot be completely standardized because custom primers need to be ordered for each individual assembly and hence diﬀerent compo- nents are required. However, the assembly method described here can be semi-standardized by two simple primer design rules (described in the ‘Materials and Methods’ section) that allow for much of the planning to be removed from primer design. The primer design tool we built will also decrease the time it takes to order primers and maximize success with In-Fusion assemblies. It would be possible to expand this method to use the same standard vector primers for every assembly reaction, but doing so would introduce large scar se- quences between parts due to the minimum amount of homology required between PCR-ampliﬁed BioBricks. These large scar sequences would most deﬁnitely cause problems for spacing between diﬀerent parts [e.g. the RBS and coding sequences (15,16), unless these sequences were already together on one part and properly spaced]. In conclusion, we have optimized the In-Fusion assembly protocol and adapted this method for BioBrick Assembly and re-engineering. We used this method to make several diverse constructs and simpliﬁed the number of experimental steps as much as possible, as illustrated by the six examples above. The cost per reaction of In-Fusion assemblies is relatively high compared with Standard Assembly, but the consistent success of our diverse assemblies and the elimination of intermediate assembly steps in complex constructions make the cost worthwhile in our experience. We hope this method can be expanded upon in the future to fuse a large number of fragments together (17,18) and be standardized to use the same laboratory components.