2626 Nucleic Acids Research, 2010, Vol. 38, No. 8 product with a linearized vector into a circular plasmid when the fragments share at least 15 bp of homology on both ends. To assemble two BioBricks, one PCR-ampliﬁed BioBrick needs to have homology on each end with the second PCR-ampliﬁed BioBrick (vector ampliﬁed with the BioBrick) to allow for the frag- ments to be fused together in the In-Fusion reaction (Figure 1b). More than two BioBricks can be fused together as shown in the examples below and four frag- ments have been successfully fused (6). This method can also be used to re-engineer BioBricks as shown in the examples below and we have also successfully used this method for vector replacement, site-directed mutagenesis and cumbersome transcriptional terminator re-engineering (results not shown). In-Fusion Assembly greatly expands the possible circuits to construct since in our experience, the individual parts we want to construct into circuits often do not exist as BioBricks on their own or in the correct order. For this reason In-Fusion provides the ﬂexibility to PCR-amplify any sequence from any part, then assemble these frag- ments together in one step. In our experience, the advan- tages of In-Fusion BioBrick assembly over Standard Assembly are that it is faster, more ﬂexible, and has a high success rate (see Supplementary Figure S1 for a timeline and schematic overview for Standard and In-Fusion BioBrick Assembly). In-Fusion Assembly is fast once primers are available since BioBricks can be im- mediately PCR-ampliﬁed from parts extracted from the BioBrick Parts Distribution plate. Standard assembly requires an initial ampliﬁcation of the BioBrick through transformation, overnight growth, and plasmid extrac- tion. Standard assembly also requires tedious extraction of restricted DNA fragments from a gel, more intermedi- ate enzymatic reactions, and more time to quantitate and optimize these reactions. In-Fusion assembly is more ﬂexible in the sense that there is more control over the exact engineered sequence and mutations can be easily introduced with mutagenic primers (6). We have found this method to have a high success rate in that nearly every assembly reaction yields the desired construct. Standard assembly we have found to be less reliable. The disadvantages of In-Fusion BioBrick assembly are that the speciﬁc assembly supplies are more expensive ( $15/assembly versus $5/assembly for Standard Assembly), custom primers are required, and occasionally there are mutations in assembled plasmids. Mutations in the construct may depend on a number of factors, including primer quality and the error rate of the polymer- ase. However, using reagents speciﬁed in the ‘Materials and methods’ section, we demonstrate here that such mu- tations are suﬃciently rare that typically only a single putative construct needs to be sequenced. In general, In-Fusion assembly is ideal when re-engineering an existing BioBrick with many parts since there are less assembly reactions to perform, or when assembling a circuit with speciﬁc parts distributed among several BioBricks. On the other hand, if the BioBricks you want to assemble already exist as digested fragments in the freezer, then Standard Assembly may be ideal for this par- ticular situation. The motivation for optimizing and adapting the In-Fusion method for BioBrick assembly is to improve the success rate, ﬂexibility, and speed for constructing genetic circuits and create a new BioBrick assembly standard (13). Small synthetic biology labs that do not perform high throughput restriction enzyme digests and ligations will especially beneﬁt from using this method. MATERIALS AND METHODS Primer design rules For the In-Fusion reaction to work, the forward primer of the ﬁrst PCR-ampliﬁed fragment must have at least 15-bp homology to the reverse primer of the second fragment, and vice versa. A longer homology length is used in this protocol because in our experience it works better than 15 bp. There are two simple primer design rules that allow for semi-standardized assembly, in the sense that even though the same components cannot be used repeat- edly as in a standardized assembly method, following these rules will remove much of the planning required for performing In-Fusion Assembly reactions. The two rules are: (i) the reverse primer for Part A (AR) should be the last 20 bases Part A + scar (if wanted) + ﬁrst 20 bases Part B (reverse complement of this entire sequence), and (ii) the forward primer for Part B (BF) should be the reverse complement of the AR primer. Since the primers are exactly complementary, this gives at least 40 bp of homology at the junction of Parts A and B in total. In contrast, the forward primer of Part A (AF) and the reverse primer of Part B (BR) do not need customization. These primers are speciﬁc to the standard Biobrick ‘preﬁx’ (immediately upstream of each part) or ‘suﬃx’ (immedi- ately downstream of each part) and may be re-used when assembling diﬀerent constructs. Of course optimization of the primers may improve chances of a clean PCR product. In the assembly examples described here using these primer design rules, we found that every PCR product ampliﬁed with a single amplicon (10/10 PCR products). One particular PCR reaction required a gradient on the primer annealing step during the PCR reaction to generate a single PCR product, but in general a 55 C annealing temperature worked well for all reactions. In general we found these primer design rules to be robust and other examples not described here have used these rules with success. Primer design software tool We have designed a software tool for In-Fusion assembly primer design (located at http://sys-bio.org/ primerdesign/). This tool allows you to input the DNA sequences for each part to assemble, the scar sequence between the parts, and the length of overlap with the ad- joining part (the default length is 20). When 20 bases of homology are added to the 50-end of each of the primers, along with the 8-base scar, this gives a total junction homology of 48 bp. The program outputs the forward and reverse primers for the Part A and Part B junction (AR and BF primers). The primer sequences generated are based on the rule described above. A future version of this