(consisting of an RBS, GFP-coding sequence and double transcriptional terminator) (Figure 3a). By amplifying the vector with R0011, the PCR product obtained (Part A) starts with the suﬃx and ends with R0011 plus the E0240 junction sequence (Figure 3b). Part B starts with the R0011 junction sequence and ends with the suﬃx (Figure 3b). Fusion of these two PCR products occurs at each end and creates a circular plasmid. Transformation of the assembly reaction into competent cells allows for the success rate to be easily determined since colonies can only ﬂuoresce if they have the correctly assembled R0011 and E0240 together in the same plasmid. We designed primers to vary the homology level from 16–24 bp in increments of 2 bp while keeping the primer length constant at 36 bases (Figure 3c). We chose 16–24 bp because the minimum homology length is 15 bp and this would allow us to determine the relationship between success rates around this minimum at a ﬁne scale. We predicted higher homology levels would increase success rate, but also possibly increase mutation rate at the same time. The 36-base primer length was chosen since it is known that longer primer lengths increases the chances of mutations occurring in the primer. We also used primers that maximized the homology and primer length with 48 bp of homology and a primer length of 48 bases. We predicted that higher homology would increase success rate since another study observed this relationship (8), but at a cost of having a higher mutation rate. We also designed primers to vary the primer length from 32 to 40 bases in increments of 2 bases since we predicted longer primers would increase mutation rate (Figure 3d). Therefore, there were a total of 10 assembly reactions per- formed in parallel: ﬁve to test homology lengths, ﬁve to test primer lengths (the 36-base length primers overlapped with the 16 base homology length assembly), and one extreme assembly of 48 bp of homology and a primer length of 48 bases. These 10 assembly reactions required 20 PCR products, one insert and one vector for each. Remarkably, all 20 PCR products ampliﬁed using an annealing tempera- ture of 55 C. Table 1 shows the results of homology length on the success rate and mutation rate of In-Fusion assembly. As homology level increases, the assembly success rate also increases. There appears to be small dif- ference between low homology (16–18 bp) and high homology (20–24 bp) with high homology increasing success by about 10% on average. The extreme case of 48 bp of homology had the highest success rate at nearly 85%. We therefore decided that 48 bp of homology should be used for our primer design rules to maximize success rates. Forty-eight base pairs should not be considered the optimal length, but is a conservative choice since no homology lengths were tested between 24 and 48 bp. Remarkably, there were no mutations in any of the junctions when we sequenced several successful clones. For all 10 assemblies, we only found one mutation among all of the clones sequenced (73 clean sequences out of 80 attempts) for the entire 1-kb genetic circuit. Table 2 shows the eﬀect of primer length on the success and mutation rates of In-Fusion assembly. There do not Nucleic Acids Research, 2010, Vol. 38, No. 8 2631 appear to be any remarkable diﬀerences between primer lengths of 32–40 bases with respect to either success or mutation rate. Therefore, the optimal homology and primer length is 48 bp because this achieves the highest success rate without the cost of high mutation rate since mutations are rare. The rare mutations in the circuit itself are because Phusion is a very high ﬁdelity polymerase (4.4 10–7 according to the product spec sheet), but we can not rule out the possibility that the mutation existed on the template DNA at a low level. Part swapping: simultaneous promoter and RBS replacement Next we wanted to construct the same R0011+E0240 circuit, but with RFP (E1010) instead of GFP (E0040). This would have taken three Standard Assembly steps: to ﬁrst construct R0011 with B0032, E1010 with B0010/ 12, then to assemble all these parts together. Instead we could simultaneously re-engineer the existing J04450 circuit with a new promoter and RBS in a single step using In-Fusion (Figure 4a). To do this, for Part A, R0011 and B0032 were ﬁrst ampliﬁed with the E1010 junction sequence and vector (Figure 4b). E1010 and B0010/12 were then ampliﬁed from J04450 with the B0032 junction sequence to make Part B (Figure 4b). Colony PCR results remarkably showed that all six colonies had the correct construct, but we were able to screen out negative colonies by the colony color for this particular assembly. Two separate colony PCR experi- ments were performed on the same six slightly red glowing colonies, one to determine the size of the insert (Figure 4c, left) and one to identify colonies that had the correct RBS (Figure 4c, right). Since the negative control J04450 plasmid (#7 in the right gel photo) did not amplify with the B0032-speciﬁc primer and the six colonies did amplify, it was assumed that all six colonies (100%) had the correct construct and three of these were veriﬁed with sequencing. Table 2. Success and mutation rates of In-Fusion assembly using dif- ferent primer lengths Primer length 32 34 36 38 40 48a Success rate 64.3% (198/308) 66.7% (246/369) 61.8% (222/359) 65.9% (245/372) 58.5% (220/376) 84.8% (495/584) Junction mutation rate 0% (0/8) 0% (0/8) 0% (0/7) 0% (0/7) 0% (0/6) 0% (0/7) Primer length is indicated with the success and mutation rates. For success rates, numbers in parentheses after the rates indicate the number of successful clones out of the number of total clones tested. For mutation rates, numbers in parentheses after the rates indicate the number of junction mutations out of the number successful clones sequenced. aHomology level is 48 bases (all others are 16 bases).