2630 Nucleic Acids Research, 2010, Vol. 38, No. 8 Table 1. Success and mutation rates of In-Fusion assembly using dif- ferent homology lengths Homology level 16 18 20 22 24 48a Success rate 61.8% (222/359) 64.6% (197/305) 73.1% (196/268) 74.2% (291/392) 74.4% (314/422) 84.8% (495/584) Junction mutation rate 0% (0/7) 0% (0/8) 0% (0/8) 0% (0/6) 0% (0/8) 0% (0/7) Homology 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. aPrimer length is 48 bases (all others are 36 bases). screening described in the previous section. In these cases, success rate is deﬁned as the percentage of correct con- structs out of the total number of colonies screened by colony PCR. Colony PCR allows us to discriminate between the desired construct and construction back- ground (PCR template plasmid that co-transforms with the newly assembled plasmid). Colony PCR was performed on at least six colonies using the VF2 (50-TGCCACCTGACGTCTAAGAA-30) and VR (50-ATTACCGCCTTTGAGTGAGC-30) primers speciﬁc to the pSB1A2 vector ( 100 bp on either side of the part) or primers speciﬁc to the desired con- struct. Negative control reactions were also performed using VF2/VR primers on the plasmid template DNA for the original PCR reaction. Thus, a colony hosting the desired construct will exhibit a mobility shift when compared with the negative control reactions. Colony PCR reactions were performed using 10 ml Fermentas PCR Master Mix per reaction in 0.2 ml PCR tubes. All 10 ml of the colony PCR products were run out on a 1% agarose gel stained with SYBR Safe (Invitrogen) with 1-kb ladder (NEB). Plasmid extraction and sequencing At least three correct colonies as identiﬁed by colony PCR were grown in test tubes with 5 ml LB supplemented with ampicillin (100 mg/ml) shaking overnight at 250 rpm. We chose three to ensure that one plasmid was correctly con- structed without mutations which tend to occur most often at the junctions (regions of homology between frag- ments). Minipreps were performed with the Qiagen Miniprep Kit using 3 ml culture volume and plasmids were eluted with 30 ml of molecular grade water. Plasmids were submitted for sequencing with VF2 and VR or custom primers to sequence the entire genetic circuit. General strategies for In-Fusion assembly We devised two general strategies for assembly depending on which BioBrick is PCR-ampliﬁed with the vector (Figure 2). Since both PCR products need to have homology to each other on both ends for the In-Fusion reaction to work, the simplest strategy was amplify each PCR product to have homology to the vector at one end and to have homology at the junction between parts at the other end. As shown in Figure 2a, to amplify the upstream part (Part A) as the insert and the downstream part (Part B) as the vector, the vector speciﬁc primers need to be upstream of each part (i.e. in the vector preﬁx). Since the forward primer for Part A (AF) and reverse primer for Part B (BR) are complementary sequences, the resulting PCR products share 25 bp of homology at the site where Parts A and B will be joined in the In-Fusion reaction. Likewise, as shown in Figure 2b, to amplify the upstream part (Part A) as the vector and the downstream part (Part B) as the insert, the vector speciﬁc primers need to be downstream of each part (i.e. in the vector suﬃx). Use of these vector-speciﬁc primers allows for BioBrick standard formats to be maintained so that new In-Fusion assembled constructs can be submitted to the Registry. These four primers described in the ‘Materials and Methods’ section can be re-used for every In-Fusion Assembly. However, custom primers need to be designed in order to provide homology at the junction between parts. The AR and BF primers shown in both Figure 2a and b are complementary, resulting in PCR products with homolo- gous ends that will be fused in the In-Fusion reaction. These primers were designed with simple rules of having a sequence speciﬁc to the template DNA of one part at the 30-end and a 50 overhang that is homologous to the other part (see ‘Materials and Methods’ section for details). Following these simple rules will allow for primers to be designed quickly and in a semi-standardized fashion. RESULTS We ﬁrst describe the success rate and mutation rate of In-Fusion assembly using diﬀerent homology and primer lengths. We then describe additional examples of In-Fusion assembly and re-engineering reactions to dem- onstrate the versatility and success of this method. These examples include assembly of two basic parts, part swapping (simultaneous promoter and RBS re- engineering), a deletion (conversion of a polycistronic transcribed sequence into a fusion protein-coding sequence), an insertion (of a degradation tag), and three- way assemblies (one to insert an antibiotic resistance gene and swap out a terminator, and another to construct a fusion protein). The insertion and deletion examples are illustrated in Supplementary Figures S2 and S3. Eﬀect of homology and primer length on success rate and mutation rate of In-Fusion assembly To understand how homology and primer length aﬀects the success rate and mutation rate of assembly reactions, we devised an assembly assay that would allow for these rates to be determined for a large number of individual clones. The two basic BioBrick parts used for the assembly assay were R0011 (lacI-regulated promoter) and E0240