2632 Nucleic Acids Research, 2010, Vol. 38, No. 8 Figure 4. Part swapping: simultaneous promoter and RBS re-engineering. (a) R0011+E0240 and J04450 plasmids are shown with the forward and reverse primers for PCR. R0011+E0240 is ampliﬁed with the vector and J04450 is ampliﬁed as the insert. (b) Detailed schematic of the assembly strategy with the forward and reverse primers. Only the promoter (R0011) and RBS (B0032) are PCR-ampliﬁed from the R0011+E0240 plasmid. Only E1010 and B0010/12 are PCR-ampliﬁed from the J04450 circuit in order to change its promoter and RBS in one assembly step. (c) Since both plasmids used as template DNA in the PCR reaction were approximately the same size as the desired construct, two colony PCR reactions were performed on the same six colonies. The gel on the left shows six colonies ampliﬁed with VF2/VR primers and the gel on the right shows the same six colonies (#1–6) and negative control J04450 plasmid (#7) ampliﬁed with the VF2 and R0011+E0240 AR primer. Correct colonies show a PCR product of about 1.1 kb for the left gel and a PCR product of about 300 bp for the right gel (correct size indicated by arrow). Three-way assembly: insertion of an antibiotic resistance gene and terminator swapping Since insertion of DNA into the middle of a multi-part construct takes several Standard Assembly reactions, we demonstrate the eﬃciency of this construction in a three-way In-Fusion reaction. We wanted to insert the B0032 RBS and kanamycin resistance gene into the T9002 (Lux receiver) circuit, while at the same time change the second transcriptional terminator of T9002 to avoid repeated sequences (3) (Figure 5a). To do this, we ﬁrst ampliﬁed Part A from the preﬁx of T9002 to upstream of the GFP-coding sequence with the B0032 junction sequence (Figure 5b). Next the B0032 and kanR-coding sequence was ampliﬁed for Part B, having the Part A and C junction sequences on either side (Figure 5b). The B0011 terminator was then ampliﬁed with the vector back to the preﬁx as Part C, having Part A and B junction sequences on either side (Figure 5b).