Validation of ABI-REC through double-resistance reporter assay. (A) The design of double resistance reporter assay. The fusion of the 1.6 kb kanR cassette (from pIRES2-EGFP) with the 2.7 kb pUC19 plasmid (ampicillin resistance) renders the new recombinant plasmid resistant to both kanamycin and ampicillin. The plasmid grows on (Kan + Amp) LB plates. An artificial ApaI restriction site is introduced into the 5′ fusion site to precisely localize the insertion site. (B) An asymmetric bridge PCR efficiently fuses the KanR cassette into the pUC19 plasmid and generates a hybrid fragment. The gradient concentration of the primer pR was assessed and identified as within the optimal range from 2 nM to 0.2 nM in the bridge PCR. A conventional two-primer PCR reaction was conducted in parallel to compare their amplification efficiency. This indicates that the quantity of pR primer is critical to the output of fused sequence in the bridge PCR reaction. (C) Double resistant colonies were found to grow on (Kan + Amp) LB plates. Bridge PCR products, purified fused sequences, two-primer PCR products (extension), and a mixture of purified KanR cassettes with pUC19 (no extension) were transformed into DH5α competent cells. Double resistant colonies were present for bridge PCR products and purified fused sequence, whereas the latter two did not produce viable colonies. This implies that intramolecular recombination occurs within the fused sequence, producing double resistant plasmids that renders cells resistant to both kanamycin and ampicillin. pUC19 and pIRES2-EGFP plasmids were not able to grow on the double-drug plates, precluding the risk of random integration of KanR cassette into the E.coli genome. (D) Plasmids of the single colonies in (Kan + Amp) LB plates were extracted and digested using SalI and ApaI. As shown in the electrophoresis gel, the 1.6 kb insert was released, indicating that the KanR cassette had been fused into pUC19 at pre-determined site. (E) Sequencing of the single colonies revealed the insertion of the KanR cassette and the presence of artificial ApaI restriction site, further proving that the insert of choice has been fused with target plasmid in a restriction- and ligase-free manner. The red rectangle represents the restriction sites.