Gibson Assembly for FMDV Cloning

The Gibson assembly (GA) reaction was used to assemble two FMDV fragments to the pKLS3 vector [22 (link)] to generate an infectious clone. The pKLS3 vector was linearized with StuI (New England BioLabs, Ipswich, MA, USA) and then purified using the HiYield™ Gel/PCR DNA Fragments Extraction Kit (RBC Bio-science, Taipei City, Taiwan). O189 F1 and F2 fragments prepared from the previous step were also purified similarly. Then, O189 F1, O189 F2, and the linearized pKLS3 vector were assembled in an isothermal GA reaction (Gibson Assembly^® Cloning Master Mix) (New England BioLabs, Ipswich, MA, USA). following an optimized protocol based on the manufacturer’s suggestion (New England BioLabs, Ipswich, MA, USA). Briefly, 25 ng each of DNA fragments (linearized pKLS3, O189 F1 and O189 F2) was mixed with 10 μL of 2X Gibson Assembly Master mixture in a 20 µL reaction and subsequently incubated at 50 °C for 1 h in a thermocycler. The assembled DNA fragments were examined by electrophoresis through 0.8% agarose gel (Cambrex Bio Science, Rockland, MA, USA) in 1X TAE buffer (Sigma-Aldrich, St. Louis, MO, USA). In addition, the GA reaction of FMDV type A (NP05) was also performed to combine the linearized pKLS3 with NP05 F1 and F2 using the Gibson Assembly kit and method as described above.