Cloning and Transformation

Fragments of bla_OXA–499 with or without the promoter region were amplified from the wild-type A. pittii A1254 strain and the CAB009 and CAB010 mutants. Additionally, bla_OXA–826 was also amplified, with or without its promoter region, but only from A. pittii A1254. The products were cloned into the BamHI and SalI-digested shuttle vector PYMAb2-Hyg^r using the ClonExpress^® II One Step Cloning Kit (Vazyme Biotech Co., Ltd., Nanjing, China) following the manufacturer’s recommendations. Cloning was performed based on recombination. Briefly, PYMAb2-Hyg^r was digested with BamHI and SalI. Using the primer pairs shown in Table 1, PCR products comprising the target fragment flanked by the recombination sequences were obtained. Then, the linearized vector, purified PCR product, buffer, and enhanced recombinase (Exnase II) were mixed and incubated for 30 min, yielding the recombinant vectors. The recombinant vectors were transformed into A. baumannii ATCC 17978, A. pittii LMG1035, and A1254 by electroporation.