Cloning of ompK36 and ompK35 genes and transformation.

The coding sequences of ompK35 and ompK36 genes from K. pneumoniae KPM1026a were amplified using the primer pairs KP-ompK35-clone (forward, 5′-CACGAAGCTTATGATGAAGCGCAATATTCTGG-3′; reverse, 5′-ACGCTCTAGATGTAGAACTGGTAAACGATACCC-3′) and KP-ompK36-clone (forward, 5′-CCGAAGCTTATGAAAGTTAAAGTACTGTCCC-3′; reverse, 5′-CGCATCTAGATTAGAACTGGTAAACCAGGC-3′), respectively, and cloned into the pCR-Blunt vector (Thermo Fisher). After verification of the nucleotide sequence, the recombinant plasmids were digested with restriction enzymes HindIII and XbaI, and the ompK35 and ompK36 coding sequences were subcloned into restriction-digested expression vector pRX1 under the IPTG (isopropyl-β-d-thiogalactopyranoside)-inducible promoter on the plasmid. The E. coli transformants were selected on Luria-Bertani (LB) agar containing gentamicin at 15 μg/ml. The recombinant plasmids were transformed into K. pneumoniae strains, which were made competent by CaCl₂ treatment. The transformants were selected on LB agar plates containing gentamicin at 15 to 50 μg/ml. The MICs for the recombinant strains were determined in the presence of IPTG at 100 μM.