Carbenicillin

Cells of P. aeruginosa PAO1 or mutants (10⁷ to 10⁹ CFU) were grown in L medium (1.0% polypeptone, 0.5% yeast extract, and 0.5% NaCl, pH 7.0) and spread onto L agar plates (1.0% polypeptone, 0.5% yeast extract, 0.5% NaCl, and 1.5% agar, pH 7.0) containing 1 × , 2 × , 4 × MIC for one of the following seven antimicrobial agents: carbenicillin, imipenem, Amikacin, gentamicin, ciprofloxacin, levofloxacin, and erythromycin. We obtained many colonies that appeared on the plates after an incubation at 37 ℃ for 24–36 h. After single-colony isolation, the drug resistance patterns of the mutants were investigated. Amikacin (Wako, cat. 014-24941), carbenicillin (Wako, cat. 037-23693), ciprofloxacin (Wako, cat. 032-18731), gentamicin (Wako, cat. 079-02973), imipenem (Wako, cat. 098-07283), levofloxacin (Fluka, cat. 28266) were purchased from indicated manufactures.

Amikacin, ampicillin, carbenicillin, chloramphenicol, ciprofloxacin, norfloxacin, and tetracycline were purchased from Wako Pure Chemicals Industries, Ltd (Osaka, Japan). Moxifloxacin and levofloxacin were purchased from LKT Laboratories, Inc. (St. Paul, MN, USA). Imipenem/cilastatin was purchased from Sandoz K.K. (Tokyo, Japan).

The OGAB blocks were amplified by PCR using the primers listed in Supplemental Table S6. PCR was performed using KOD DNA polymerase (Toyobo). An A-protrusion at the 3’ end was added to the obtained PCR fragment using A-attachment Mix (Toyobo) according to the instruction manual. The obtained DNA was ligated into pMD19 (simple) (Takara) using Mighty Mix ligation mixture (Takara), and then was used to transform each of the E. coli strains TOP10, JM109, and DH5α. Transformants of these plasmids were cultivated with LB medium supplemented with 100 μg/ml of carbenicillin (Wako, Japan). OGAB plasmid was purified from 1 ml of the culture using QIAprep Miniprep Kit (Qiagen) according to the instruction manual. The obtained crude plasmid solution was further purified enzymatically using Plasmid Safe DNase (Epicenter) as follows. Crude plasmid (5 μg/50 μl) was added to 6 μl 10 × buffer for Plasmid Safe DNase, 2.4 μl of 25 mM ATP, and 2 μl Plasmid Safe DNase. This reaction mixture was incubated at 37 °C for 1 h, then incubated at 70 °C for 30 min to inactivate the enzymes. The resulting solution was cleaned up by using a conventional column-based cleanup kit and then eluted in a small volume of TE (pH 8.0) (<25 μl).