Penicillin g

HCT116 cells and 293T cells (American Type Culture Collection) were cultured in DMEM with 10% fetal bovine serum (FBS; Invitrogen, Thermo Fisher Scientific, Inc.), penicillin G (100 U/ml; Sangon Biotech Co., Ltd.) and streptomycin (100 mg/ml; Sangon Biotech Co., Ltd.) at 37°C in a humidified atmosphere containing 5% CO₂.

According to the requirements of the Clinical and Laboratory Standard Institute (CLSI), the antimicrobial susceptibility changes of the strains were detected by using Mueller–Hinton (MH) (hopebiol, Qingdao, China) medium dilution antimicrobial susceptibility tests. The concentration that completely inhibits the growth of the strain is defined as the minimum inhibitory concentration (MIC). The experiment was repeated 3 times.The stock solutions of the following antibiotics were prepared, and their classes are listed in parentheses: ofloxacin (fluoroquinolone, Sangon) at 20 mg/mL; erythromycin (macrolide, Sangon) at 30 mg/mL; Kanamycin (aminoglycoside, Sangon) at 50mg/mL; penicillin G (β-lactams, Sangon) at 100mg/mL.
Based on previous methods (Yu et al., 2020 (link)), the antibiotic susceptibility of the strains was determined by antibacterial activity assays. The overnight cultured strains were diluted into fresh MH containing 30 μg/mL chloramphenicol and incubate in 37°C for 2 h with shaking. After incubation, different antibiotics (final concentration 1/2MIC) were added separately and incubate for another 2 h. Then, they were diluted continuously by 10-fold and three appropriate dilutions (0.1 mL) were dropped onto LB agar plates and colony counts (CFU/mL) were counted after incubation at 37°C. The experiments were repeated 3 times.

Chemicals and solvents were purchased from standard suppliers and used without further purification. Ampicillin, penicillin G and piperacillin were purchased from Sangon Biotechnology Co. Ltd (Shanghai, China); cefuroxime, ceftizoxime, cefpiramide, ceftazidime, cefoxitin, cefaclor, cephalexin, nitrocefin, cefepime, cephalothin, cefotaxime, imipenem and meropenem were purchased from TCI (Shanghai, China); inhibitors were purchased from Sigma-Aldrich Corporation (USA). The host strain Escherichia coli BL21 (DE3), E. coli JM109 [recA supE hsR Δ(lac-pro)] and the expression plasmid pET-28a (+) were obtained from Novagen (Madison, WI, USA). LATaq DNA and PrimeSTAR DNA polymerase were from TakaRa (Dalian, China). The T-vector, restriction enzymes, T₄-ligase, plasmid miniprep kit, and agarose gel DNA purification kit were supplied by TaKaRa Biotechnology (Otsu, Japan).