Phsg396

Three plasmid DNA (pUC19, pHSG298, and pHSG396) were purchased from Takara Biotechnology (China) Co., Ltd. The plasmids were suspended in the solution of 10 mM Tris–HCl (pH 8.0) and 1 mM EDTA at a concentration of 0.5 μg μl^-1. pUC19 (2686 bp), pHSG298 (2675 bp), and pHSG396 (2238 bp) carry ampicillin, kanamycin, and chloramphenicol resistance genes, respectively (Supplementary Table S1). NaCl, tryptone, and yeast extract in biotechnology grade were purchased from Oxoid (England) Co, Ltd. Agar powder, ampicillin sodium salt, kanamycin, and chloramphenicol were purchased from Solarbio Science and Technology (China) Co., Ltd. CaCl₂⋅2H₂O and propidium iodide were purchased from Sigma–Aldrich (St. Louis, MO, United States).

Cloning was performed by amplifying the suspected carbapenemase gene using a primer set targeting the first and last 20 bases of the gene with added restriction sites XbaI and EcoRI. The resulting fragment was cleaned using the commercial kit Nucleospin gel and PCR cleanup (MACHEREY-NAGEL GmbH & Co, Düren, Germany). The clean fragment was subjected to cutting by XbaI and EcoRI restriction enzyme (New England Biolabs, Ipswich, MA, USA) and cleanup, as stated above. The vector pHSG396 (Takara Bio, Saint-Germain-en-Laye, France) was subjected to the same restriction enzymes and separated by agarose gel electrophoresis, followed by Nucleospin gel and PCR cleanup kit. The vector and insert were ligated by T4 DNA Ligase (New England Biolabs). The circular plasmid with the gene was inserted into MAX Efficiency™ DH10β Competent Cells (Invitrogen, Waltham, MA, USA). The resulting plasmids were extracted using NucleoSpin^® Plasmid EasyPure (MACHEREY-NAGEL). The success of cloning was evaluated by positive PCR for the target gene. The modified carbapenem inactivation method was used on the C. freundii isolate, the transformant E. coli DH10β with the suspected carbapenemase ORF, and E. coli DH10β with the pHSG396 plasmid but no insert (as a negative control).