Chloramphenicol

The DNA fragments of tBLIP-II, tBLIP-II-CPP, and CPP-tBLIP-II were commercially synthesized and cloned into pETDuet-1 by Genscript (Piscataway, NJ, USA). It should be noted that the expression of BLIP-II may inhibit β-lactamase, which could affect the functionality of ampicillin used as a selection marker. To address this, all constructs were additionally cloned into another vector, pACYCDuet-1, which carries a chloramphenicol-resistant gene as the selection marker. Polymerase chain reaction (PCR) was employed to introduce the BamHI and HindIII restriction sites for the tBLIP-II and tBLIP-II-CPP constructs, and the NcoI and HindIII restriction sites for the CPP-tBLIP-II construct, using the primers listed in Table 1. All specific primers were ordered from Integrated DNA Technologies, Inc. (Coralville, IA, USA). The PCR products and pACYCDuet-1 were digested with the corresponding restriction enzymes and then ligated together using T4 DNA ligase (New England Biolabs Inc., Ipswich, MA, USA). The resulting ligation mixtures were transformed into NovaBlue Escherichia coli competent cells from Novagen (Darmstadt, Germany) and selected on Luria-Bertani (LB) agar plates supplemented with 34 μg/mL of chloramphenicol (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan). The presence of the inserted gene was verified by DNA sequencing.

All the high purity organic solvents were purchased from Merk. Certified reference materials of ethyl paraben (GBW(E) 100064) with a certificate value of 99.7% as an internal standard for qNMR measurement and chloramphenicol (GBW(E)060907) with a certificate value of 99.8% were supplied by National Institute of Metrology (NIM), China. Methanol-D₄ was obtained from Sigma-Aldrich. The powder of chloramphenicol material with a labeled purity of 99.8% was purchased from Tokyo Chemical Industry (TCI) Co., Ltd., Japan.