Escherichia coli dh5α

A 3.0 kb plasmid DNA pBluescript II SK(+) was obtained from Stratagene Corp., San Diego, CA, USA. Escherichia coli DH5α (Nippon Gene Co. Ltd., Tokyo, Japan) was used as the host for the plasmid and grown at 303 K on an LB medium supplemented with the ampicillin antibiotic. The test solution was prepared by the following three steps: alkaline lysis of E. coli containing plasmid DNA, the addition of CaCl₂ for the removal of high molecular weight RNA [25 (link)], and the addition of ethanol for the concentration of nucleic acid. The plasmid DNA-containing sediment was dissolved in 10 mM Tris-HCl buffer (pH 5), and this solution, free of impurities such as proteins, was used for a two-stage affinity microfiltration experiment. The ligand employed in the experiments was α-Fe₂O₃ (particle size: 0.5 μm) provided by the Kojundo Chemical Lab. Co. Ltd., Saitama, Japan. A microelectrophoresis Mark II apparatus (Rank Brothers Ltd., Cambridge, UK) was used to determine the zeta potential of α-Fe₂O₃ particles.

S. mutans strain MT8148 (serotype c) was used in the present study [10 (link)]. Bacterial organisms were grown at 37°C in brain-heart infusion (BHI) medium or Todd-Hewitt (TH) medium, as well as on Mitis Salivarius agar, each obtained from Becton Dickinson and Co. (Franklin Lakes, NJ, USA). When spectinomycin- or erythromycin-resistant S. mutans strains were cultured, spectinomycin (1 mg/ml) or erythromycin (10 μg/ml) was supplemented as necessary.
Escherichia coli DH5α (Nippon Gene, Tokyo, Japan), used as the host strain for transformation of plasmid DNA, was cultured in Luria-Bertani (LB; 1% tryptone, 0.5% yeast extract, 0.5% NaCl) medium while LB agar plate was prepared by adding 1.5% agar. Ampicillin sodium (100a μg/ml) was added to the medium for subcloning using a pGEM-T Easy Vector (Promega Co., Madison, WI, USA), while erythromycin (500 μg/ml) was added for use of pVA838 [22 (link)]. All antibiotics were obtained from Wako Pure Chemical Industries (Osaka, Japan).