E coli dh10b cells

A BAC library using pESAC13-Apramycin (Bio S&T, Canada) was constructed. The vector DNA was digested with BamHI. The partially digested high-molecular-weight DNA was size-selected on 1% (w/v) pulsed-field agarose gels in 0.5X TBE on a CHEF DRIII (Bio-Rad, Canada). Partially digested size-selected DNA fragments were ligated to the vector DNA in a volume of 50 μl with 1X ligase buffer and three units of ligase (USB, Canada) at 14°C for overnight incubation. Two microliters of ligation mix were used to transform 20 μl of E. coli DH10B cells (Invitrogen, USA) by electroporation using a CellPorator equipped with a voltage booster (Invitrogen). The cells were then selected on LB medium supplemented with 5% sucrose plus Apramycin by incubation at 37°C overnight. The clones were selected and arrayed in 6 × 384-well plates in a medium containing freezing medium supplemented with 10% (v/v) glycerol and Apramycin. The average insert size was evaluated using randomly selected clones. The miniprep of PAC clones and digestion of PAC DNA with DraI was done according to standard procedure. Pulsed-field gel electrophoresis (PFGE) was conducted to check the insert sizes.

The construction of metagenomic libraries and their subsequent amplification was accomplished as previously described (Mirete et al., 2007 (link); González-Pastor and Mirete, 2010 ). Briefly, the metagenomic DNA was partially digested using Sau3AI, and fragments from 1 to 8 kb were collected directly from a 0.8% low-melting-point agarose gel with the QIAquick extraction gel (QIAGEN) for ligation into the dephosphorylated and BamHI-digested pSKII⁺ vector. DNA (100 ng) excised from the gel was mixed with the vector at a molar ratio of 1:1. Ligation mixtures were incubated overnight at 16°C using T4 DNA ligase (Roche) and used to transform E. coli DH10B cells (Invitrogen) by electroporation with a Micropulser (Bio-Rad) according to the manufacturer’s instructions.

Since the qnrD-positive clinical isolates may have additional mechanisms conferring quinolone resistance, including chromosomal mutations, we determined the quinolone resistance of both the original Proteeae strains and the transformants obtained after transfer of qnrD-bearing plasmids into competent E. coli DH10B cells (Invitrogen, Cergy-Pontoise, France). Plasmid DNA was extracted from the qnrD-positive strains using the QIamp DNA mini Kit (Qiagen, Courtaboeuf, France) and introduced by electroporation (Gene Pulser, Biorad, Marnes la Coquette, France). Transformants were selected on BHI agar plates containing ciprofloxacin (0.015 µg/ml, 0.03 µg/ml, 0.06 µg/ml and 0.12 µg/ml) or ofloxacin (0.03 µg/ml, 0.06 µg/ml, 0.12 µg/ml, and 0.25 µg/ml). In order to distinguish transformants and quinolone resistant mutants growing on quinolone containing media, we sequenced the QRDRs in gyrA and parC, in addition to qnrD detection by PCR.
Minimal inhibitory concentrations (MICs) of nalidixic acid, ciprofloxacin, levofloxacin, moxifloxacin, norfloxacin, and ofloxacin were determined by the agar dilution method using Mueller–Hinton agar plates containing serially twofold-diluted antibiotics. Plates were inoculated with a Steers-type multiprong device with ca. 10⁴ CFU per spot, and were read after incubation for 18 hours at 37°C.

The BAC plasmid pBeloBAC11 [40 (link)], kindly provided by H. Shizuya (California Institute of Technology, Pasadena, CA, USA), was used to assemble the ZIKV-RGN infectious cDNA clone. This plasmid is a synthetic low-copy-number plasmid (one copy per cell) based on the Escherichia coli (E. coli) F-factor [41 (link)] that minimize the toxicity problems in the bacteria of exogenous sequences. E. coli DH10B cells (Invitrogen, ThermoFisher Scientific) were used to amplify the BAC plasmids. Electrocompetent DH10B cells (Invitrogen, ThermoFisher Scientific) were transformed by electroporation using a MicroPulser unit (Bio-Rad, Madrid, Spain), according to the manufacturer’s instructions. BAC-based plasmids were isolated and purified using the Large-Construct kit (Qiagen, Hilden, Germany), following the manufacturer’s specifications.