Omega ezna bacterial dna kit

The identified E. coli isolates were kept in brain heart infusion broth (BHI) medium with 20% glycerol at -80°C and genomic DNA (gDNA) was purified using Omega EZNA Bacterial DNA Kit (Omega Bio-tek, GA, USA). All the E. coli isolates were subjected to genomic DNA extraction in accordance with the manufacturer’s protocol of E.Z.N.A. Bacterial DNA Kit (Omega Bio-Tek, Norcross, GA, USA).

We previously published the design and construction of these variants followed by whole genome sequencing for mutant and off-target validation (Sanders et al., 2023 (link)). In short, we designed ssDNA oligonucleotides to insert cusS mutations (R15L, T14P, T17P and N279H) into the chromosome of Escherichia coli K12 MG1655 using the Mage Oligo Design Tool (MODEST) (Bonde et al., 2014 (link)). We then followed a standard protocol for recombineering (Sawitzke et al., 2013 ). The only experimental deviation was in the final step, as our desired cusS mutations had been shown to lead to silver resistance, we performed a 10-fold serial dilution and plated on 20 μg/mL of silver nitrate to screen for mutations. We then extracted genomic DNA using the OMEGA E.Z.N.A.® Bacterial DNA Kit (Omega Bio-tek, Inc., GA, USA), PCR amplified the cusS gene and sent for sequencing (ETON Biosciences, Durham, NC, USA). After mutations were confirmed, the plasmid was cured by serial plating on LB alone, typically it took only 1–2 overnight platings to cure the plasmids. We then extracted genomic DNA from the cured populations and performed whole genome Illumina sequencing at the Microbial Genome Sequencing Center (MiGS Center) at the University of Pittsburgh (Sanders et al., 2023 (link)).

Each isolate was grown in brain heart infusion (BHI) broth (HopeBio, Qingdao, China) at 37°C, and genomic DNA (gDNA) was purified using an Omega EZNA Bacterial DNA kit (Omega Bio-Tek, GA, USA). Genomic DNA was extracted with the sodium chloride-Tris-EDTA (STE) methods. The harvested DNA was detected by agarose gel electrophoresis and quantified by a Qubit 2.0 fluorometer (Thermo Fisher Scientific, USA).