Ecori

To validate and characterize the transgenic events, Southern blot analysis was conducted using genomic DNAs isolated from the leaves of transgenic and non-transgenic control plants. Genomic DNAs were digested with EcoRI (New England Biolabs, Ipswich, MA) which cuts once within the T-DNA construct, but not within the region of hptII resistance gene used for the probe (Fig. 4). EcoRI-digested genomic DNAs of each line were electrophoresed through a 1% (w/v) agarose gel and blotted onto a positively charged nylon membrane (Amersham Hybond-N+, GE Healthcare Life Sciences, Pittsburgh, PA) as described in the DIG Application Manual for Filter Hybridization (2008 by Roche Diagnostics GmbH, Mannheim, Germany). The DIG labeled probe (specific to hptII gene) was synthesized using the PCR DIG Probe Synthesis Kit. All labeling, hybridization and detection were carried out in accordance to the instructions in the DIG Application Manual for Filter Hybridization (2008 by Roche Diagnostics GmbH). All labeling, hybridization and detection reagents were purchased from Sigma-Aldrich (St. Louis, MO). Hybridization was carried out at 42 °C overnight. The chemiluminescent substrate CDP-Star-ready-to-use was used for detection, according to manufacturer’s instructions (Roche Diagnostics GmbH). The signal was detected by exposing the hybridized blot to X-ray film.

Genomic DNA isolated from S. aureus strain Newman was used as the template for all PCR experiments with oligonucleotide primers described in Supplementary Table 1 of the supplementary figure, restriction enzyme cleavage sites are underlined. For His-vWbp and Coa, PCR products were digested with BamHI and PstI (New England BioLabs) and then ligated into pRSET-A vector (Invitrogen). For GST-vWbp, PCR products were digested with BamHl and EcoRI and ligated into pGEX-5x-1 (GE Healthcare). The ligation mixture for vWbp was transformed in E. coli strain TG1 cells (Zymo Research). Cloning of GST-Coa was described previously (Ko et al., 2016 (link)). These were grown on LB agar plates containing 100 μg/ml ampicillin to select for transformants. Insertions were confirmed by DNA sequencing.