Evaluating PCB Accumulation in hmox1 and ho1su1

To evaluate the accumulation of PCB in hmox1 and ho1su1, the cyanobacterial bilin reporter NpF2164g5 from Nostoc punctiforme was fused with a twin-strep tag (STII) and inserted into the p322-PsbDp-AtpAp.aadA vector (Duanmu et al., 2013 (link)). The resulting plasmid was used for chloroplast transformation experiments with hmox1 and ho1su1 using particle bombardment with the PDS-1000/He system (BioRad, California, United States). The bombarded cells were transferred to a solid TAP medium containing 400 μg/mL spectinomycin. Positive transformants that expressed NpF2164g5-STII were identified using PCR and immunoblotting analyses and were further selected with at least 4∼5 rounds of growth on solid TAP medium supplemented with 400 μg/mL spectinomycin to achieve chloroplast DNA homoplasmy. Transgenic lines were named hmox1:NpF2164g5-STII and ho1su1:NpF2164g5-STII, respectively. NpF2164g5-STII purification was performed using Strep-tag II magnetic beads (Beaver, Suzhou, China) following the protocol recommended by the manufacturer. Purified NpF2164g5-STII protein was concentrated and used for zinc-dependent fluorescence assays to evaluate the presence or absence of covalently bound PCB as described previously (Zhang et al., 2018 (link)).