Phage Display Screening of PhuR Binders

Colonies of E. coli XL-1 Blue bacteria containing individual phagemid clones from round 5 of phage display were used to inoculate 400 µL of 2×YT media supplemented with ampicillin (100 µg/mL) and M13-KO7 helper phage (10⁹ PFU/mL). Phage were amplified overnight in 96-well deep well blocks at 37°C with shaking at 280 RPM. Amplified phage were diluted into ELISA buffer (selection buffer with 0.5% BSA) and then tested against PhuR-loaded or empty biotinylated E3D1 nanodiscs. All ELISA experiments were performed on 96-well ELISA plates (Nunc) coated overnight with 2 µg/mL neutravidin and blocked for at least two hours with selection buffer. ELISA were performed as previously described and bound phage were detected with TMB substrate (Thermo Fisher Scientific) following a 30-min incubation with HRP-conjugated anti-M13 monoclonal antibody (Antibody Design Laboratories)^{19 (link)}. Absorbance was measured at 450 nm following quenching with 1 M HCl. Wells containing empty biotinylated E3D1 nanodiscs were used to determine non-specific binding. Specific binders were determined based on their signal/background ratio.