E coli 8739

Colicin N was purified from the supernatant by affinity chromatography using a nickel-sepharose HisTrap™ HP affinity column (GE Healthcare Technologies, West Milwaukee, WI, USA) in a Fast Protein Liquid Chromatography (FPLC) ÄKTA Start machine (GE Healthcare Technologies, West Milwaukee, WI, USA). The column was pre-equilibrated with the binding buffer and the supernatant was applied via sample pump. The column was washed with the washing buffer (50 mM sodium phosphate buffer; pH 8.0, 300 mM NaCl and 20 mM imidazole) then colicin N was eluted with a buffer containing 50 mM sodium phosphate buffer (pH 8.0), 300 NaCl and 250 mM imidazole. The protein-containing fractions were lyophilized and resuspended in PBS. The protein concentration was measured by Pierce™ BCA kit (Thermo Fisher Scientific, Waltham, MA, USA). The purity colicin N were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Moreover, antibacterial activity of expressed colicin N was confirmed by broth microdilution assay in microplate format, with LB media for the growth of E. coli 8739™ (ATCC, Manassas, VA, USA).

Hexafluoroisopropanol (HFIP) was purchased from Oakwood Chemicals, Estill, SC, USA. Tributylammonium chloride (TBAC), tert-butanol, potassium carbonate, bromohexane, iodomethane, Tween 20, N-3-dimethylaminopropyl-N-ethylcarbondiimide (EDC), N-hydroxy succinimide (NHS), 2-N-morpholino ethanesulfonic acid (MES), hexanes and fluorescent latex beads (L5155, L9904, L5530, L9654, L9529) were purchased from Sigma-Aldrich, St. Louis, MO, USA. Trypticase soy broth, trypticase soy agar, phosphate buffered saline (PBS), and dey engley broth were purchased from Fisher, Waltham, MA, USA. E. coli (8739) and S. aureus (6538) were obtained from ATCC, Manassas, VA, USA. Vantocil 100 (B-1018-04) was purchased from Lonza, Allendale, NJ, USA. Lupasol WF (25,000 g/mol) was purchased from BASF, Florham Park, NJ, USA.