4.6. Bactericidal Activity of Free ClyF and Its SiBP-Fused Variants

Antibacterial activity of ClyF and its SiBP-fused variants were evaluated by log killing assay as described previously [14]. Briefly, S. aureus strains (Table S3) were inoculated in 5 mL of LB broth at 37 °C. Next day, 50 µL of overnight grown culture was inoculated to 5 mL of fresh LB media for 3–4 h at 37 °C with shaking. Cells were then harvested, washed, and resuspended in PBS to an optical density of OD₆₀₀ = 0.6. Bacterial suspension was treated with an equal molar concentration (final concentration of 0.7 μM, corresponding to ClyF concentration of 20 μg/mL) of ClyF and its SiBP-fused variants for 1 h at 37 °C. The turbidity of each treatment was monitored by a Synergy H1 microplate reader and viable bacterial quantification was determined by plating serial 10-fold PBS-diluted dilutions on LB agar. Parallel suspensions that treated with an equal volume of PBS were used as controls.