Predator Stock Lysate Preparation

Predator stock lysates were made by coculturing the predator and the prey (E. coli LF82, 10⁸ CFU/mL) inoculating pieces of YPSC medium double-layered plate in Diluted Nutrient Broth 2× (DNB2×) (Bacto Nutrient Broth 1.6 g/L, yeast extract 0.1 g/L, casaminoacids 0.5 g/L, CaCl₂ × 2H₂O 0.3 g/L, MgCl₂ × 6H₂O 0.6 g/L) [28 (link)]. The coculture was then incubated at 30 °C on a rotary shaker for at least 72 h, until the cultures cleared. The fresh co-culture was filtered three times with 0.45-μm pore-size filters (Millex^®, Merck KGaA, Darmstadt, Germany) to eliminate the prey cells. In order to ensure the effective elimination of the LF82, aliquots (10 µL) of the filtered co-culture were plated onto BHI agar plates. Further, the filtrated co-culture was washed three times at 29,000× g for 45 min (Sorvall LYNX 4000 centrifuge, Thermo Fisher Scientific Inc), re-suspending the pellet in 10 mL of phosphate-buffered saline (PBS) (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) for two cycles, and then in 2 mL of PBS after the last step. The predator concentration was then evaluated by counting the plaque-forming units (PFU) and seeding the B. bacteriovorus preparation onto a double-layered plate of YPSC medium as described above. We obtained a PFU count of between 5 × 10⁸ and 5 × 10⁹ PFU/mL. The predator stock was prepared fresh for each experiment.