Isolation and Purification of S-CREM1 Phage

S-CREM1 phage suspensions were inoculated into 2 L of exponentially growing cultures of Synechococcus sp. CB0101 at an MOI of 0.1. After host cell lysis, RNase A and DNase I were added to the lysates both at a final concentration of 2 µg mL⁻¹, and they were treated at room temperature for 1 h. Afterward, the NaCl concentration of phage lysates was adjusted to 1 M, and the lysates were ice-bathed for 0.5 h. To remove the remaining cells and debris, the phage lysates were centrifuged at 12,000× g at 4 °C for 20 min and further filtered through 0.22 µm filters (Millipore, Bedford, MA, USA). The filtrates were treated with PEG8000 (w/v 10%) and kept at 4 °C for 24 h [22 (link),33 (link)]. The PEG-treated phage suspensions were centrifuged at 12,000× g at 4 °C for 1 h to precipitate phage particles and then resuspended with 6 mL of TM buffer. Concentrated phage particles were then purified by CsCl density gradient ultracentrifugation (gradient density 1.45, 1.5, 1.55, and 1.6 g mL⁻¹, 200,000× g at 4 °C, 6 h) in a SW 41Ti rotor (Beckman Optima L-100XP, Beckman Coulter, CA, USA) [8 (link),34 (link)]. The visible phage band was extracted and then desalted using a 30 kDa centrifugal ultrafiltration unit. The purified high-titer phages were stored at 4 °C.