Propagation and Purification of Phage Vectors

The T7-wild-type (WT), phage 54 and phage 74 were propagated in E. coli BL21 (18 (link)) containing the pEGFP-C1-HA2-AS eukaryotic expression plasmid to generate T7-WT/pEGFP-C1-HA2-AS, phage 54/pEGFP-C1-HA2-AS and phage 74/pEGFP-C1-HA2-AS particles, respectively. Briefly, 500 mL of freshly cultured E. coli BL21 (OD_{600 nm}=1.0) was infected with phages at a multiplicity of infection (MOI) of 0.001 and agitated until complete lysis of bacteria was observed. DNase I and RNase A (Takara, Dalian Bio, China) were added 30 min before harvesting the phages from the culture medium. The lysate was centrifuged for 15 min at 6000 rpm (Avanti ^® J-26 XPI, JLA-162500 Rotor) to separate the bacterial debris from the phage particles. Polyethylene glycol 8000 was added to the supernatant at a final concentration of 10% to allow cross-linking of phage particles. The pellet from the secondary centrifugation was resuspended in 50 mL Tris-buffered saline (TBS) buffer, followed by three extractions with 0.1% Triton-114 to remove endotoxin. The endotoxin residue was assayed using the ToxinSensor™ Chromogenic LAL Endotoxin Assay Kit (Genscript, China). Purified phage particles were examined by titering, Western-blotting and internalized pEGFP-C1-HA2-AS plasmid DNA was quantified by real-time fluorescent quantitative PCR.