Strains and growth conditions
All bacterial and phage strains used in this study are listed in Supplementary Table 2. E. coli strains were routinely grown at 37 °C in Luria–Bertani (LB) medium for cloning and maintenance. Phages were propagated by infecting a culture of E. coli MG1655 at an optical density at 600 nm (OD 600 ) of approximately 0.1–0.2 with a multiplicity of infection of 0.1. Cleared cultures were pelleted by centrifugation to remove residual bacteria and filtered through a 0.2-μm filter. Chloroform was then added to phage lysates to prevent bacterial growth. Antibiotics were used at the following concentrations (liquid; plates): carbenicillin (50 μg ml−1; 100 μg ml−1) and chloramphenicol (20 μg ml−1; 30 μg ml−1).
Plasmid construction
All plasmids are listed in Supplementary Table 3. All primers and synthesized gene sequences are listed in Supplementary Table 4.
For the pEssential-gp77 construct, the coding sequence of gene 77 from phage SECΦ27 was codon optimized for expression in E. coli, and commercially synthesized by Integrated DNA Technology as a gBlock (TZ-1) and assembled into pEssential vector amplified with TZ-2 and TZ-3 by Gibson assembly.
For the pBAD33-gp77 constructs, gp77 was PCR amplified from phage SECΦ27 using primers TZ-4 and TZ-5 and inserted into pBAD33 linearized with primers TZ-6 and TZ-7 by Gibson assembly. To add a C-terminal HA tag, primers TZ-10 and TZ-11 were used to PCR amplify pBAD33-gp77 followed by Gibson assembly.
For the pBAD33-portal protein constructs, gp8T7, gp31T3, gp21SECΦ18 or gp52 SECΦ27 was PCR amplified from the corresponding T7, T3, SECΦ18 or SECΦ27 phage using primers TZ-12 to TZ-17, or TZ-23 and TZ-24, respectively. Amplified fragments were inserted into pBAD33 linearized with TZ-6 and TZ-7 using Gibson assembly. To add a C-terminal HA tag, primers TZ-20 to TZ-22 were each used in combination with TZ-10 to PCR amplify the corresponding pBAD33-portal protein constructs followed by Gibson assembly.
For the pBAD33-razr construct, razr was PCR amplified from pLAND-razr using primers TZ-18 and TZ-19 and inserted into pBAD33 linearized with TZ-6 and TZ-7 by Gibson assembly.
For the pET-razr–His 6 and pET-gp77–His 6 constructs: razr–His 6 or gp77–His 6 was PCR amplified from pLAND-razr or pBAD33-gp77 using primers TZ-27 to TZ-30, respectively. Amplified fragments were inserted into pET vector linearized with TZ-25 and TZ-26 by Gibson assembly.
For pACYC constructs, to construct pACYC-gp77–HA, gp77–HA was PCR amplified from pBAD33-gp77–HA using primers TZ-33 and TZ-34 and inserted into pACYC linearized with primers TZ-31 and TZ-32. To construct pACYC-gp8T7–Flag, first gp8T7–HA was PCR amplified from pBAD33-gp8T7–HA using primers TZ-34 and TZ-35 and inserted into pACYC linearized with primers TZ-31 and TZ-32. To replace the C-terminal HA tag with a Flag tag, primers TZ-36 and TZ-37 were used to amplify pACYC-gp8T7–HA followed by Gibson assembly.
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