Rebooting Synthetic Phage-Inducible Chromosomal Islands: One Method to Forge Them All
Effects of phage packaging and SaPI transfer on SaPIbov1 engineered mutations. Upper panel: The 80α (a) or ϕ11 (b) mediated induction, replication, and packaging of the islands was analyzed by analysis of the DNA extracted from the purified SaPI and phage lysates. In these experiments, two bands are observed, corresponding to the phage-sized or SaPI-sized capsids. The lower panel is a Southern blot using a probe for the SaPIbov1 tetM cassette. Sample P represents the parent strain containing the helper phage and SaPIbov1 tst::tetM while samples RB represent the rebooted versions of SaPIbov1 WT, SaPIbov1 terS, SaPIbov1 ppicpmA, and SaPIbov1 ppicpmAterS. Null (∅) represents a lysate generated from a strain containing only the helper phage as a control. (c, d) The lysates of each SaPIbov1 version obtained after induction of the helper phages 80α (c) or ϕ11 (d) were analyzed for phage titer (PFU ml-1) and SaPIbov1 transduction titer (TFU ml-1), using RN4220 as recipient strain. The SaPIbov1-mediated phage interference was tested by infecting strains containing the different SaPIbov1 derivative islands with lysates of phage (e) 80α or (f) ϕ11. Statistical analysis was performed using two-way ANOVA followed by Dunnett’s multiple comparisons test with the parent strain 80α SaPIbov1 tst::tetM or ϕ11 SaPIbov1 tst::tetM as controls and as reference for all comparisons, respectively (). Adjusted values for phage titers using helper phage 80α, WT ns ,terS ns ,ppicpmA , ppicpmAterS and (-) ; and for SaPI titers WT ns ,terS , ppicpmA ns ,ppicpmAterS . Adjusted values for phage titers using helper phage ϕ11, WT ns ,terS , ppicpmA , ppicpmAterS and (-) ; and for SaPI titers WT ns ,terS , ppicpmA , ppicpmAterS.