Host cell cultures, lagoons, media, and the PACE apparatus were as previously described9 (link). Recombined selection phage harboring gene III (rSP) will poison a PACE experiment by outcompeting the evolving SP. We have noted that the likelihood of rSP occurrence in an SP stock increases with extended standing culture growth during the initial SP stock preparation. To reduce the likelihood of rSP formation, all SPs are repurified prior to any continuous evolution experiments. Briefly, SPs were plaqued on S2208 cells. A single plaque was picked into 2 mL 2xYT (United States Biological) supplemented with the appropriate antibiotics and grown until the culture reached mid log-phase (OD600 0.5–0.8). The culture was centrifuged using a tabletop centrifuge for 2 min at 10,000 G, followed by supernatant filtration using a 0.22 μm PVDF Ultrafree centrifugal filter (Millipore). This short growth period routinely yields titers of 106–108 pfu/mL and was found to minimize the occurrence of rSP during PACE experiments.
To prepare the PACE strain, the AP and MP were co-transformed into electrocompetent S1030 cells (see above) and recovered using Davis rich media9 (link) (DRM) to ensure MP repression. Transformations were plated on 1.8% agar-2xYT containing 50 μg/mL carbenicillin, 40 μg/mL chloramphenicol, 10 μg/mL fluconazole, 10 μg/mL amphotericin B, 100 mM glucose (United States Biological) and grown for 12–18 h in a 37 °C incubator. Following overnight growth, four single colonies were picked and resuspended in DRM, then serially diluted and plated on 1.8% agar-2xYT containing 50 μg/mL carbenicillin, 40 μg/mL chloramphenicol, 10 μg/mL fluconazole, 10 μg/mL amphotericin B, and either 100 mM glucose or 100 mM arabinose (Gold Biotechnology) and grown for 12–18 h in a 37 °C incubator. Concomitant with this plating step, the dilution series was used to inoculate liquid cultures in DRM supplemented with 50 μg/mL carbenicillin, 40 μg/mL chloramphenicol, 10 μg/mL tetracycline, 50 μg/mL streptomycin, 10 μg/mL fluconazole, 10 μg/mL amphotericin B and grown for 12–18 h in a 37 °C shaker at 230 rpm. Following confirmation of arabinose sensitivity using the plate assay, cultures of the serially diluted colonies still in log-phase growth were used to seed a 25-mL starter culture for the PACE chemostat.
Once the starter culture had reached log-phase density (OD600 0.5–0.8), the 25-mL culture was added directly to 175 mL of fresh DRM in the chemostat. The chemostat culture was maintained at 200 mL and grown at a dilution rate of 1.5–1.6 vol/hr as previously described9 (link). Lagoons flowing from the chemostats were maintained at 40 mL, and diluted as described for each experiment. Lagoons were supplemented with 25 mM arabinose to induce the MP for 8–16 h prior to infection with packaged SP. Samples were taken at the indicated time points, centrifuged at 10,000 G for 2 min, then filtered with a 0.2 μm filter and stored overnight at 4°C. Phage aliquots were titered by plaque assay on S2208 cells (total phage titer) and S1030 or S2060 cells (rSP titer) for all time points.
To prepare the PACE strain, the AP and MP were co-transformed into electrocompetent S1030 cells (see above) and recovered using Davis rich media9 (link) (DRM) to ensure MP repression. Transformations were plated on 1.8% agar-2xYT containing 50 μg/mL carbenicillin, 40 μg/mL chloramphenicol, 10 μg/mL fluconazole, 10 μg/mL amphotericin B, 100 mM glucose (United States Biological) and grown for 12–18 h in a 37 °C incubator. Following overnight growth, four single colonies were picked and resuspended in DRM, then serially diluted and plated on 1.8% agar-2xYT containing 50 μg/mL carbenicillin, 40 μg/mL chloramphenicol, 10 μg/mL fluconazole, 10 μg/mL amphotericin B, and either 100 mM glucose or 100 mM arabinose (Gold Biotechnology) and grown for 12–18 h in a 37 °C incubator. Concomitant with this plating step, the dilution series was used to inoculate liquid cultures in DRM supplemented with 50 μg/mL carbenicillin, 40 μg/mL chloramphenicol, 10 μg/mL tetracycline, 50 μg/mL streptomycin, 10 μg/mL fluconazole, 10 μg/mL amphotericin B and grown for 12–18 h in a 37 °C shaker at 230 rpm. Following confirmation of arabinose sensitivity using the plate assay, cultures of the serially diluted colonies still in log-phase growth were used to seed a 25-mL starter culture for the PACE chemostat.
Once the starter culture had reached log-phase density (OD600 0.5–0.8), the 25-mL culture was added directly to 175 mL of fresh DRM in the chemostat. The chemostat culture was maintained at 200 mL and grown at a dilution rate of 1.5–1.6 vol/hr as previously described9 (link). Lagoons flowing from the chemostats were maintained at 40 mL, and diluted as described for each experiment. Lagoons were supplemented with 25 mM arabinose to induce the MP for 8–16 h prior to infection with packaged SP. Samples were taken at the indicated time points, centrifuged at 10,000 G for 2 min, then filtered with a 0.2 μm filter and stored overnight at 4°C. Phage aliquots were titered by plaque assay on S2208 cells (total phage titer) and S1030 or S2060 cells (rSP titer) for all time points.
