Figure 3

Investigation of cell-wall-weakening and osmoprotection on electrotransformation of C. pasteurianum . A. Investigation of cell-wall-weakening agents. Cells were grown to early exponential phase (OD₆₀₀ 0.3-0.4) and glycine (gly) or dl-threonine (thr) was added along with 0.25 M sucrose (suc). For lysozyme and penicillin G treatments, additives were supplemented to buffer SMP prior to electroporation and incubated anaerobically at 37°C for 30 minutes. An untreated culture was included as a control. The OD₆₀₀ of each culture at time of harvest is shown (ο). Pulse duration was unaffected between samples. B. Investigation of glycine and sucrose concentrations. Six cultures were grown to an OD₆₀₀ of 0.4 and glycine was added to a final concentration of 0.75, 1.0, or 1.25% together with sucrose at 0.25 (light shading) or 0.4 M (dark shading). Growth was minimally affected between samples, as all cultures attained a final OD₆₀₀ of 1.2-1.5. Pulse duration was unaffected between samples. C. Investigation of glycine concentration and duration of exposure. Two cultures were grown to an OD₆₀₀ of 0.4 and glycine was added to a final concentration of 0.75 or 1.25% together with 0.4 M sucrose. An additional control culture was prepared without either glycine or sucrose supplementation. Cells were harvested, washed, and electroporated at either 2.5 (light shading) or 4 hours (dark shading) following supplementation with glycine and sucrose. Pulse duration was unaffected between samples. D. Effect of sucrose concentration within the wash and electroporation buffer. Cultures were washed and electroporated in SMP buffer containing either isotonic (0.27 M) or hypertonic sucrose (0.5 M). Pulse duration was unaffected between samples. E. Effect of sucrose concentration within the outgrowth medium. Cultures were electroporated and resuspended and grown in 2×YTG medium containing either 0.2 or 0.4 M sucrose.