Fig. 6: Interchanging regulatory control of assembly factor proteins and shell protein expression resulted in limited mitigation of proteotoxic stress.

a Schematic representation of the interchanged dual-inducer system for independent control of GV shell and assembly proteins. The shell protein gene is driven by an IPTG--inducible T7 promoter, whereas assembly protein genes are under a TetR-repressed, aTc-inducible promoter. b, d Schematic representation of the genetic construct for GV expression under the interchanged dual-inducer transcriptional system. Cultures were induced with varying concentrations of IPTG while maintaining aTc at a constant concentration (b), or with varying concentrations of aTc while maintaining IPTG at a constant concentration (d). Growth profiles of E. coli cultures expressing GVs under the interchanged dual-inducer system after being induced with inducing strategies in (b) (c) and (d) (e). The growth profile of WT 20 μM IPTG served as a control (WT). The vertical gray dashed line indicates the time point at which IPTG was introduced. The horizontal black dashed line is the same reference line as in Fig. 5c, d. f, g CFU measurements of E. coli cultures expressing GVs under the interchanged dual-inducer system. Cultures were induced with strategy in (b) (f) or in (d) (g). Controls are the same in Fig. 5e, f. Representative plates are shown in Supplementary Figs. 10a and 11a. h Heatmap of cell viability of E. coli cultures expressing GVs under the interchanged dual-inducer system. The cells were harvested at predetermined time points post-induction and analyzed by flow cytometry (SYTO-9 vs. PI). Comprehensive flow cytometry plots are shown in Supplementary Figs. 10d and 11d. Absorbance values (y-axis) in (c, e) are reported in absorbance units (AU). Data in (c, e) represent mean ± s.d. for n = 6 biologically independent samples, and in (f, g) represent mean ± s.d. for n = 3 biologically independent samples for all groups.