Fig. 5: Sequential expression of assembly factor proteins prior to shell protein synthesis mitigates proteotoxic stress and restores normal growth in E. coli.

Schematic representation of the genetic construct for GV expression under the dual-inducer system. Cultures were induced with varying concentrations of IPTG while maintaining a constant aTc concentration (a) or induced with varying concentrations of aTc while maintaining a constant IPTG concentration (b). Growth profiles of E. coli cultures expressing GVs under the dual-inducer system with inducing strategies in (a) (c) and (b) (d). The growth profile of wild-type pNL29 GV-operon-expressing E. coli cultures induced with 20 μM IPTG (WT) served as the control. The vertical gray dashed line indicates the time point at which aTc was introduced. The horizontal black dashed line indicates the average OD₆₀₀ value of the MBP control from Figs. 1d and 2c at 24 h post-induction, representing the maximal OD₆₀₀ achieved by the E. coli culture expressing MBP. e, f CFU measurements of E. coli cultures expressing GVs under the dual-inducer system. Cultures were induced with strategy in (a) (e) or in (b) (f). As controls, CFU measurements were performed on cultures expressing wild-type pNL29 GVs or MBP under 20 µM IPTG induction. Representative plates are shown in Supplementary Figs. 8a and 9a. g Heatmap of cell viability of E. coli cultures expressing GVs under the 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. 8d and 9d. Absorbance values (y-axis) in (c, d) are reported in absorbance units (AU). Data in (c, d) represent mean ± s.d. for n = 6 biologically independent samples, and in (e, f) represent mean ± s.d. for n = 3 biologically independent samples for all groups.