Fig. 4

Quantitative analysis of sfGFP expression and enrichment in E. coli parent cells and minicells. A The ΔminCD strain harboring pSEVA234-sfGFP was cultured overnight in LB medium with 50 μM IPTG. Parent cells and minicells were isolated from the culture, and whole-cell proteins were extracted from equal masses of each fraction. The relative accumulation of fluorescent proteins in parent cells and minicells was analyzed through western blot using an anti-GFP antibody, with DnaK serving as the loading control. B The reporter strain was also cultured with varying concentrations of IPTG (0, 5, 10, 20, and 50 µM), and the fluorescence intensities were measured in both isolated parent cells and minicells. C The fluorescence signals from each fraction were used to estimate the relative enrichment of the reporter protein in minicells compared to parent cells, expressed as fold enrichment. Fold enrichment in minicells is calculated as the fluorescence intensity in minicells divided by that in parent cells. No significant (NS) fold enrichment was observed, regardless of the GFP expression level in parent cells. D Correlation of GFP accumulation between parent cells and minicells. Error bars represent mean ± SD (n = 3), (R² = 0.9937)