Fig. 2

Optimizing the production of the membrane protein YidC using pReX. BL21(DE3) cells harboring pReXyidCgfp and Lemo21(DE3) cells harboring pETyidCgfp were cultured in LB medium at 30 °C at varying concentrations of l-rhamnose as indicated. All measurements were done 8 h after the addition of IPTG to induce the expression of t7rnap. a Left panel: to assess the effect of different l-rhamnose concentrations on the production of YidC-GFP in the cytoplasmic membrane, we monitored the fluorescence (relative fluorescence unit, RFU) per milliliter of culture. Right panel: the effect of YidC–GFP production at different l-rhamnose concentrations on biomass formation was monitored by measuring the A₆₀₀. The production of YidC-GFP under Lemo21(DE3) and pReX-based optimal conditions corresponds to 14.0 and 14.7 mg of protein produced per liter of culture, respectively [25]. b Top panel: the integrity of the YidC–GFP fusion produced in the cytoplasmic membrane at different l-rhamnose concentrations was monitored using in-gel fluorescence. Bottom panel: accumulation levels of IbpB in cells producing YidC–GFP at different l-rhamnose concentrations were monitored by immuno-blotting using an antibody against IbpB. c Using flow cytometry the amount of YidC-GFP fusion produced in the cytoplasmic membrane per cell was assessed. Traces of BL21(DE3)/pReXyidCgfp and Lemo21(DE3)/pETyidCgfp cells cultured at the optimal l-rhamnose concentration (maximal amount of fluorescent protein per milliliter of culture) are in red. Traces of the same cells cultured in the absence of l-rhamnose (control) are in gray