Fig. 3

Characterizing YidC-GFP and GltP-GFP production. BL21(DE3) cells harboring either a pET-based yidC-gfp or gltP-gfp expression vector were cultured in LB medium at 30 °C in the absence and presence of IPTG (final concentration 0.4 mM). a The integrity of the in the cytoplasmic membrane produced YidC-GFP and GltP-GFP fusions (double asterisk) was monitored in whole-cell lysates using in-gel fluorescence 24 h after the addition of IPTG. 0.05 A₆₀₀ units of cells were loaded per lane. b The ratio of the cytoplasmic membrane inserted to non-inserted YidC-GFP and GltP-GFP was monitored 24 h after the addition of IPTG. Levels of non-inserted (asterisk; see also Fig. 1) and inserted (double asterisk; see also Fig. 1) membrane protein-GFP fusions in whole-cell lysates were analyzed by means of SDS-PAGE followed by immuno-blotting using an antibody recognizing the His-tag at the C-terminus of the GFP moiety (top panels). Note that the inserted membrane protein-GFP fusions correspond with the fluorescent bands detected using in-gel fluorescence; both are marked with double asterisk. Protein folding/aggregation stress in the cytoplasm was monitored by determining the levels of IbpB in whole-cell lysates using immuno-blotting (bottom panels). 0.05 A₆₀₀ units of cells were loaded per lane. c The production of YidC-GFP and GltP-GFP in the cytoplasmic membrane was monitored on-line by measuring GFP fluorescence every 5 min in cells cultured in the presence and absence of IPTG in a 96-well plate in a spectrofluorometer. Cells cultured in the presence of IPTG are represented in grey and cells cultured in the absence of IPTG are represented in black