Fig. 4

Relationship of the expression level of the target gene egfp in ORF1 and the reporter gene mcherry in ORF2. a Time course of the fluorescence intensity of eGFP and mCherry. BL21(DE3)pLysS harboring pGFP-RFP, pGFPI-RFP, and pGFPII-RFP were grown for 6 h in LB media after 0.5 mM IPTG induction. b Comparison of the co-expression levels of eGFP and mCherry in three recombinant E. coli at 6 h after IPTG induction. The fluorescence values of eGFP and mCherry in pGFP-RFP were defined as 100%, and the fluorescence values in pGFPI-RFP and pGFPII-RFP were normalized by dividing the actual fluorescence values by the fluorescence values of eGFP and mCherry in pGFP-RFP. Data are representative of three independent experiments and values are expressed as mean ± SEM. c Fluorescence microscopic analysis of E. coli BL21(DE3)pLysS/pGFP-RFP after 4 h induction with 0.5 mM IPTG. E. coli cells were visualized using a fluorescence microscope (×400 or ×1000 magnification). The fluorescence imaging of eGFP upon excitation at 475–490 nm and emission detection between 505 and 535 nm, and fluorescence imaging of mCherry upon excitation at 545–565 nm and emission detection between 580 and 620 nm. The same setting was applied for imaging of both eGFP and mCherry. At least three independent preparations were analyzed and representative images are shown here