Fig. 3
Characterization of the evolved mutants. a The growth curves of the five mutants. WT stands for the strain harboring the parental promoter. b The time-course GFP expression levels of these mutants were determined using the FI. c The expression levels of GFP of the five mutants were compared to the WT to reveal the superior mutant for further characterization and application. The FI values of the five mutants after the 28-h culture in the shake flask condition were adopted to compare the differences in GFP expression. The data are shown as the mean ± S.D. All the experiments were performed independently in triplicate. d SDS-PAGE analysis confirmed the expression levels of GFP of the five mutants. The whole-cell proteins of each sample were separated by 12% SDS-PAGE and were then stained with Coomassie Brilliant Blue R-250. e RT-qPCR was carried out to analyze the relative expression level of GFP under the control of WT and the variant promoter PBH4 at 8 h- and 24 h-culture. The data are calculated by the 2−ΔΔCt method. All the data were independently repeated in triplicate