Fig. 4

Influence of synonymous amino acid substitutions and N39D replacement on the in vivo performance of sfGFP in P. thermoglucosidasius. a Flow cytometry-based comparison of the signal intensities of mid-log DSM 2542 cells transformed with sfGFP(Sp), orange; sfGFP(iGEM), blue; sfGFP(N39D, A179A, H231H), red; sfGFP(N39D/A179A) (green) grown at 60 °C in TGP broth. C, control cells containing the pNW-Ppta-3TER plasmid without gfp gene (black). Representative histograms of three independent biological replicates are shown. b Analysis of temperature-dependent functionality of the four sfGFP variants at mid-exponential growth phase in DSM 2542 as assessed by flow cytometry. The MFI is presented in relative fluorescence units (R.F.U.) and is the average of three independent biological replicates with error bars showing standard deviations. Asterisks denote the detection of fluorescence signals marginally above the autofluorescence of mock cells as shown in control (c) in a