Figure 2.

TcI39 simultaneously activates and represses, serving as a temperature-controlled state switch. (a,b) Circuit diagram of TcI39 switch construct with state of regulation arcs indicated at low (a) and high (b) temperature. TcI39 activates expression of mWasabi (GFP) from the PRM promoter and represses expression of mRFP1 (RFP) from the PR–PL tandem promoter. (c) Thermal profile of GFP and RFP co-expression. Central plot: bivariate kernel density estimation for RFP channel and GFP channel. Marginal plots: summed frequency histograms for RFP channel (right) and GFP channel (top). NF indicates nonfluorescent control measured in each channel (not shown in central plot for visual clarity; overlaps with 32.0, 33.3, 35.1 °C histograms in RFP channel). (d) Thermal profile of mean population fluorescence of GFP and RFP expressed in E. coli containing the TcI39 switch construct. Eight hours of incubation, n = 4 biological replicates. Error bars represent ± SEM. (e) Diagram of experiment illustrating differential gene expression with temperature. We drew images on two agar plates using a glycerol stock of E. coli containing the TcI state switch construct. We incubated each plate at a different temperature overnight before performing fluorescence imaging. (f) Overlay of GFP (green) and RFP (magenta) fluorescence images of E. coli containing the TcI switch construct, cultured on agar plates at 37 °C (left) and 44 °C (right). (g) GFP fluorescence image of plates in (f). (h) RFP fluorescence image of plates in (f). Color map limits were adjusted for each fluorophore to make the relative fluorescence levels of the two plates apparent. Parts of the figure were created with BioRender.com.