Fig. 1: Design and characterization of thermosensitive bifunctional bio-switch (T-switch).

a Circuits of T-switch using a NOT gate design enables bifunctional gene expression, of which the input is the repressor PhlF related to a temperature-controlled system based on cI⁸⁵⁷(cI), and the output is a reporter sfgfp encoding sfGFP under the promoter P_phlF. Another output is a reporter mrfp encoding mRFP placed downstream the phlF gene (phlF-mrfp) for achieving bifunctional control of gene expression. b Promoter activity and orthogonality of the CI⁸⁵⁷-regulated panel are characterized based on two expression cassettes: sfgfp as a reporter alone in a and phlF-mrfp cluster. c Data generated using the constructs from part a via cytometry analysis are used to characterize the bidirectional temperature-response functions of the T-switch. d Data gathered onto a single transfer function between two reporters, namely, sfGFP and mRFP, are identical to those shown in c from the same experiments. Each point represents a condition of cultural temperature from 30 ℃ to 37 ℃. e The fluorescence intensity was measured from each temperature from 30 °C to 37 °C for controlling the expression levels of sfgfp and phlF-mrfp. The fluorescence ratios between sfGFP and mRFP expression from each temperature point collapse onto a single function for linear regression analysis with R² = 0.9868 and slope close to 1 (1.065), which was plotted in log-log coordinates (log₁₀). Data are presented as mean ± s.d. of three replicates. FI, Fluorescence Intensity in arbitrary unit (a.u.).