Fig. 3: Design and testing of autonomous microbial TNT sensors in liquid culture.

A TNT activates the synthetic genetic circuit by binding to the RS14 riboswitch, which activates the expression of the Int2 recombinase. The Int2 recombinase binds to the att recognition sites and flips the state-switchable promoter’s orientation, which activates the expression of a mRFP1 reporter (output module). Designed sense promoters control the transcription rate of the mRNA encoding the RS14 riboswitch and Int2 recombinase. Designed anti-sense promoters control the reduction in mRNA levels, due to transcriptional interference. Numbers are predicted transcription and translation rates. B Average mRFP1 fluorescence levels of engineered B. subtilis cells during exponential growth in liquid culture carrying the most performant synthetic genetic circuit in response to varied TNT concentrations (0, 15, 25, and 35 µM). Bars and error bars are the mean and standard deviation of 3 biological replicates. Dots are individual data points. Numbers are activation ratios with statistical significance (two-tailed T test p-values are 0.0263 and 0.0112). C The single-cell mRFP1 fluorescence distributions are shown for these engineered B. subtilis cells responding to each TNT concentration versus a wild-type control (no mRFP1). D Average mRFP1 fluorescence levels of engineered B. subtilis cells carrying different synthetic genetic circuits growing in the same conditions, comparing responses at 0 µM TNT (blue bars) versus responses at 35 µM TNT (red bars). Bars and error bars are the mean and standard deviation of 3 biological replicates. Dots are individual data points. Numbers are activation ratios with statistical significance (two-tailed T-test p-values are 0.0337, 0.0329, 1.2E-05, 0.000196, 2.8E-06, 0.0195). All stars denote the statistical significance of comparisons based on p-value thresholds (*0.05, **0.01, ***0.001). Quantitative models of (E) transcriptional interference and (F) riboswitch regulation show how changes in sense promoter and antisense promoter transcription rates alter Int2 mRNA levels and translation activation. Transcription rate predictions are shown using the Promoter Calculator v1.0 scale. RFU is a relative fluorescence unit.