Fig. 4: Long-term testing of an autonomous microbial TNT sensor in wild soil.

A The experimental workflow and measurement timelines for characterizing the function and persistence of the TNT-sensing autonomous microbial sensor in wild soil. Measurements on soil samples included quantitative PCR (qPCR), colony-forming units (CFUs), flow cytometry (FLOW), and next-generation sequencing (NGS). B Measured red fluorescence levels of the TNT-sensing autonomous microbial in wild soil at 0 mg TNT / kg soil (blue circles), 4.5 mg TNT / kg soil (green squares), 45 mg TNT / kg soil (red diamonds), and 110 mg TNT / kg soil (tan stars) over a 28-day period. C Measured red fluorescence levels of WT B. subtilis cells (gray triangles), mRFP1 only B. subtilis cells (orange diamonds), and the no added cells control (purple octagons) in wild soil without TNT over a 28-day period. D Measured mRFP1 activation ratios of the TNT-sensing autonomous microbial sensor in wild soil with 4.5 mg TNT / kg soil (green bars), 45 mg TNT / kg soil (red bars), and 110 mg TNT / kg soil (tan bars) over a 28-day period. Bars and error bars are the mean and standard deviation of 3 biological replicates. Dots are individual data points. The statistical significance of the starred comparisons is quantified by two-tailed T test p-values (0.000003, 0.0001, 0.0021, 0.000049, 0.0000047, 0.00021, 0.000091, 0.0000006, 0.000495, 0.01198, 0.00060, 0.037026, 0.009956, 0.00448). E The percentage of autonomous microbial sensors in wild soil with varied TNT amounts over a 28-day period. F The percentage of auto-fluorescent control cells in wild soil without TNT over a 28-day period. G The percentage of other natural microbial cells in wild soil with varied conditions across a 28-day period. H Measured cell viability counts on selective agar plates (LB/Cm5) from wild soil samples containing the autonomous microbial sensor with varied TNT amounts. I Measured cell viability counts on selective (LB/Cm5) or non-selective (LB) agar plates from wild soil samples containing controls. J Measured copy number of autonomous microbial sensor genomes in wild soil with varied TNT amounts as quantified by qPCR. Data points and error bars are the mean and standard deviation of (B–I) 3 or (J) 2 biological replicates (independent soil containers), each with (B–I) 3 or (J) 2 technical replicates (samples per container). RFU is a relative fluorescence unit.