Rational Engineering of Explosive-Sensing Soil Bacteria

Buried explosives are a serious danger to civilians and warfighters, whether they are left over from 75 years ago or recently buried by an enemy combatant. Sites containing old, discharged, degraded, or buried ordnance will have surrounding soil with elevated levels of DNT and TNT due to leakage. We have engineered soil bacteria to detect these explosive compounds, letting out an odor humans and dogs can smell from stand-off distances. Our system design is broken down into two functional sections insulated by flanking double terminators. The first section is the sensing/signal input of the circuit which includes a library of promoters, a chemical-sensing riboswitch, and a site-specific integrase. The downstream section is the signal output provides a measurable response that contains the integrase-specific attB and attP sites harboring a state-switchable promoter, a synthetic RBS, and the fluorescent protein/odorant-producing enzyme. We have designed, constructed, and characterized 30 sensor circuits in Bacillus subtilis, varying promoter transcription rates and concentrations of DNT and TNT followed by measuring their RFP reporter protein levels using spectrophotometry and flow cytometry. To verify the orientation of the state-switchable promoter, we performed quantitative PCR experiments with specifically designed primers to determine if the promoter is in the forward or reverse direction. Using the results from these characterizations, 8 of the original 30 sensor circuits were selected for construction using the odorant-producing enzyme. By inserting the VvOMT3 odorant-producing enzyme and adding DNT and TNT, our sensor circuits can produce a detectable amount of 2-methoxy-3-isobutylpyrazine (IBMP) in our proposed soil systems. We measured growth and persistence of these strains in wild soil, confirming sustained growth and persistence for at least 4 weeks to simulate in field use conditions.