Rational Engineering of Explosive-Sensing Soil Bacteria
Restricted (Penn State Only)
- Author:
- Essington, Erin
- Graduate Program:
- Chemical Engineering (MS)
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- July 16, 2021
- Committee Members:
- Howard M Salis, Thesis Advisor/Co-Advisor
Esther Gomez, Committee Member
Phillip Savage, Program Head/Chair
Amir Sheikhi, Committee Member - Keywords:
- buried explosives
Bacillus subtilis
genetic circuits - Abstract:
- 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 2,4-dinitrotoluene (DNT) and 2,4,6-trinitrotoluene (TNT). To detect buried ordnance, we have engineered sense-and-respond genetic circuits in soil bacteria to detect DNT and TNT and respond by producing an odorant molecule that is readily detectable by humans and canines from stand-off distances. To do this, we engineered genetic circuits with several components, including constitutive or chemical-sensing promoters, chemical-sensing riboswitches or ribosome binding sites, site-specific recombinases, switchable promoters, and output modules expressing either a fluorescent protein or an odorant-producing enzyme. We have designed and constructed 30 genetic circuit variants, varying component design and usage, followed by integration into the genome of the soil bacteria Bacillus subtilis. We then characterized the functionalities of these genetic circuit variants, measuring fluorescent protein expression levels across several conditions and carrying out quantitative PCR to interrogate the state of the switchable promoters. From these results, we identified 8 high-performance genetic circuit variants. For these variants, we then inserted the odorant-producing enzyme in place of the fluorescent protein reporter to produce 2-methoxy-3-isobutylpyrazine (IBMP) as the output signal. We also measured the growth and persistence of engineered Bacillus subtilis strains in wild soil, confirming that growth continued, and functionality persisted for at least 4 weeks. Genetically engineered in-soil sense-and-respond bacteria provide a new countermine capability and a platform for detecting specific metabolites inside soil systems.