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Biology subjects

Cetnar, D. P.

Publications and source records attributed to Cetnar, D. P..

2 recordsLinked to original sources

An Autonomous Microbial Sensor Enables Long-term Detection of TNT Explosive in Natural Soil

Microbes can be engineered to detect target chemicals, but when they operate in real-world environments, it remains unclear how competition with natural microbes affect their performance over long time periods. We engineered sensors and memory-storing genetic circuits inside Bacillus subtilis to sense and respond to the TNT explosive, using predictive models for rational design. We characterized their ability to detect TNT in a natural soil system, measuring single-cell and population-level behavior over a 28-day period. The autonomous microbial sensor activated its response by 14-fold when exposed to low TNT concentrations and maintained stable activation for over 21 days, exhibiting exponential decay dynamics at the population-level with a half-life of about 5 days. Our results show that engineered soil bacteria can carry out long-term detection of an important chemical in natural soil with competitive growth dynamics serving as additional biocontainment.

synthetic biology↗

Systematic Quantification of Sequence and Structural Determinants Controlling mRNA stability in Bacterial Operons

mRNA degradation is a central process that affects all gene expression levels, and yet the determinants that control mRNA decay rates remain poorly characterized. Here, we applied a synthetic biology, learn-by-design approach to elucidate the sequence and structural determinants that control mRNA stability in bacterial operons. We designed, constructed, and characterized 82 operons, systematically varying RNAse binding site characteristics, translation initiation rates, and transcriptional terminator efficiencies in the 5 UTR, intergenic, and 3 UTR regions, and measuring their mRNA levels using RT-qPCR assays. We show that introducing long single-stranded RNA into 5 UTRs reduced mRNA levels by up to 9.4-fold and that lowering translation rates reduced mRNA levels by up to 11.8-fold. We also found that RNAse binding sites in intergenic regions had much lower effects on mRNA levels. Surprisingly, changing transcriptional termination efficiency or introducing long single-stranded RNA into 3 UTRs had no effect on upstream mRNA levels. From these measurements, we developed and validated biophysical models of ribosome protection and RNAse activity with excellent quantitative correspondence. We also formulated design rules to rationally control a mRNAs stability, facilitating the automated design of engineered genetic systems with desired functionalities.

synthetic biology↗