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ABSTRACT Microbial biosensors that convert environmental information into real-time visual outputs are limited in their sensing abilities in complex environments, such as soil and wastewater, due to optical inaccessibility. Biosensors that could record transient exposure to analytes within a large time window for later retrieval represent a promising approach to solve the accessibility problem. Here, we test the performance of recombinase-memory biosensors that sense a sugar (arabinose) and a microbial communication molecule (3-oxo-C12-L-homoserine lactone) over 8 days (~70 generations) following analyte exposure. These biosensors sense the analyte and trigger the expression of a recombinase enzyme which flips a segment of DNA, creating a genetic memory, and initiates fluorescent protein expression. The initial designs failed over time due to unintended DNA flipping in the absence of the analyte and loss of the flipped state after exposure to the analyte. Biosensor performance was improved by decreasing recombinase expression, removing the fluorescent protein output, and using quantitative PCR to read out stored information. Application of memory biosensors in wastewater isolates achieved memory of analyte exposure in an uncharacterizedPseudomonasisolate. By returning these engineered isolates to their native environments, recombinase-memory systems are expected to enable longer duration andin situinvestigation of microbial signaling, cross-feeding, community shifts, and gene transfer beyond the reach of traditional environmental biosensors.IMPORTANCEMicrobes mediate ecological processes over timescales that can far exceed the half-lives of transient metabolites and signals that drive their collective behaviors. We investigated strategies for engineering microbes to stably record their transient exposure to a chemical over many generations through DNA rearrangements. We identify genetic architectures that improve memory biosensor performance and characterize these in wastewater isolates. Memory biosensors are expected to be useful for monitoring cell-cell signals in biofilms, detecting transient exposure to chemical pollutants, and observing microbial cross-feeding through short-lived metabolites within cryptic methane, nitrogen, and sulfur cycling processes. They will also enablein situstudies of microbial responses to ephemeral environmental changes, or other ecological processes that are currently challenging to monitor non-destructively using real-time biosensors and analytical instruments. 

The rapid diversification of synthetic biology tools holds promise in making some classically hard-to-solve environmental problems tractable. Here we review longstanding problems in the Earth and environmental sciences that could be addressed using engineered microbes as micron-scale sensors (biosensors). Biosensors can offer new perspectives on open questions, including understanding microbial behaviors in heterogeneous matrices like soils, sediments, and wastewater systems, tracking cryptic element cycling in the Earth system, and establishing the dynamics of microbe-microbe, microbe-plant, and microbe-material interactions. Before these new tools can reach their potential, however, a suite of biological parts and microbial chassis appropriate for environmental conditions must be developed by the synthetic biology community. This includes diversifying sensing modules to obtain information relevant to environmental questions, creating output signals that allow dynamic reporting from hard-to-image environmental materials, and tuning these sensors so that they reliably function long enough to be useful for environmental studies. Finally, ethical questions related to the use of synthetic biosensors in environmental applications are discussed.