Long duration environmental biosensing by recording analyte detection in DNA using recombinase memory

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. Alternative reporter outputs are needed that stably record the presence of analytes. Here, we test the performance of recombinase-memory biosensors that sense a sugar (arabinose) and a microbial communication molecule (3-oxo-C12- homoserine lactone) over 8 days ([~]70 generations) following analyte exposure. These biosensors use analyte sensing to trigger the expression of a recombinase 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 qPCR to read out stored information. Application of memory biosensors in wastewater isolates achieved memory of analyte exposure in an uncharacterized Pseudomonas isolate. By returning these engineered isolates to their native environments, recombinase-memory systems are expected to enable longer duration and in situ investigation of microbial signaling, community shifts, and gene transfer beyond the reach of traditional environmental biosensors. IMPORTANCELiving microbial sensors can monitor chemicals and biomolecules in the environment in real-time, but they remain limited in their ability to function on the week, month, and year timescales. To determine if environmental microbes can be programmed to record the detection of analytes over longer timescales, we evaluated whether the sensing of a microbial signaling molecule could be recorded through a DNA rearrangement. We show that off-the-shelf DNA memory is suboptimal for long-duration information storage, use iterative design to enable robust functioning over more than a week, and demonstrate DNA memory in an uncharacterized wastewater Pseudomonas isolate. Memory biosensors will be useful for monitoring the role of quorum sensing in wastewater biofilm formation, and variations of this design are expected to enable studies of ecological processes in situ that are currently challenging to monitor using real-time biosensors and analytical instruments.