skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

Explore Research Products in the PAR
It may take a few hours for recently added research products to appear in PAR search results.


Title: 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, 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.  more » « less
Award ID(s):
2237052
PAR ID:
10572231
Author(s) / Creator(s):
; ; ;
Editor(s):
Rudi, Knut
Publisher / Repository:
ASM
Date Published:
Journal Name:
Applied and Environmental Microbiology
Volume:
90
Issue:
4
ISSN:
0099-2240
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; (, Applied Microbiology and Biotechnology)
    AbstractThe animal gut microbiome is a complex system of diverse, predominantly anaerobic microbiota with secondary metabolite potential. These metabolites likely play roles in shaping microbial community membership and influencing animal host health. As such, novel secondary metabolites from gut microbes hold significant biotechnological and therapeutic interest. Despite their potential, gut microbes are largely untapped for secondary metabolites, with gut fungi and obligate anaerobes being particularly under-explored. To advance understanding of these metabolites, culture-based and (meta)genome-based approaches are essential. Culture-based approaches enable isolation, cultivation, and direct study of gut microbes, and (meta)genome-based approaches utilizeinsilicotools to mine biosynthetic gene clusters (BGCs) from microbes that have not yet been successfully cultured. In this mini-review, we highlight recent innovations in this area, including anaerobic biofoundries like ExFAB, the NSF BioFoundry for Extreme & Exceptional Fungi, Archaea, and Bacteria. These facilities enable high-throughput workflows to study oxygen-sensitive microbes and biosynthetic machinery. Such recent advances promise to improve our understanding of the gut microbiome and its secondary metabolism. Key points• Gut microbial secondary metabolites have therapeutic and biotechnological potential• Culture- and (meta)genome-based workflows drive gut anaerobe metabolite discovery• Anaerobic biofoundries enable high-throughput workflows for metabolite discovery Graphical abstract 
    more » « less
  2. ; ; ; ; ; ; ; ; ; (, Nature Microbiology)
    Abstract Development of microbial communities is a complex multiscale phenomenon with wide-ranging biomedical and ecological implications. How biological and physical processes determine emergent spatial structures in microbial communities remains poorly understood due to a lack of simultaneous measurements of gene expression and cellular behaviour in space and time. Here we combined live-cell microscopy with a robotic arm for spatiotemporal sampling, which enabled us to simultaneously acquire phenotypic imaging data and spatiotemporal transcriptomes duringBacillus subtilisswarm development. Quantitative characterization of the spatiotemporal gene expression patterns revealed correlations with cellular and collective properties, and phenotypic subpopulations. By integrating these data with spatiotemporal metabolome measurements, we discovered a spatiotemporal cross-feeding mechanism fuelling swarm development: during their migration, earlier generations deposit metabolites which are consumed by later generations that swarm across the same location. These results highlight the importance of spatiotemporal effects during the emergence of phenotypic subpopulations and their interactions in bacterial communities. 
    more » « less
  3. ; ; ; ; ; (, Frontiers in Microbiology)
    null (Ed.)
    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. 
    more » « less
  4. ; ; ; ; ; (, bioRxiv)
    Abstract Anthropogenic pollution affects environments differently depending on proximity to pollution source, exposure route, and species ecology. Thus, understanding organism’s ecological role and exposure route to contaminants is central to assessing pollution impact. Treated municipal wastewater releases contaminants into waterways and alters microbial communities. Plants absorb contaminants and expose animals through foraging and nest-building activities. Nesting ecology differences of ground vs wood cavity-nesting bees offers insight into niche-specific susceptibility to pollution. Because contaminants bind to soil strongly, ground-nesting bees near wastewater are likely most impacted, while wood cavity-nesting bees likely less impacted since plants’ ability to uptake contaminants are species dependent. We compared gut microbiomes of directly exposed soil-nestingHalictus ligatusand indirectly exposed wood-nestingCeratinaspp. upstream/downstream of wastewater. We collected bees, flowers, and soil, and analyzed their bacteria microbiomes (16S rRNA). Wastewater altered ground-nestingH. ligatusmicrobiome >18 times greater than wood cavity-nestingCeratinaadults.Ceratinalarvae and pollen provisions showed significant but smaller shifts. Conversely, soil and flower microbiomes remained stable, indicating higher resilience. These results demonstrate that exposure routes drive contaminants susceptibility, with animal-associated microbes most vulnerable. Because bees are important pollinators and biodiversity contributors, these disruptions point to broader ecological risks in increasingly contaminated landscapes. Abstract Figure 
    more » « less
  5. ; (, Frontiers in Bioengineering and Biotechnology)
    Engineered probiotic bacteria have been proposed as a next-generation strategy for noninvasively detecting biomarkers in the gastrointestinal tract and interrogating the gut-brain axis. A major challenge impeding the implementation of this strategy has been the difficulty to engineer the necessary whole-cell biosensors. Creation of transcription factor-based biosensors in a clinically-relevant strain often requires significant tuning of the genetic parts and gene expression to achieve the dynamic range and sensitivity required. Here, we propose an approach to efficiently engineer transcription-factor based metabolite biosensors that uses a design prototyping construct to quickly assay the gene expression design space and identify an optimal genetic design. We demonstrate this approach using the probiotic bacterium Escherichia coli Nissle 1917 (EcN) and two neuroactive gut metabolites: the neurotransmitter gamma-aminobutyric acid (GABA) and the short-chain fatty acid propionate. The EcN propionate sensor, utilizing the PrpR transcriptional activator from E. coli , has a large 59-fold dynamic range and >500-fold increased sensitivity that matches biologically-relevant concentrations . Our EcN GABA biosensor uses the GabR transcriptional repressor from Bacillus subtilis and a synthetic GabR-regulated promoter created in this study. This work reports the first known synthetic microbial whole-cell biosensor for GABA, which has an observed 138-fold activation in EcN at biologically-relevant concentrations. Using this rapid design prototyping approach, we engineer highly functional biosensors for specified in vivo metabolite concentrations that achieve a large dynamic range and high output promoter activity upon activation. This strategy may be broadly useful for accelerating the engineering of metabolite biosensors for living diagnostics and therapeutics. 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email