Genomic Science Program
U.S. Department of Energy | Office of Science | Biological and Environmental Research Program

Synthetic Biology Tools to Reliably Establish and Monitor Microbial Invasions in the Rhizosphere


Ilenne Del Valle Kessra* (, Joshua K. Michener, Carrie A. Eckert, Paul E. Abraham


Biosciences Division, Oak Ridge National Laboratory



The Secure Ecosystem Engineering and Design (SEED) Science Focus Area (SFA), led by Oak Ridge National Laboratory, combines unique resources and expertise in the biochemistry, genetics, and ecology of plant-microbe interactions with new approaches for analysis and manipulation of complex biological systems. The long-term objective is to develop a foundational understanding of how non-native microorganisms establish, spread, and impact ecosystems critical to U.S. DOE missions. This knowledge will guide biosystems design for ecosystem engineering while providing the baseline understanding needed for risk assessment and decision-making.


Precise manipulation of natural or managed ecosystems can improve ecosystem resilience and productivity benefiting biosecurity and the bioeconomy. Successful, targeted ecosystem alterations are increasingly feasible by deliberately introducing non-native or genetically modified plants and microbes. However, scientists currently lack the knowledge to successfully predict and introduce beneficial alterations, prevent undesired modifications, or predict the risks of proposed ecosystem biodesign efforts.

Today, microbes are routinely used as active ingredients in commercial biofertilizers and biopesticides to improve plant sustainability and productivity. However, these non-native microbes often fail to establish and spread, requiring frequent reapplication. Successful establishment, dispersal, and beneficial impact of these microbes relies on the interaction of multiple phenotypic traits with the environment and resident microbial community. Identifying the genetic determinants of these complex traits requires a genome-wide interrogation of gene function. Moreover, the ability to monitor the movement, activity, and persistence of microbes in the environment is limited primarily to destructive approaches such as meta-sequencing technologies. Therefore, new techniques for in situ or nondestructive measurements and imaging of environmental microbial activities are needed to interrogate the dynamics of microbial invasions.

To this end, the project has developed a workflow to rapidly enhance transformation efficiency and genetic part characterization in nonmodel bacteria to engineer genome-wide libraries for high-throughput CRISPR interference (CRISPRi) screens. Researchers are developing biodesign tools for real-time in situ detection and quantification of microbial activity in ecosystems. These tools are currently being deployed in the plant growth–promoting bacteria Bacillus velezensis, a strong candidate for improving Populus sp. resistance to the pathogenic fungus Sphaerulina musiva.

First, the team is using CRISPRi to study how perturbation of gene expression impacts microbial establishment in the soil, rhizosphere, and in planta. Researchers built a 40,000-gRNA library targeting 10 gRNAs per annotated coding region in the genome and are performing growth assays to measure gRNA enrichment/depletion using next-generation sequencing under different selective conditions, such as during growth with root exudates and in different soil types. These functional assays will allow researchers to identify genetic perturbations affecting microbial establishment and inform engineering targets for future rhizosphere microbiome manipulation.

Second, the team is engineering a gas-based biosensor into B. velezensis and S. musiva to monitor the dispersal and activity of engineered strains belowground. These genetically engineered microorganisms will use an enzyme called methyl halide transferase to continuously produce methyl halide gas in vegetative cells. This indicator gas can be easily detected using gas chromatography–mass spectrometry without sample disruption from laboratory-to-field scales. Monitoring the location and activity of B. velezensis and S. musiva will aid in tracking and controlling the spread of microbes within and between select environments.

Collectively, these studies will provide new tools to study, engineer, and optimize targeted beneficial alterations to microbial communities in managed ecosystems.

Funding Information

The SEED SFA is sponsored by the GSP, U.S DOE, Office of Science, BER program, under FWP ERKPA17. Oak Ridge National Laboratory is managed by UT-Battelle, LLC for the U.S. DOE under contract no. DE-AC05-00OR45678.