SAGE enables high-efficiency, multi-site integration of genetic cargo with recyclable genetic containers into bacteria. This eases introduction of DNA-carrying genetic circuits and metabolic pathways to engineer microorganisms for enhancing crop growth and for making bioproducts.
SAGE enables high-efficiency, multi-site integration of genetic cargo with recyclable genetic containers into bacteria. This eases introduction of DNA-carrying genetic circuits and metabolic pathways to engineer microorganisms for enhancing crop growth.
Image courtesy of Nathan Johnson, Pacific Northwest National Laboratory

The Science

Genetic engineers use synthetic biology to provide novel functions in microbes by introducing new genes. Synthetic biology involves modifying biological parts and systems to create new ones. Its techniques include introducing a small number of genes into bacteria using small DNA molecules called plasmids. Scientists also use an approach called homologous recombination to introduce genes into the bacterial chromosome. Researchers have now developed a more efficient method called Serine recombinase-Assisted Genome Engineering (SAGE). SAGE borrows components from bacterial viruses to aid in the insertion of genes into bacterial chromosomes. This new tool has the potential to work well in many species of bacteria. Moreover, SAGE genome integration components can be recycled. This will make it easier to deliver multiple pieces of genetic cargo into bacterial cells.

The Impact

By studying different natural environments, scientists constantly discover new microbes. They can then use synthetic biology to genetically modify these microbes. This can help address challenges ranging from converting plant biomass into renewable fuels to reducing crops’ need for fertilizer. However, harnessing the potential of these microbes faces a challenge. Scientists lack sophisticated genetic manipulation tools for novel bacteria. A major problem with traditional tools is the instability of the DNA introduced into bacterial cells. This is especially important for newly discovered bacteria that must grow outside controlled laboratory conditions. In contrast, SAGE is a universal and efficient genetic manipulation system for bacteria. These features will help accelerate synthetic biology research for bioenergy.


Scientists have developed a new method to rapidly introduce genetic material into bacteria. The team included researchers from Pacific Northwest National Laboratory, Oak Ridge National Laboratory, the University of Tennessee, Knoxville, the University of California, Berkeley and the United States Department of Agriculture Agricultural Research Service. The technique, called Serine recombinase-Assisted Genome Engineering (SAGE), allows the iterative introduction of as many as 10 DNA molecules into bacteria. SAGE uses serine recombinases that can stably install genetic cargo on the chromosome of potentially any bacterial species. The high-efficiency and multi-cycle integrations enable scientists to establish large genetic programs into cells, endowing them with, for example, new biosynthetic properties. This is possible by deploying different recombinases and by recycling the genetic markers used to select engineered strains.

The researchers successfully applied SAGE to five taxonomically diverse bacterial hosts that have attractive traits related to plant rhizosphere colonization and plant growth production, hydrogen production, bioremediation of hydrocarbons, and industrial bioproduction. In these hosts, SAGE provided genetic transformation efficiency equivalent to or better than standard plasmid transformation for each host. Further, the integration of a library of taxonomically diverse genetic regulation elements via SAGE revealed promoters with consistent behaviors across multiple environmental growth conditions. In this way, SAGE will expand the number of environmental and industrial microbes that are compatible with high-throughput synthetic biology for biotechnological applications.    


Robert Egbert
Pacific Northwest National Laboratory

Joshua Elmore
Pacific Northwest National Laboratory

Adam Guss
Oak Ridge National Laboratory


Funding was provided in part by the Department of Energy (DOE) Office of Science, Biological and Environmental Research program to Pacific Northwest National Laboratory’s Secure Biosystems Design Science Focus Area, "Persistence Control of Engineered Functions in Complex Soil Microbiomes" and to the Oak Ridge National Laboratory Center for Bioenergy Innovation. Additional support was provided by the DOE Office of Energy Efficiency and Renewable Energy, Bioenergy Technologies Office to the Agile BioFoundry, and by the Defense Advanced Research Projects Agency Synergistic Discovery & Design program.


Elmore, J. R., et al., High-throughput genetic engineering of non-model and undomesticated bacteria via iterative site-specific genome integrationScience Advances 9, 10 (2023). [DOI: 10.1126/sciadv.ade1285]