Beneficial microbes have great potential to improve plant growth, health, and resilience in agricultural settings, but many microbial treatments fail to establish or persist long enough to deliver their intended benefit. These microbes often struggle to compete with native soil microorganisms or survive long enough to influence the plant. This project explores a new strategy to improve colonization by supporting a selective association between plants and beneficial bacteria, enabling more reliable delivery of microbial benefits. The research will also investigate the biochemistry of the enzymes that produce the molecules supporting these specific plant-microbe associations. This work may lead to more predictable and efficient use of microbial technologies in agriculture. The project will also train undergraduate and graduate students in biology and engineering. In addition, we will engage K–12 students through interactive science outreach activities, and will lead a one-day professional development workshop for local elementary teachers focused on synthetic biology topics related to plants, food, and agriculture. This project will investigate the engineering of opine-mediated mutualistic plant-microbe interactions. Opines are small molecules naturally produced in some plant-microbe systems that can serve as a carbon and nitrogen source for specific bacteria. We will assess the genetic requirements for opine utilization in plant-beneficial bacteria, characterize the enzymes