** AWARDS ISSUED PRIOR TO JANUARY 20, 2025, WERE FUNDED UNDER PREVIOUS ADMINISTRATIONS AND MAY NOT REFLECT THE PRIORITIES AND POLICIES OF THE CURRENT ADMINISTRATION.** WE PROPOSE NEW METHODS TO EVALUATE GENETIC CONTAINMENT STRATEGIES FOR ENGINEERED MICROORGANISMS (GEMS) IN PLANTS AND SOILS, FULFILLING THE PROGRAM GOAL TO EVALUATE MANAGEMENT, MONITORING, AND MITIGATION METHODOLOGIES. GENETIC CONTAINMENT OF CELL VIABILITY ('KILL SWITCH' CIRCUITS) OFFER SAFEGUARDS AGAINST THE UNCONTROLLED PROLIFERATION OF GEMS OUTSIDE THEIR INTENDED USE. THE RISK OF EVASION DEPENDS ON MULTIPLE INTERACTING ENVIRONMENTAL AND ECOLOGICAL FACTORS THAT INFLUENCE KILL SWITCH EFFICACY AND THE FITNESS OF SURVIVOR POPULATIONS. THEREFORE, AN EVALUATION OF EVASION RISK REQUIRES EXPERIMENTATION AND MODELING IN NATURALISTIC SETTINGS THAT CAPTURE THESE DYNAMICS. WE PROPOSE TO DEVELOP A MODEL SYSTEM TO EVALUATE THE EFFICACY OF KILL SWITCHES USING A MODEL LEGUME AND BACTERIAL SYMBIONT, WHERE INFECTION AND NODULE FORMATION IS USED TO ASSESS RISK. OUR FIRST OBJECTIVE IS TO ESTABLISH THE MOLECULAR METHODS NEEDED TO QUANTIFY KILL SWITCH EFFICACY AND THE PREVALENCE OF ESCAPE MUTANTSIN SOIL AND PLANT SAMPLES. SUBSEQUENTLY, WE WILL USE THE MODEL TO STUDY THE FACTORS IMPACTING EVASION RISK IN DIFFERENT PLANT AND SOIL COMPARTMENTS (OBJECTIVE 2), AND THE RELATIVE IMPORTANCE OF ENVIRONMENTAL STRESS, RESOURCES, AND MICROBIAL COMPETITION ON EVASION RISK (OBJECTIVE 3). OUR AIM IS TO LINK EVASION RISK TO FACTORS RELEVANT TO MANAGEMENT PRACTICES, SUCH AS NUTRIENT MANAGEMENT AND SOIL TYPE, TO INFORM THE USE OF KILL SWITCHES IN AGROECOSYSTEMS. OVERALL, OUR PROJECT WILL PROVIDE RESEARCHERS AND BIOENGINEERS WITH A MODEL FOR RISK ASSESSMENT TO VALIDATE NEW GENETIC CONTAINMENT STRATEGIES AND ADVANCE OUR UNDERSTANDING OF THE ENVIRONMENTAL AND ECOLOGICAL FACTORS IMPACTING CONTAINMENT.