**AWARDS ISSUED PRIOR TO JANUARY 20, 2025, WERE FUNDED UNDER PREVIOUS ADMINISTRATIONS AND MAY NOT REFLECT THE PRIORITIES AND POLICIES OF THE CURRENT ADMINISTRATION.** WITH ADVANCES IN SYNTHETIC BIOLOGY TECHNIQUES, MANY MICROBES HAVE BEEN GENETICALLY MODIFIED TO ENHANCE THEIR NATIVE ABILITY OR ENDOW THEM WITH A NEW ABILITY. MANY PRACTICAL APPLICATIONS INVOLVE USE OF THESE GENETICALLY ENGINEERED (GE) MICROORGANISMS THAT ARE OFTEN EXPECTED TO BE RELEASED INTO THE ENVIRONMENT. FOR EXAMPLE, RESEARCHERS HAVE BEEN DEVELOPING GE PROBIOTICS AS PATHOGEN KILLERS IN THE GUT OF LIVESTOCK, PROVIDING NEW PATHOGEN REDUCTION STRATEGIES FOR FARMERS, BUT ADMINISTERED GE PROBIOTICS WILL BE RELEASED TOGETHER WITH FECES INTO THE ENVIRONMENT. THE CONSEQUENCES OF SUCH RELEASES ARE DIFFICULT TO ASSESS, AND THIS BIOSAFETY CONCERN SHOULD BE ADDRESSED. IF GE MICROBES ARE PROGRAMMED TO COMMIT SUICIDE AFTER THEY ACCOMPLISH THEIR MISSION, SUCH AS PATHOGEN KILLING IN THE GUT OF LIVESTOCK AND REMEDIATION OF CONTAMINATED SITES, WE CAN PREVENT THEIR PROLONGED SURVIVAL IN THE ENVIRONMENT.THE LONG-TERM GOAL OF THIS PROJECT IS TO DEVELOP A GENERALIZABLE GENETIC KILL-SWITCH THAT CAN BE USED IN A VARIETY OF APPLICATION-RELEVANT GE MICROBES. TO ACHIEVE THIS LONG-TERM GOAL, THIS PROJECT WILL ADDRESS THREE MAIN CHALLENGES. FIRST, ALTHOUGH GENETIC KILL-SWITCHES THAT RESPOND TO LAB CHEMICALS HAVE BEEN DEVELOPED, FOR REAL-WORLD APPLICATIONS, THE MICROBE'S VIABILITY SHOULD BE CONTROLLED IN RESPONSE TO APPLICATION-RELEVANT CUES. THIS PROJECT WILL ADDRESS THIS ISSUE BY ENGINEERING APPLICATION-RELEVANT GENETIC SENSORS THAT ALLOW SUICIDE TO BE TRIGGERED IN RESPONSE TO SPECIFICALLY DESIGNED SIGNALS, INSTEAD OF LAB CHEMICALS. ANOTHER KEY CHALLENGE IS TO MEET THE REGULATORY CRITERIA FOR BIOCONTAINMENT. ONE INDICATOR FOR THE BIOCONTAINMENT EFFICIENCY IS THE ESCAPE RATE, DEFINED AS THE RATIO OF CELL NUMBERS IN THE KILLING CONDITION TO THE NON-KILLING CONDITION. THE NIH GUIDELINE IS A RATIO OF LESS THAN 1/100,000,000, WHICH HAS BEEN ACHIEVED BY USING LAB CHEMICALS AS SUICIDE SIGNALS. THIS PROJECT WILL DEMONSTRATE APPLICATION-RELEVANT KILL-SWITCHES THAT MEET THE REGULATORY CRITERIA FOR BIOCONTAINMENT. LASTLY, FOR REAL-WORLD APPLICATIONS, WE MUST DEVELOP KILL-SWITCHES WITH LONG-TERM MUTATIONAL STABILITY TO ENSURE SUICIDE AFTER THE MISSION IS ACCOMPLISHED. TO THIS END, GE MICROBES WILL CONTAIN MULTIPLE FUNCTIONALLY-REDUNDANT KILL-SWITCHES THAT PROVIDE BACKUP IN CASE OF MUTATION. TO OUR KNOWLEDGE, THIS PROPOSAL IS THE FIRST PROJECT TO COLLECTIVELY ADDRESS ALL THREE ISSUES THAT EACH OF THE STATE-OF-THE-ART KILL-SWITCHES HAS PARTIALLY ADDRESSED BUT FAILED TO ADDRESS FULLY. ONCE SUCCESSFULLY DEVELOPED, THE GENERALIZABLE GENETIC KILL-SWITCH WILL HELP MAXIMIZE THE POTENTIAL BENEFITS OF SYNTHETIC BIOLOGY WHILE AVOIDING POTENTIAL RISKS OF GE MICROBES RELEASED INTO THE ENVIRONMENT.