** AWARDS ISSUED PRIOR TO JANUARY 20, 2025, WERE FUNDED UNDER PREVIOUS ADMINISTRATIONS AND MAY NOT REFLECT THE PRIORITIES AND POLICIES OF THE CURRENT ADMINISTRATION.** BOVINE MASTITIS IS A CONSTANT PROBLEM IN THE DAIRY INDUSTRY. MILK QUALITY IS AFFECTED, PRODUCTION IS REDUCED, AND ANTIBIOTICS ARE NEEDED FOR TREATMENT WHEN DAIRY COWS ARE AFFECTED BY MASTITIS. ONE OF THE MOST PREVALENT MASTITIS CAUSING BACTERIA IS STAPHYLOCOCCUS AUREUS (S. AUREUS). A POSSIBLE ALTERNATIVE TO ANTIBIOTICS IS THE USE OF BACTERIOPHAGES (PHAGES), WHICH ARE VIRUSES THAT ONLY INFECT BACTERIA. A MAJOR LIMITATION FOR PHAGES TO BE USED AS A TREATMENT FOR BOVINE MASTITIS IS WHEY PROTEIN'S ANTIVIRAL ACTIVITY. THE OVERALL GOAL IS TO INCREASE BACTERIOPHAGE (PHAGE) INFECTIVITY, THUS INCREASING ITS THERAPEUTIC POTENTIAL, OF MASTITIS CAUSING S. AUREUS BY REDUCING ITS INTERACTION WITH MILK ANTIVIRAL MECHANISMS. WE WILL DEVELOP A CELL-FREE TRANSCRIPTION-TRANSLATION (TXTL) SYSTEM DERIVED FROM STAPHYLOCOCCUS AUREUS. THE TXTL SYSTEM DOES NOT PRODUCE ENDOTOXINS OR EXOTOXINS DURING PHAGE PRODUCTION, WHICH IS A MAJOR PROBLEM IN THE CURRENT IN VIVO METHOD OF PHAGE PRODUCTION. IT WILL BE USED TO PRODUCE GREEN FLORESCENT PROTEIN AND WHOLE PHAGES FROM CIRCULARIZED PLASMID DNA. MILK WHEY PROTEIN ANTIVIRAL ACTIVITY IS A HINDRANCE TO PHAGE TREATMENT FOR MASTITIS. WE WILL BE USING THE CRISPR-CAS9 ENGINEERING METHOD TO GENETICALLY MODIFY THE SURFACE OF PHAGE CAPSIDS TO OBTAIN THE POLYMER POLYETHYLENE GLYCOL (PEG) TO CONCEAL THE PHAGE FROM THE ANTIVIRAL ACTIVITY IN RAW MILK LONG ENOUGH TO ELIMINATE S. AUREUS CONTAMINATION.THE RESULTS OF THIS PROJECT WILL BE INSTRUMENTAL IN ADVANCING THE USE OF PHAGES AS AN ANTIMICROBIAL THERAPY IN ANIMAL HUSBANDRY. THE TXTL SYSTEM WILL INCREASE THE SPEED IN WHICH WE CAN PRODUCE AND EVALUATE VIRULENT PHAGES FOR THE USE OF TREATMENT. THE ENGINEERED PHAGES FOR THE TREATMENT OF S. AUREUS IN RAW MILK SAMPLES WILL BRING US ONE STEP CLOSER TO REDUCING THE USE OF ANTIBIOTICS IN THE USE OF TREATING INFECTIONS IN THE DAIRY INDUSTRY AND REDUCE THE SPREAD OF ANTIMICROBIAL RESISTANT BACTERIA.