GENETIC MODIFICATION OF BACTERIOPHAGE K TO EVADE ANTIVIRAL ACTIVITY IN MILK TO TREAT BOVINE MASTITIS

The overall goal of this project is to increase bacteriophage (phage) infectivity, thus increasing its therapeutic potential, of mastitis causing Staphylococcus aureus (S. aureus) by reducing its interaction with milk antiviral mechanisms. To accomplish this overall goal, two objects will be pursued. Objective 1 is to develop a cell-free genetic engineering system for S. aureus phage K. The short-term milestones of this objective are to verify pure S. aureus TXTL cytoplasmic lysate production of GFP; phage K DNA verification of size, purity, and genomic sequence; and verification of phage K production by S. aureus TXTL cytoplasmic lysate. Objective 1 would remove the need for gRNA testing if using CRISPR-Cas9 method of genetic engineering, remove the process of removing exotoxins and enterotoxins from the phage solution, and should increase the speed of genetic engineering of phage K. Objective 2 is to engineer phage K for low protein adsorption in milk. The short-term milestones of this objective are to verify mSA-capsid sequence using Sanger sequencing; verify phage production using CRISPR-Cas9 method or S. aureus TXTL method from Objective 1 if completed by the time of phage production; ensure exotoxins and enterotoxins have been removed from the phage lysate; ensure the biotinylated Polyethylene glycol (PEG) has attached to the mSA-phage K; and ensure PEG-mSA-phage K's infectivity, surface charge, and solubility43. Objective 2 would potentially allow for phages to get past the antiviral activity of bovine whey protein thus increasing its therapeutic potential of mastitis caused by S. aureus.