Rhodococcus, a species of bacteria commonly found in the soil, is an under-explored producer of small bioactive compounds including siderophores, pigments and antibiotics. MTM3W5.2 is a strain of Rhodococcus that was previously discovered to produce an antibiotic-like compound that has inhibitory effects on other Rhodococcus strains, including the veterinary pathogen, R. equi. The biosynthetic gene cluster responsible for production of the antibiotic has been identified, and a small gene, BTZ20_3964 at the start of the operon is believed to be a luxR solo regulator of the gene cluster. The goal of this project was to determine this gene’s status as a regulator for the gene cluster. Merodiploids were constructed using the deletion construct, pEX18Km3964AD to obtain a double crossover recombination event to replace the functional gene with the deletion construct. However, evidence indicates that an illegitimate recombination event occurred to produce a merodiploid strain.