All cells must accurately and rapidly duplicate their chromosomes as an essential step for cell division. Consequently, our treatment of human pathogens and cancer hinges on a better understanding of DNA replication. Most of what is known about microbial replication has been derived from studies in a single organism, Escherichia coli, a Gram negative human pathogen. However a majority of human pathogens are Gram positive bacteria that are separated from E. coli by a billion years of evolution. While many features of DNA replication are certainly conserved, important differences may exist. The goal of this research is to reconstitute the first complete replication system in a Gram positive organism, Bacillus subtilis. B. subtilis has been chosen because it is a well characterized and genetically tractable organism. This work will provide valuable insights into the differences and similarities in mechanisms between E. coli and the distantly related B. subtilis potentially identifying novel antibiotic targets.