The stability of recombinant DNA (plasmid) in bacteria used for commercial scale fermentations is of great concern. Bacillus subtilis is a potentially useful microorganism for the production of recombinant DNA products. However, instability of plasmids in B. subtilis has limited its usefulness as an industrial cloning host. The overall objective of this study is to analyze genetic, nutritional and environmental factors that affect the stability of recombinant DNA in B. subtilis. The PIs propose to quantitate the parameters that govern the kinetics of plasmid loss, i.e., to measure - The frequency with which segregational events or structural changes in the plasmid occur. - The growth advantage the modified cells have over parental cells. Chemostat studies will be employed as a unique tool for delineating the relative effects of segregational and structural plasmid instabilities. Using state-of-the-art fermentor monitoring and estimation methodologies, the on-line detection of revertants in the recombinant culture will be attempted at an early stage to allow for the application of stabilization strategies in fed-batch and continuous culture.
