Stabilization of Recombinant Plasmids in Bacillus Subtilis

用于工业规模发酵的重组DNA(质粒)在细菌中的稳定性是非常令人关注的。枯草芽孢杆菌是一种潜在的生产重组DNA产物的有用微生物。然而，枯草芽孢杆菌表达载体的不稳定性限制了其作为工业克隆宿主的有效性。本研究的总体目标是分析影响枯草杆菌重组DNA稳定性的遗传、营养和环境因素。PI建议对控制质粒丢失动力学的参数进行量化，即测量质粒中分离事件或结构变化发生的频率。-修饰细胞比亲本细胞具有生长优势。恒化器研究将被用作描述分离和结构质粒不稳定的相对影响的独特工具。利用最先进的发酵罐监测和评估方法，将在早期阶段尝试在线检测重组培养中的回复菌株，以便在补料分批和连续培养中应用稳定策略。

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.