? Our goal is to improve the versatility, safety and effectiveness of E. coli as a platform for commercial production of DNA, proteins and metabolites. The natural E. coli genome will be reduced to its essentials by removing unnecessary, detrimental and potentially harmful genes through our Scarless Genome Reduction (SGR) technology. The result will be a robust, benign, environmentally friendly organism which can be modified for production as needed. DNA applications include vaccines, gene therapy and RNAi based therapeutics. Protein applications include drugs, hormones, industrial enzymes and some antibiotics. Metabolites include many antibiotics, vitamins, amino acids as well as industrial chemicals and fuels such as ethanol and hydrogen. As the technical ability to manipulate genomes grows, it will be possible for reduced genome ?. coli to take on many applications previously reserved for other organisms or chemical processes. In the Phase I work, the E. coli genome was reduced to the point that there were no more IS transposable elements. These elements have the capacity to hop around the genome and inactivate genes including the genes for products. The new IS-free strains have remarkably improved properties, including a greater tolerance for high level product expression, greater cloning ability, and better stability. IS hopping is one of the ways E coli defends itself from the stress of production which, from the bacterial point of view, resembles an infection. For production of medicines, the cleanness of the genome is a benefit because purification of product is less difficult and good quality assurance and quality control are more readily attainable. For injectable DNA preparations, the elimination of IS hopping is a tangible safety benefit, which will avoid the risk of transposition into a chromosome of a patient. Finally, the cells grow robustly to high density in industrial fermentation conditions using inexpensive minimal salts media, alleviating concern about prion contamination from bovine-derived media additives. This Phase II project will explore a variety of commercial applications which have been brought to our attention by companies interested in our product. ? ? ?