Development of a Bacillus subtilis cell-free transcription-translation system

Cell-free transcription-translation systems were originally conceived as foundational tools to understand the fundamentals of molecular biology, cellular biochemistry and also for in vitro protein production. More recently, synthetic biology is enabling these systems to be used within a systematic design context for prototyping DNA regulatory elements, genetic logic circuits and cell-free metabolic engineering. The Gram-positive soil bacterium, Bacillus subtilis, is an established model organism of biotechnological importance yet, there are few reports of B. subtilis cell-free transcription-translation systems and those that have been reported were largely encumbered by endogenous cell extract protease activity and/or the availability of inefficient cell-free energy systems. To this end, we developed several B. subtilis-based cell-free systems to be applied towards regulatory element prototyping.