A Streamlined and High-Osmolarity Bacillus subtilis Cell-Free Platform for Rapid Characterization of Genetic...

Lysate-based cell-free systems (CFS) mimic cellular functions in a controllable environment, making them versatile synthetic biology tools used from fundamental research to protein production and synthetic circuit prototyping. Here, we establish a streamlined and robust Bacillus subtilis CFS by systematically re-evaluating the traditional extract preparation and reaction environment. We show that the conventionally required runoff incubation and labor-intensive dialysis steps can be omitted without compromising performance. A key advance is the reformulation of the reaction buffer: increasing ionic strength with potassium glutamate concentration to 400 mM and adding the osmoprotectant betaine significantly enhanced translational capacity, yielding 2-3 μM sfGFP. Benchmarking against established in vivo promoter library data sets revealed that the relative hierarchy of promoter strengths is preserved, validating the platform as a predictive proxy for bacterial physiology. We further reconstituted complex genetic regulations by demonstrating functional repression of a cre-box-containing promoter by the catabolite repressor CcpA in complex with HPr, activated through a phosphorylation cascade within the CFS. Finally, we show that this high-glutamate framework is beneficial for another Gram-positive bacterium, Lactobacillus gasseri, suggesting a broad-host compatibility. Together, the simplified preparation and optimized buffer composition provide a versatile toolkit for developing CFS in diverse, nonmodel bacterial species.