Robust Cell Free Extract from Bacillus Subtilis to Prototype Engineered Spore Circuitry

B. subtilis is a common, gram-positive bacterium capable of forming spores which our lab is exploiting to create engineered spore devices such as embedded sensors, time-delayed expression vehicles, and hydrolytic logic incorporated in advanced materials. Transformation of these cells is much less efficient than work in other model organisms, such as E. coli. To prototype spore-based circuitry we turn to cell free reactions. Cell free protein expression has been shown previously in B. subtilis[1] but we found extract made from tip-sonication batches to be highly variable in performance (much more sensitive than E. coli extract preparation). We demonstrate improved homogeneity in extract prepared from large-batch continuous homogenization (single piston cycle) followed by lyophilization, as we have done previously for E. coli extract [2]. Using this robust extract, we demonstrate the expression of beta-galactosidase and super folding green fluorescent protein. We also show utility of cell-free prototyping, by testing a panel of 6 riboswitch circuits demonstrating varying levels of translation control (expression of red fluorescent protein) when exposed to the theophylline analyte.

[1] Kelwick R, Webb AJ, MacDonald JT, Freemont PS (2016) Development of a Bacillus subtilis cell-free transcription-translation system for prototyping regulatory elements. Metabolic Engineering, 38:370–381. https://doi.org/10.1016/j.ymben.2016.09.008

[2] Dopp JL, Reuel NF (2018) Process optimization for scalable E. coli extract preparation for cell-free protein synthesis. Biochemical Engineering Journal, 138:21–28. https://doi.org/10.1016/j.bej.2018.06.021