Automation and factorial experimental design together with cell-free in vitro transcription-translation systems offers a new route to the precise characterization of regulatory components. This now presents a new opportunity to illuminate the genetic circuitry from arcane microbial chassis, which are difficult to assess in vivo. One such host, Bacillus megaterium, is a giant microbe with industrial potential as a producer of recombinant proteins at gram per litre scale. Herein, we establish a B. megaterium cell-free platform and characterize a refactored xylose-repressor circuit using acoustic liquid handling robotics to simultaneously monitor 324 reactions in vitro. To accurately describe the system, we have applied a Bayesian statistical approach to infer model parameters by simultaneously using information from multiple experimental conditions. These developments now open up a new approach for the rapid and accurate characterization of genetic circuitry using cell-free reactions from unusual microbial cell chasses for bespoke applications.