Fig. 1.

Model two-component system NarX-L is readily integrated and active in synthetic membranes. (A) Cell-free reactions were assembled by combining proteins required for gene transcription and translation, plasmids encoding T7 RNAP, NarX, NarL, a reporter gene (nanoluciferase), a membrane mimetic (DMPC liposomes), and nitrate. Upon expression and binding to nitrate, NarX can phosphorylate NarL. Phosphorylated NarL then dimerizes, binds to the promoter, and initiates transcription of the reporter gene, nanoluciferase. (B) The highest luminescence is achieved when all components of the sensor are present. Components were systematically removed to characterize downstream luciferase expression. (C) Cell-free expressed NarX-L has higher nitrate-induced expression of luciferase in the presence of a membrane mimetic, DMPC. (D) Sensor activity as reported by luminescence and (E) fold change of NarX-L can be tuned by altering the DNA ratio of NarX and NarL. By increasing the DNA ratio of NarX: NarL, the overall luminescence signal is decreased, but the fold change in luminesce in response to nitrate is increased relative to the 1:10 NarX:NarL plasmid ratio. The sum of NarX and NarL plasmid concentrations were kept at 6.6 nM. All error bars represent the SEM for n = 3 independent replicates.