Vesicles of varying composition alter NarX and NarL expression and subsequent activity. (A) By modulating the composition of vesicles that are doped into cell-free reactions, we can characterize how membrane physical features affect NarX cotranslational insertion and activity. Vesicles were composed of 100% DMPC, 100% POPC, 100% DOPC, 30% POPE/70% POPC, 30% POPG/70% POPC, and 100% E. coli polar lipid extract. (B) NarX-L activity in membranes of different compositions in the absence and presence of 1 mM nitrate. Fold change in luminescence is indicated over each membrane composition. (C) NarX expression varies with membrane composition as determined by western blot band intensity. (D) Western blot band intensity of NarX is correlated with luminescence and performance of the sensor. (E and F) The viscosity of each membrane measured via DPH anisotropy correlates with protein expression as determined by western blot. All error bars represent the SEM for n = 3 independent replicates. For western blots, n = 6 as band intensities in the absence and presence of nitrate were grouped for each membrane condition.