Fig. 4.

Mechanosensitive and biosensing synthetic cell system. (A) Schematic depicting the reversible transformation between the fluorescent and non-fluorescent states of G-GECO protein in presence of calcium and EGTA. (B) Fluorescence images of G-GECO expression with calcium addition inside vesicles (1 nM P70a-G-GECO, 1.5 mM calcium chloride was added to TXTL reaction after 1.5 hour incubation prior to encapsulation). (C) (i), (ii)-Three plasmid expression under different external conditions. Concentrations of P70a-S28, P28a-MscL and P70a-G-GECO in cell-free reaction were fixed at 0.2 nM, 1.4 nM and 0.6 nM respectively. The outer solutions for all conditions contained 10 mM calcium chloride. (D) Box plot showing the relative fluorescence intensities from vesicles in hypo-osmotic media with different external calcium concentrations. Each box corresponds to intensity values from ten vesicles. Plasmid concentrations of P70a-S28, P28a-MscL and P70a-G-GECO were 0.4 nM, 1.3 nM and 1 nM respectively. For both (C) and (D), the lipid vesicles were introduced into hypo-osmotic solution immediately after encapsulation following a 1.5 hour incubation period. EGTA was used at a concentration of 1 mM in all cell-free reactions. The osmolarity difference between iso-osmotic and hypo-osmotic solutions was measured at 100 mOsm. All experiments were repeated three times under identical conditions. Scale bars: 50 µm.