4.

Evolution of fuel-dependent synthetic cells at high degree of functionalization (A) Schematic depiction of the experimental design and the observed droplet morphologies at DF = 70 and 100%. (B) DF-dependent turbidity traces at λ = 600 nm plotted against time after EDC addition to a mixture of RG₂D (12.3 mM), pSS (20.0 mM), N₃–Arginine (1.0 mM), CuSO₄ (1.0 mM), ascorbic acid (4.0 mM), and the BTA copolymers (total c = 1.0 mM, DF based on mol %) in 5-vol % EtOH/200 mM MES buffer (pH = 5.3) with the extracted lifetime based on an optical density threshold of ΔOD < 0.25, with the control experiment in the absence of any supramolecular polymer or CuAAC component. Standard deviations are based on triplicate experiments. (C) Time-dependent snapshots of confocal microscopy studies conducted under the same experimental conditions as the turbidity traces shown in (B). Droplets are stained using NBD-functionalized RG₂N (c = 0.5 μM), all images share the same scale bar. (D) Snapshots of FRAP experiments of Cy3-labeled pSS (c = 0.5 μM, 10.8 kDa) at DF = 100% (all images share the same scale bar corresponding to 10 μm) with average FRAP intensity traces for DF = 0 and 100% and the extracted diffusivity shown below (3 technical replicates of 3 chemical replicates, n = 9), using identical experimental conditions as in (B). Statistical analysis was performed using a two-tailed t test assuming unequal variance; ns, not significant (p > 0.05), *p < 0.05, **p < 0.01, and ***p < 0.001.