Fig. 2: Characterization of protein expression and reaction compartmentalization in membrane-less protocells.

a Schematic of single-well and multi-well protocell reactions and their preparation methods. A small volume of Ficoll or dextran solution containing reagents for cell-free expression of GFP was pipetted into a bulk phase solution of PEG to form a protocell. Subsequent reactions were incubated at 37 °C for 3 h. b Fluorescence of Ficoll and dextran protocell reactions in single-protocell format were measured with a plate reader with 485/528 nm excitation/emission wavelengths. For detectable protein expression, energy buffer must be supplemented in the bulk phase. c Fluorescence image of protocell arrays obtained by a ChemiDoc MP imager (0.5 s exposure time, 530/28 nm filter) for three biological replicates. Contents of individual protocells are indicated with colored circles: blue for Ficoll polymer-encapsulated reactions, red for dextran-encapsulated reactions. Bright colors indicate CFE reactions containing GFP plasmid for expression, and faded colors indicate negative controls with no plasmid. Scale bar is 1 mm. d Quantitative assessment of fluorescence image in c, with pixel intensity quantified by image processing software (Fiji). Data are presented as mean values ± SD of 9 replicates (3 biological replicates × 3 technical replicates). Solid-filled circles represent the mean of each biological triplicate, and hollow circles represent all data points. Details on CFE lysate used and plasmid concentrations are provided in Supplementary Table 1. Note, due to different measurement instruments being used for b and d, the arbitrary units on the y-axes are different scales.