Fig. 1: Assembly and characterization of enzyme-decorated coacervate vesicles.

a Scheme showing design and construction of enzyme-decorated coacervate vesicles. Step I: a suspension of positively charged PDDA/DNA coacervate (Coac) micro-droplets is added to a DOPC/ethanol solution to produce phospholipid-based coacervate vesicles (DOPC-CVs). Step II: The DOPC-CVs are surface-decorated with palmitic acid-modified enzymes (PA-enzyme) to give molecularly crowded protocells with reactive outer membranes (Enzyme-CVs). b Types of enzyme-decorated coacervate vesicles produced; PA-glucose oxidase (GOx-CVs), PA-horseradish peroxidase (HRP-CVs) and PA-catalase (CAT-CVs). Labels marked inside the CVs (GOx, HRP and CAT) identify the different types of CVs; in each case, the PA-enzymes are attached to the outer membrane surface and not encapsulated. Bright field (c) and corresponding red fluorescence (d) images of undecorated DOPC-CVs (PDDA: DNA = 0.6: 1, w/w; DOPC: coacervate = 0.1: 1, w/w) stained with the lipophilic dye Dil (1,1^/-dioctadecyl-3,3,3^/,3^/-tetramethyl indocarbocyanine perchlorate; red fluorescence, Ex: 549 nm, Em: 565 nm). Each coacervate droplet is encased in a continuous phospholipid membrane; scale bar, 10 μm. Optical phase contrast (e) and dark field (f) images of closely packed multiple layers of undecorated DOPC-CVs showing absence of coalescence. Optical (g), red fluorescence (h), green fluorescence (i), and merged red/green fluorescence (j) images of a single FITC-labelled PA-GOx-CV after staining with Dil. DOPC (red) and GOx (green) are co-located at the DOPC-CV membrane. A representative fluorescence intensity profile (k) corresponding to the dashed line in (j), and associated 3D reconstructed image (l), of single PA-GOx-decorated DOPC-CV. The DOPC membrane and enzyme surface attachment is uniform and continuous; 3 times each experiment was repeated independently with similar results (c–j, l). Scale bars (g–j), 5 μm.