Fig. 2: BUD-ELMs contain a de novo protein matrix and display a hierarchical structure.

a Confocal microscopy of ELMs stained with SpyCatcher-GFP at increasing magnifications, showing a hierarchical structure. The bottom images show individual fluorescent channels: GFP (matrix) on the left and mKate2 (cells) on the right. Scale bars are, from left to right, 100, 10, and 5 µm, respectively. b Percentage of overlapping pixels between cell-free and stained regions, confirming the absence of lipids in the BUD protein matrix. Error bars are centered on the mean value (cross) and represent the standard deviation; the red line indicates the median value; the boxes show the interquartile range (25–75%). The analysis has been performed on 16 images for the protein and 11 for the lipid staining, each obtained from three independent samples. Source data are provided as a Source Data file. c AFM images of single cells at early (left) and late (right) stages of BUD-ELM formation, showing a difference in surface morphology. d High-resolution AFM images of single-cell surfaces at early (left) and late (right) stages of BUD-ELM formation, showing differences in surface layer thickness. Scale bars are 100 nm. e Immunoblot of BUD protein was detected in the growth media of culture grown in static (left) and shaking (right) conditions, showing a similar amount of protein in both conditions. BUD proteins were stained with the ANTI-FLAG® antibody. Source data are provided as a Source Data file. f Comparison between the ∆ELP₆₀ and ∆rsaA_1–250 BUD-ELM strains, showing differences in morphology and cell content. Each panel shows the genetic constructs (top), a representative image of BUD-ELMs at low (bottom, left), and high (bottom, right) magnification. For each panel, scale bars are 1 cm (bottom, left) and 50 µm (bottom, right).