Fig. 5

Compartmentalization of self-encoded DNA replication inside liposomes. Images in a and b display fluorescence confocal micrographs of PUREfrex-containing phospholipid vesicles labeled with a membrane dye (red). a Fluorescence emitted by the YFP synthesized from its gene (7.4 nM bulk concentration of dsDNA) is visualized in green (overlaid channels). Assuming that the entrapped DNA molecules follow a Poisson distribution, liposomes with a diameter of 4 µm contain ~140 DNA copies on average. Thousands of gene-expressing liposomes can be imaged per sample. About 30% of the liposomes produce YFP at a detectable level. This functional heterogeneity is probably a consequence of the compositional diversity of the biochemical network within vesicles. Scale bar is 20 µm. b Following the IVTTR reaction scheme shown in Fig. 3a, 5 nM of the oriLR-p2-p3 DNA template along with the purified p5 and p6 proteins were co-encapsulated with or without dNTPs during liposome formation. After gene expression and liposome immobilization, the DNA staining fluorophore acridine orange (green channel) was injected. Amplification of DNA in the lumen of individual vesicles is accompanied by a higher fluorescence signal of acridine orange in the form of bright spots. We noticed that acridine orange can stain the liposome membrane, presumably due to the hydrophobic nature of its aromatic groups. Nonetheless, the DNA and membrane signals can easily be discriminated by using the red membrane dye for co-localization analysis, so that the lumen signal from amplified DNA can unambiguously be ascribed. The fluorescence images show representative fields of view from three independent experiments. Five different fields of view of similar liposome density were analysed per experiment to quantify the number of ‘nucleoids’. Comparing + dNTPs and –dNTPs (+/–) in the three experimental repeats, 350/9, 130/2, and 773/58 nucleoid-like structures were identified. Scale bars represent 20 µm. c Line intensity profiles from eight liposomes framed in b. In the images from the –dNTPs sample, we deliberately chose liposomes exhibiting green spots to show that they co-localize with the membrane dye, demonstrating that they are of different nature than those triggered by DNA replication. Color coding is the same as in b. a.u., arbitrary units