Fig. 3. Semi-continuous evolution of mod-ori-p2p3.

a Schematic illustration of the experimental set-up for a semi-continuous evolution approach in liposomes. Step (i) in-liposome IVTTR (ii) 100-fold dilution of old vesicle suspension in a suspension of fresh vesicles, (iii) liposome fusion-fission promoted by cycles of F/T. b Trajectories of mod-ori-p2p3 concentrations in liposomes in the semi-continuous evolutionary campaign, Cont-WT, as measured by qPCR. Each IVTTR was incubated for 16 h and liposomes were diluted 100 times with feeding vesicles. The target region for qPCR quantification ( ~ 200 bp) belongs to the p2 gene. c Size analysis of PCR-amplified DNA during Con-WT by agarose gel electrophoresis. The arrowhead indicates the full-length replicator. d Schematic illustration of the experimental set-up for the bulk serial transfer campaign (Bulk-WT). Bulk IVTTR reactions were incubated for 16 h. The next round of IVTTR was started after 10-fold dilution of the pre-ran IVTTR reaction in a fresh PURE system complemented with DNA replication factors. e Trajectories of mod-ori-p2p3 concentrations in a bulk reaction, Bulk-WT, as measured by qPCR. f Size analysis of PCR-amplified DNA during Bulk-WT by agarose gel electrophoresis. The arrowhead indicates the full-length replicator. g Amplification of DNA in the two evolution campaigns. h,i Quantitative comparison of the abundance of different DNA regions throughout the evolutionary rounds in Con-WT (h) and Bulk-WT (i). DNA presence was assayed by quantitative PCR using standard curves for each primer pair. The inset cartoon is a schematic illustration of mod-ori-p2p3 self-replicator regions that were targeted by qPCR. Full-length replicator persists in liposome but is outcompeted by DNA that does not contain p2 gene in bulk IVTTR. Source data are provided as a Source Data file.