Fig. 1
From: Self-replication of DNA by its encoded proteins in liposome-based synthetic cells
Basic elements of the DNA self-replication strategy. a Flow of genetic information reconstituted in batch mode reaction or inside a liposome. The Φ29 virus-inspired DNA replication mechanism is implemented in the PUREfrex transcription-translation system. A DNA replication cycle is completed when the DNA template expressing the Φ29 proteins is also the replicating DNA. Alternatively, the expressing DNA does not contain the Φ29 origin sequences (oriLR- in brackets) and a different target DNA is used as a replication substrate (solid colored line). Some essential reaction substrates are indicated. b Schematic depicting the mechanism of replication initiation by the Φ29 system. c Schematic of the Φ29 genome and four de novo designed DNA constructs used in this study (Supplementary Table 1). The most relevant regulatory elements are depicted: the T7 promoter (arrows), the vesicular stomatitis virus (VSV) internal terminator47 or the T7 terminator at the 3′ end (T, for both terminators), the genes (rectangles) and the Φ29 origins of replication. Their termination efficiency was experimentally estimated (Supplementary Fig. 2). d, e Analysis of translation products on polyacrylamide protein gels. PUREfrex solution was supplemented with BODIPY-Lys-tRNALys (GreenLys) to introduce fluorescent lysine residues in the synthesized proteins and distinguish them from the IVTT protein background (e). Coomassie Blue staining was also performed to visualize the purified proteins and the total protein content in PUREfrex reactions (d). Amounts of purified proteins: 180 ng p2, 2 µg p5, 180 ng p3, and 2 µg p6. The estimated concentrations are 1.0 µM for DNAP, 4.0 µM for TP, 5.0 µM for p5 and 1.7 µM for p6, when all genes are separately expressed (Supplementary Note 4 and Supplementary Fig. 3). The production of all four full-length proteins was confirmed when the oriLR-p2-p3 and oriLR-p6-p5 templates were co-expressed in equimolar amounts (1:1) or with an excess of the oriLR-p6-p5 DNA (∼1:13), the latter ratio being used in replication experiments, where larger amounts of p5 and p6 are required. Note also the generation of truncated translation products, in particular for p2 and p3. Predicted molecular masses are 12 kDa for p6, 13.3 kDa for p5, 31 kDa for p3, and 66 kDa for p2
