Fig. 1: PURErep enables efficient transcription–translation coupled DNA replication.
From: In vitro self-replication and multicistronic expression of large synthetic genomes
a Map of pREP. The plasmid encodes the gene for Phi29-DNAP under the control of a T7 promotor (T7p) and a bidirectional T7-terminator (T7t). IVT of the DNAP gene is increased by a T7 g10-L leader sequence. A zeocin-resistance gene and a pUC origin allow selective propagation in E. coli. b Replication of pREP in PURExpress (grey) or PURErep (blue) after 6 h at 30 °C. Fold changes were determined by qPCR relative to the pREP input levels (4 nM). Bars show the means with their 68% confidence intervals (CI) from biological triplicates using different PURErep/PURExpress batches. c Image of representative agarose gels loaded with TTcDR samples of pREP (8 nM input DNA) after MluI treatment. Samples were tested in three biological replicates. d Relative changes in compound levels between PURErep and PURExpress (log2-scale). Estimated compound levels for PURExpress are based on the numbers from Kuruma and Ueda40 (TF translation factors, AAs amino acids, DTT dithiothreitol, 10-THF 10-Formyltetrahydrofolate, SP spermidine, CP creatine phosphate). e TTcDR of pREP at different input concentrations. Fold changes relative to input levels were measured by qPCR (means with 68% CI, biological triplicates using different PURErep batches for each concentration). f pREP propagation over repeated passages of serial transfer. After each overnight TTcDR reaction in PURErep, 4% of the volume was transferred into a freshly prepared, plasmid-free PURErep reaction. Fold changes relative to the initial concentration (4 nM) were used to approximate the concentrations before and after each generation (mean ± 68% CI, technical triplicates). Source data are available in the Source Data file.
