Fig. 2: Validation of DNArep and PLsyn protein activity inside gene-expressing liposomes at different incubation temperatures.

Percentage of liposomes with active DOPS synthesis (a) and active DNA replication (b) under 30 °C, 37 °C and 34 °C incubation temperatures. Flow cytometry data are SSC-A vs. dsGreen for DNA replication and SSC-A vs. LactC2-mCherry for DOPS synthesis. Data points represent biological repeats and bar height the mean value. Raw data from individual replicates can be found in Supplementary Fig. 5. NC refers to samples, where DNA was omitted but the solutions were incubated at the indicated temperature. c Percentage of liposomes exhibiting dual dsGreen and LactC2-mCherry signals at 30 °C, 37 °C, and 34 °C incubation temperatures. Joint phenotype populations were selected from LactC2-mCherry vs dsGreen scatter plots. Raw data from individual replicates can be found in Supplementary Fig. 5. d Flow cytometry scatter plots from liposome samples displaying four regions of interest (ROI 1-4) at all tested temperatures: DNArep-active liposomes are in ROI 1, PLsyn-active liposomes in ROI 4, and liposomes with both active DNArep and PLsyn modules are in ROI 2. Vertical and horizontal dashed lines indicate intensity threshold values that have been defined using control samples (see Supplementary Fig. 5). Data from additional biological repeats can be found in Supplementary Fig. 5. e Absolute DNA quantification by qPCR of samples incubated at 30 °C, 37 °C, and 34 °C. qPCR target regions (∼200 bp) are from pssA and p2 genes. The negative control (NC) represents calculated DNA values at initial incubation points (0 h). DNA concentration changes between 0 h (NC) and 16 h were assessed using a two-sided log-ratio paired t-test. Log-transformed ratios of 16-h to 0-h values were calculated for each replicate and each corresponding gene (pssA and p2). A one-sample t-test was then performed to determine if the mean log-ratio significantly differed from zero (p < 0.05). * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001. Exact p values for the genes p2/pssA are: 0.0173/0.0178 (30 °C), 0.003/0.00397 (37 °C), and 0.00162/0.00035 (34 °C). f Amplification fold of DNArep-PLsyn DNA calculated from qPCR data in panel e: end-point (16 h) DNA concentration / DNA concentration at time zero. Data points represent biological repeats and bar height the mean value. g LC-MS detection of DOPS and PLsyn intermediate enzymatic products before and after expression of the DNArep-PLsyn genome. Peak area for each compound was normalized to that of DOPG. Additional biological repeats and negative controls can be found in Supplementary Fig. 8. Source data are available for this figure in the Source Data file.