Fig. 5: Synthesis of ribosomes in vitro.
A Scheme showing in vitro synthesis of both SSU and LSU in a single reaction space. B Successful detection of the nascent artificial SSU and LSU translational activity in the bulk assay. In the first reaction, the concentrations of the native ribosomes, artificial rRNA operon with or1-oASD, C1192U spectinomycin resistance (SpcR), and A2058U clindamycin resistance (CldR), and 54 r-protein genes were 80, 0.9, and 0.01 nM each, respectively. The second reactions using the bulk assay were conducted with the or1-oSD–LacZ reporter and spectinomycin for the nascent artificial SSU, the improved LacZ reporter and clindamycin for the nascent artificial LSU, the or1-oSD–LacZ reporter, spectinomycin, and clindamycin for the nascent artificial ribosomes. a.u., arbitrary unit. Mean ± SD (n = 3, biological replicates). **, p < 0.01; n.s., not significant; two-tailed Welch’s t-test. C Successful detection of the nascent artificial ribosome translational activity. The nascent artificial SSU (SpcR) and LSU (CldR) with streptavidin-binding aptamer (Sb-aptamer) were synthesized by the in vitro SSU and LSU biogenesis, respectively, and purified using streptavidin resin under the subunit dissociation condition (1 mM Mg2+), as described in Supplementary Fig. 9C. We observed translational activity under the double-antibiotic condition only when we mixed the purified nascent artificial SSU and LSU. NC, negative control prepared using the same production and purification procedure without expressing the artificial rRNA operon with Sb-aptamer. Violin plot represents the mean fluorescence intensity values of droplets from three independent experiments. ****, p < 0.0001; one-way ANOVA with Dunnett’s test against NC. Scale bars = 10 μm. The p-value showing p  <  0.0001 is p  =  1.0 × 10−5. Source data are provided as a Source Data file.
