Fig. 3: SSU biogenesis in vitro.
A Experimental scheme for SSU biogenesis in vitro. B Exploring optimal conditions for the first reaction using a simplex-lattice design. In the first reaction, the concentrations of the native ribosomes, the artificial rRNA operon with or1-oASD and C1192U spectinomycin resistance (SpcR), and 21 SSU r-protein genes were 0–100, 0–3, and 0–0.5 nM each, respectively. The second reaction was conducted with the or1-oSD–LacZ reporter and spectinomycin using the droplet assay. The data represent the mean fluorescence intensity of droplets. a.u., arbitrary unit. C Follow-up optimization of the first reaction. In the first reaction, the concentrations of the native ribosomes, the artificial rRNA operon, and 21 SSU r-protein genes were 0–240, 0–0.9, and 0–0.15 nM each, respectively. The second reaction was conducted with the or1-oSD–LacZ reporter and spectinomycin using the droplet assay. The optimal reaction condition was a native ribosome concentration of 120 nM. However, in the following experiments, we selected suboptimal reaction conditions with 80 nM native ribosomes to reduce reagent costs. D Successful detection of the nascent artificial SSU translational activity using the bulk assay under the optimized reaction condition. In the first reaction, the concentrations of the native ribosomes, the artificial rRNA operon, and 21 SSU r-protein genes were 80, 0.3, and 0.05 nM each, respectively. The second reaction was conducted with the or1-oSD–LacZ reporter and spectinomycin using the bulk assay. Mean ± SD (n = 3, biological replicates). ****, p < 0.0001; **, p < 0.01; n.s., not significant; one-way ANOVA with Dunnett’s test against the negative control without native ribosomes. The p-values showing p  <  0.0001 and p  >  0.9999 are p  =  1.0 × 10−5 and p  =  9.9999 × 10−1, respectively. Source data are provided as a Source Data file.
