Fig. 5: Experimental construction and validation of CAD-engineered tRNA abundances in a synthetic system expressing a fail-safe encoded gene.
From: Engineering tRNA abundances for synthetic cellular systems
A Schematic overview of TINA with CD-CAD. We designed synthetic tRNA sequences that we sourced via direct RNA synthesis. We used CD-CAD to design tRNA abundance distributions and combined our synthetic tRNA as specified. We built in vitro translation systems by supplementing PUREΔtRNA with synthetic tRNA distributions plus synthetic initiator tRNA. Colors and color bars represent concentration of each tRNA species per reaction mixture. Icons adapted with permission from The Noun Project under a Creative Commons CC BY license. B CD-CAD specified tRNA distributions optimized for faster (synFast, above) and slower (synSlow, below) translation of RED20-encoded GFP. Color bars represent concentration of each tRNA species per reaction mixture. (C–K) Experimental data from tRNA batch #1 (C–E, n = 3 technical replicates), tRNA batch #2 (F–H, n = 4 technical replicates), and tRNA batch #3 (I–K, n = 5 technical replicates). Protein expression for three tRNA distributions is measured and analyzed: synFast (green), synSlow (orange), and uniform (blue, I–K only) along with a negative control, PUREΔtRNA (black, labeled as (-)). Colors are used consistently across (C–K). Traces of GFP fluorescence over time (C, F, I), numerically computed derivatives of these traces (D, G, J) and calculated protein synthesis rates (E, H, K). In (C, F, I), solid lines represent the mean trace across replicates for each distribution. Shaded regions represent the 95% confidence interval in the estimate of the mean across replicates within each condition. Dashed lines represent the mean of traces individually smoothed with a Gaussian filter (see “Methods”). In (D, G, J), solid lines represent the mean of smoothed derivatives across replicates for each distribution. Shaded regions represent the 95% confidence interval in the estimate of the mean. In (E, H, K), error bars represent 95% confidence interval in the estimate of the mean and colors bars are mean values; colored dashed vertical lines are CD-CAD predicted rates. Solid black lines with labels show statistical significance (n.s. is not significant, *p < 0.05, **p < 0.01); one-sided Kolmogorov–Smirnov (KS) tests used for comparing synFast to all others, and a two-sided KS test used for comparing synSlow to Uniform. Source data are provided via a Zenodo repository (https://doi.org/10.5281/zenodo.7953836).
