Extended Data Fig. 5: Growth and fluorescence of strains with readthrough reporter.
From: Engineering a genomically recoded organism with one stop codon
a, Schematic representation of YFP:mCherry fluorescent reporter used to assess release activity at the three stop codons (X = UAG, UGA, or UAA) relative to sense codon (GCG) within a peptide linker between mCherry and YFP. Release activity at codon X prevents downstream YFP expression and fluorescence without compromising mCherry. Meanwhile, stalling and degradation results in loss of mCherry and YFP fluorescence. High YFP fluorescence suggests readthrough. A colour key for sections b-c is located below. Schematic representation of expected fluorescent outcomes resulting from the mCherry-YFP readthrough assay is to the right. b-d, OD-normalized b, mCherry (top) and c, YFP (middle) fluorescence (corresponds to Fig. 2e,f), along with d, strain OD at time of measurement (bottom) resulting from YFP:mCherry fluorescent assay presenting readthrough and Ser suppression at GCG, UAG, UGA, and UAA in the absence and presence of UAG- or UGA-suppressing SupD tRNAs. Error bars display standard errors of the mean, n = 3 biological replicates. e, Comparison of RF variant stop codon readthrough: RF2.B2 (S205P) and RF2.B4 (E170K) with WT-RF2.B0). Normalized mCherry and YFP readthrough fluorescence (same assay as Fig. 2d – “No Suppressor”) interrogating the impacts of E170K or S205P mutations in RF2.B3 release activity. mCherry (left) and YFP (right) fluorescence resulting from YFP:mCherry fluorescent assay presenting translation termination or native suppression (readthrough) at GCG, UAG, UGA, and UAA, normalized to GCG ( + control) mCherry or YFP fluorescence, respectively. Statistical significance is displayed compared to rEc∆1.∆A. Unpaired t-tests (n = 3), * = p-value < 0.05, ** = p-value < 0.01. Error bars display standard errors of the mean, n = 3 biological replicates.
