Fluorescence assays

For orthogonal translation assays, S2060 chemically competent cells were transformed with the E. coli O-rRNA plasmid and the relevant orthogonal reporter plasmid. Transformants were streaked on 1.8% agar-2xYT plates supplemented with kanamycin and carbenicillin. Plates were grown in a 37 °C incubator for 16 h. Colonies were picked into 500 µL DRM (United States Biological)⁶⁰ (supplemented with kanamycin, carbenicillin, 1 mM IPTG + /- 1000 ng mL⁻¹ aTc) in deepwell plates (VWR) and grown at 37° C with shaking at 900 RPM for 20 hours. To assay heterologous O-rRNA function, chemically competent cells carrying the sfGFP reporter plasmid were prepared (S2060.sfGFP) and transformed with the appropriate O-rRNA plasmid. E. coli O-rRNA was always transformed alongside experimental O-rRNAs as a positive control. Transformants were streaked out and picked into media as above.

To assay r-protein effects on heterologous O-rRNA function, S2060.sfGFP chemically competent cells were co-transformed with the appropriate O-rRNA plasmid and r-protein plasmid. As a positive control, E. coli O-rRNA was transformed alongside an mCherry expression plasmid. In the absence of r-protein supplementation, heterologous O-rRNAs were transformed with mCherry to maintain consistent growth rates and antibiotic selection markers. Transformants were streaked on 1.8% agar-2xYT plates supplemented with kanamycin, carbenicillin, chloramphenicol, and 200 mM glucose, picked into DRM supplemented with kanamycin, carbenicillin, chloramphenicol, 1 mM IPTG, 1000 ng mL⁻¹ aTc, +/- 10 mM arabinose, and grown up as above. For assays using the erythromycin-dependent reporter, DRM was supplemented with 100 µg mL⁻¹ erythromycin in addition to the above inducers and antibiotics.

To quantify fluorescence output, 150 µL of each culture were aliquoted into a 96-well black wall, clear bottom plate (Costar). OD₆₀₀ and the appropriate excitation and emission wavelengths were used for fluorescence measurements (Supplementary Table ⁵) using either a SpectraMax M3 (Molecular Devices) or Spark (Tecan) plate reader running SoftMax Pro v6.4 or SparkControl v2.3, respectively. Fluorescence was normalized to OD₆₀₀ after blank media subtraction. Data were normalized to E. coli O-rRNA sfGFP/OD₆₀₀ and expressed as a percentage. When assaying the effects of r-protein complementation, data were normalized to E. coli O-rRNA (sfGFP signal) upon mCherry control plasmid induction.