Liquid culture NanoLuc assay

Cells transformed with individual plasmids were spread on LB agar with antibiotics and allowed to form colonies overnight. LB supplemented with ampicillin and chloramphenicol was inoculated with a single colony to grow an overnight culture. Overnight cultures were diluted into fresh LB containing antibiotics to OD₆₀₀ = 0.01 and incubated in a 37 °C shaker at 220 rpm. Cell densities were monitored with a plate reader (ThermoMax Microplate Reader, Molecular Devices). At OD₆₀₀ = 0.2–0.4, cultures were induced with 500 μM L-rhamnose and 500 μM IPTG for 1 h. Cell densities were measured and 100 μl cultures were put onto a white round-bottom 96-well plate for luminescence measurement. All NanoLuc assays in this study were carried out with Nano-Glo^® Live Cell Assay from Promega (N2011) according to manufacturer instructions. Luminescence was measured on a GloMax^® Navigator Microplate Luminometer (Promega). Signals were linearly normalized to OD₆₀₀ = 0.4. Each data set was acquired using identical instrument settings to allow comparison between samples. The integration time chosen such that the highest signal did not exceed 1E9 RLU to avoid saturating the detector. For visualization of translation products by Western blotting, Streptavidin-HRP was used to detect biotinylated proteins in whole-cell lysates after ribonuclease treatment. Uncropped images of the Western blots shown in Fig. 2 and Supplementary Fig. ² are provided in the Source Data file.