With inducible PylRS expression confirmed, the functionality of PylRS–tRNAPyl in L. lactis was determined with a GFP amber suppression reporter. The detailed experimental setup is available in the Supplementary Material, Section 1. In short, the fluorescence of the GFP variants produced in the presence and absence of BocK was determined by FACS and compared to cells expressing WT GFP as positive control and cells expressing only PylRS–tRNAPyl as negative control. The experiments revealed a small, but reproducible increase of the intact cell fluorescence in the presence of BocK, documenting the functionality of the OTS resulting in incorporation of BocK into GFP (Supplementary Figure S1).