Fig. 2. PylRS mutants for Acd incorporation. Top: fluorescence measurements of sfGFP reporter to evaluate RS efficiency. Orange, blue, and yellow represent fluorescence from colonies induced in media containing no ncAA, 1 mM Acd, or 1 mM Npf, respectively. Cultures of 500 μL were grown for 48 hours before 4-fold dilution of suspended cells with buffer for fluorescence measurement at 528 nm (485 nm excitation) using a plate reader. RSs enabling high sfGFP fluorescence in Acd conditions with low fluorescence in no ncAA or Npf conditions were selected for further characterization. Additional data showing sfGFP fluorescence for all clones are shown in ESI, Fig. S10.† Bottom: a homology model of Mb AcdRS 82 based on the X-ray crystal structure of Mm PylRS (PDB ID: 2ZIN)³⁴ with Acd docked in the active site using Rosetta. Key points of interaction with Acd are shown from two angles. Sites of mutation relative to parent Mb PylRS are labeled in red boxes. A model of Mb AcdRS 41 is shown in ESI, Fig. S11.†.