In Vitro Cell-Free Protein Synthesis

The iSAT reaction was performed as described previously (16 (link),17 (link)) with slight modifications on the protein concentration and the operon of plasmid. Briefly, the iSAT reaction was performed in 57 mM Hepes-KOH, 1.5 mM spermidine, 1 mM putrescine, 10 mM Mg(Glu)₂, and 150 mM KGlu at pH 7.5 with 2 mM DTT, 0.33 mM NAD, 0.27 mM CoA, 4 mM sodium oxalate, 2% w/v PEG-6000, 2 mM amino acids (Sigma-Aldrich), 1 nM pY71sfGFP plasmid encoding superfolder GFP (M. Jewett), 0.1 mg/mL T7 RNA polymerase, 42 mM phosphoenolpyruvate (Roche) and NTP+ mix (1.6 mM ATP (Sigma), 1.15 mM of GTP, CTP and UTP each (Sigma), 45.3 μg/μL tRNA from E. coli MRE 600 (Roche), 227.5 μg/μL Folinic acid. The above components were premixed. A 5.36 μL aliquot of the premix was pipetted into 5 μl of S150 extract. Ribosomal proteins and plasmid encoding the engineered rrnB operon were added to a final concentration of 0.6 μM and 4 nM respectively. iSAT reactions of 15 μL each were performed in 96 well plates (Applied Biosystems) and incubated in a CFX Connect Real-time System (BIORAD) at 37 °C for variable time. sfGFP production was detected by fluorescence measurement at 5 min intervals (excitation: 450–490 nm, emission: 510–530 nm).