In Vitro Synthesis of Fluorescent Protein

iSAT reactions of 15 μl were set-up as previously described (44 (link)). Briefly, reactions were prepared in polymerase chain reaction tubes with optically clear flat caps and incubated at 37°C in a CFX96 real-time thermal cycler (Bio-Rad). iSAT reactions contained reporter protein plasmids encoding superfolder GFP (sfGFP). Green fluorescence of sfGFP was monitored using the CFX96real-time thermal cycler as (excitation: 450–490 nm, emission: 510–530 nm). Additives were included at the described final concentrations. Specifically, crowding agent (2% PEG-6000) and reducing agent (2 mM DTT) were added to each reaction. iSAT reactions for S150 extracts were optimized for concentrations of magnesium glutamate to maximize reaction productivity and minimize consumption of parts (Supplementary Figure S12). sfGFP quantification was performed as previously reported (43 (link)), using measurements of relative fluorescence units (RFU) from CFX96 real-time thermal cycler (BioRad, Hercules, CA, USA) and BioTek Synergy 2 plate reader (Winooski, VT, USA). RFU values were converted to molar concentration using a linear standard curve made in-house by expressing ¹⁴C-leucine labeled sfGFP in E. coli PANOx CFPS reactions and relating RFUs to trichloracetic acid precipitable soluble protein yield.