Refer to the detailed description for the preparation of the cell-free expression experiment included in the Supplemental Note and the Supplemental Experimental Design Spreadsheet. The final CFE reaction mixture is composed of the following reagents: 10–20 mM magnesium glutamate; 10 mM ammonium glutamate; 130 mM potassium glutamate; 1.2 mM ATP; 0.850 mM each of GTP, UTP, and CTP; 0.034 mg/mL folinic acid; 0.171 mg/mL yeast tRNA; 2 mM amino acids; 30 mM PEP; 0.33 mM NAD; 0.27 mM CoA; 4 mM oxalic acid; 1 mM putrescine; 1.5 mM spermidine; 57 mM HEPES; 30% CFE extract by volume; plasmid DNA to the desired concentration (refer to Table S1 for plasmid DNA concentrations used in this study); and water. For reactions involving T7 RNAP expression, in-house purified T7 RNAP was doped into the reaction at 0.10 mg/mL. The optimal magnesium concentration was determined for each reporter construct and was found to be 16 mM magnesium glutamate in nearly all cases (for exceptions to this, as well as the inducer concentrations in Figure 5, refer to Table S1).
All kinetic CFE reactions were prepared on ice in triplicate at the 10 μL scale. 33 μL of a mixture containing the desired reaction components was prepared and then 10 μL was pipetted into three wells of a 384-well plate (Corning, 3712), taking care to avoid bubbles. Plates were sealed (Thermo Scientific, 232701) and sfGFP fluorescence (emission/excitation: 485/520 nm) was monitored every 5 min on a BioTek Synergy Him plate reader for 8 h at 30 °C. For the bulk endpoint experiments in Figure 1, reactions were prepared to the 15 μL scale in triplicate, pipetted into the bottom of a 2.0 mL microcentrifuge tube, and incubated without shaking at 30 °C overnight for 15 h. Final protein titers were calculated from a previously developed plate reader correlation to either sfGFP (high-yield overnight experiments) or FITC (low-yield kinetic experiments) (see below). For the malachite green experiments in Figure 3, fluorescence (emission/excitation: 615/650 nm) was measured every 3 min. For all experiments, a no-DNA negative control was prepared in triplicate for every extract being tested. All reported fluorescence values have been baseline-subtracted by the no-DNA condition, and all error bars include the propagated error from the no-DNA condition.