Figure 2.

Postlysis processing steps in extract preparation enhance cell-free gene expression yields from E. coli σ⁷⁰ promoters. Extracts prepared with runoff and dialysis (green) increase the yield of an sfGFP reporter across constructs containing a range of synthetic (A) ribosome binding site and (B) E. coli σ⁷⁰ promoter strengths, compared to extracts prepared without these steps (gray). (C) Titration of reporter construct DNA that contains a strong promoter and RBS in both extracts suggests that protein synthesis saturates above 10 nM of reporter template DNA in the processed extract but continues to increase in the nonprocessed extract. (D) Kinetics at the 20 nM reporter template DNA concentration show that despite having equal endpoint sfGFP production levels, protein production is 3× faster for the processed extract. Endpoint data in (A–C) are from 8 h experiments incubated and measured in a plate reader at 30 °C. Error bars represent the standard deviation of the mean across three technical replicates drawn from a single batch of extract.