Uncovering cell-free protein expression dynamics by a promoter library with diverse strengths

Cell-free protein expression systems have been widely used for synthetic biology and metabolic engineering applications in recent years. Yet little is known about protein expression in the cell-free systems. Here we take a systems approach to uncover underlying dynamics of cell-free protein expression. We construct a set of T7 promoter variants to express proteins at different transcription rates in a reconstituted and E. coli extract-based cell-free systems. We find that the maximum expression level and the rate of protein synthesis as responses to the transcription rate change are different in the two cell-free systems, suggesting they are driven by different expression dynamics. We confirm this by constructing a simple mathematical model for each cell-free system, which well reproduce the experimental results and also identify different limiting factors for better protein expression in the two cell-free systems. In particular, they revealed there is a negative feedback effect in the mRNA-protein translation by the PURE system and also identified different limiting factors for better protein expression in the two cell-free systems.