Gene position more strongly influences cell-free protein expression from operons than T7 transcriptional promoter strength.

The cell-free transcription-translation of multiple proteins typically exploits genes placed behind strong transcriptional promoters that reside on separate pieces of DNA so that protein levels can be easily controlled by changing DNA template concentration. However, such systems are not amenable to the construction of artificial cells with a synthetic genome. Herein, we evaluated the activity of a series of T7 transcriptional promoters by monitoring the fluorescence arising from a genetically encoded Spinach aptamer. Subsequently the influences of transcriptional promoter strength on fluorescent protein synthesis from one, two, and three gene operons were assessed. It was found that transcriptional promoter strength was more effective at controlling RNA synthesis than protein synthesis in vitro with the PURE system. Conversely, the gene position within the operon strongly influenced protein synthesis but not RNA synthesis.