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To facilitate the construction of cell-free genetic devices, we evaluated the ability of 17 different fluorescent proteins to give easily detectable fluorescence signals in real-time from in vitro transcription-translation reactions with a minimal system consisting of T7 RNA polymerase and E. coli translation machinery, i.e., the PUREsystem. The data were used to construct a ratiometric fluorescence assay to quantify the effect of genetic organization on in vitro expression levels. Synthetic operons with varied spacing and sequence composition between two genes that coded for fluorescent proteins were then assembled. The resulting data indicated which restriction sites and where the restriction sites should be placed in order to build genetic devices in a manner that does not interfere with protein expression. Other simple design rules were identified, such as the spacing and sequence composition influences of regions upstream and downstream of ribosome binding sites and the ability of non-AUG start codons to function in vitro.

Fluorescent Proteins and in Vitro Genetic Organization for Cell-Free Synthetic Biology