An <i>E. coli</i> Cell-Free Expression Toolbox: Application to Synthetic Gene Circuits and Artificial Cells

Abstract

Cell-free protein synthesis is becoming a powerful technique to construct and to study complex informational processes <i>in vitro</i>. Engineering synthetic gene circuits in a test tube, however, is seriously limited by the transcription repertoire of modern cell-free systems, composed of only a few bacteriophage regulatory elements. Here, we report the construction and the phenomenological characterization of synthetic gene circuits engineered with a cell-free expression toolbox that works with the seven <i>E. coli</i> sigma factors. The <i>E. coli</i> endogenous holoenzyme E₇₀ is used as the primary transcription machinery. Elementary circuit motifs, such as multiple stage cascades, AND gate and negative feedback loops are constructed with the six other sigma factors, two bacteriophage RNA polymerases, and a set of repressors. The circuit dynamics reveal the importance of the global mRNA turnover rate and of passive competition-induced transcriptional regulation. Cell-free reactions can be carried out over long periods of time with a small-scale dialysis reactor or in phospholipid vesicles, an artificial cell system. This toolbox is a unique platform to study complex transcription/translation-based biochemical systems <i>in vitro</i>