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

TLDR

Cell‑free protein synthesis is a powerful technique for constructing and studying complex informational processes in vitro, but current systems are limited by a small set of bacteriophage regulatory elements. The study constructs and characterizes synthetic gene circuits using a cell‑free toolbox that employs all seven E. coli sigma factors. The toolbox uses the E.

Abstract

Cell-free protein synthesis is becoming a powerful technique to construct and to study complex informational processes in vitro. 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 E. coli sigma factors. The E. coli endogenous holoenzyme E(70) 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 in vitro.