An E. coli 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 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.

Sealable Femtoliter Chamber Arrays for Cell-free Biology

“…Recently cell-free systems have begun to gain a foothold as viable platforms for synthetic biology [8][9][10] . The appeal of such approaches is that they free synthetic biology from the resource sharing and 'extrinsic noise' that affects reaction dynamics in living cells.…”

Protocols for Implementing an <em>Escherichia coli</em> Based TX-TL Cell-Free Expression System for Synthetic Biology

“…Mechanisms of regulation and transcription exogenous to E. coli, such as lac/tet repressors and T7 RNA polymerase, can be supplemented. 6 Endogenous properties, such as mRNA and DNA degradation rates, can also be adjusted. 7 The TX-TL cell-free expression system has been demonstrated for large-scale circuit assembly, exploring biological phenomena, and expression of proteins under both T7-and endogenous promoters.…”

Construction de cellules synthétiques

“…Des auto-assemblages biologiques complexes programmés par l'ADN peuvent désor-mais être réalisés dans des tubes à essais (Figure 2A, B) [46]. Au regard de ce résultat, la construction de réseaux génétiques synthétiques in vitro reste limitée mais elle progresse rapidement ( Figure 2C, D) [47,48]. Le développement de systèmes d'expression encore plus puissants est nécessaire à la construction de cellules minimales.…”