Deconstructing Cell-Free Extract Preparation for <i>in Vitro</i> Activation of Transcriptional Genetic Circuitry

TLDR

Cell‑free gene expression systems are widely used for rapid prototyping of genetic circuits, yet the many existing extract‑preparation protocols create confusion about how preparation methods influence extract performance, especially the activity of native E. coli RNA polymerase, which varies and is often low in conventional crude extracts. The study aims to determine how extract preparation affects transcription of genes under bacterial σ70 promoters and to identify processing steps that relieve transcriptional constraints. By applying a ribosomal runoff reaction followed by dialysis and a streamlined sonication‑based protocol, the authors generate extracts that support robust transcription from σ70 promoters and enable expression of diverse protein and RNA sensors.

Abstract

Recent advances in cell-free gene expression (CFE) systems have enabled their use for a host of synthetic biology applications, particularly for rapid prototyping of genetic circuits and biosensors. Despite the proliferation of cell-free protein synthesis platforms, the large number of currently existing protocols for making CFE extracts muddles the collective understanding of how the extract preparation method affects its functionality. A key aspect of extract performance relevant to many applications is the activity of the native host transcriptional machinery that can mediate protein synthesis. However, protein yields from genes transcribed in vitro by the native Escherichia coli RNA polymerase are variable for different extract preparation techniques, and specifically low in some conventional crude extracts originally optimized for expression by the bacteriophage transcriptional machinery. Here, we show that cell-free expression of genes under bacterial σ70 promoters is constrained by the rate of transcription in crude extracts, and that processing the extract with a ribosomal runoff reaction and subsequent dialysis alleviates this constraint. Surprisingly, these processing steps only enhance protein synthesis in genes under native regulation, indicating that the translation rate is unaffected. We further investigate the role of other common extract preparation process variants on extract performance and demonstrate that bacterial transcription is inhibited by including glucose in the growth culture but is unaffected by flash-freezing the cell pellet prior to lysis. Our final streamlined and detailed protocol for preparing extract by sonication generates extract that facilitates expression from a diverse set of sensing modalities including protein and RNA regulators. We anticipate that this work will clarify the methodology for generating CFE extracts that are active for biosensing using native transcriptional machinery and will encourage the further proliferation of cell-free gene expression technology for new applications.