Deconstructing cell-free extract preparation forin vitroactivation of transcriptional genetic circuitry

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 designed as 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 method by which an extract is prepared affects its functionality. Specifically, a key goal toward developing cell-free biosensors based on native genetic regulators is activating the transcriptional machinery present in bacterial extracts for protein synthesis. However, protein yields from genes transcribed in vitro by the native Escherichia coli RNA polymerase are quite low in conventional crude extracts originally optimized for expression by the bacteriophage transcriptional machinery. Here, we show that cell-free expression of genes under bacterial σ 70 promoters is constrained by the rate of transcription in crude extracts and that processing the extract with a ribosomal run-off reaction and subsequent dialysis can alleviate 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 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 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 and will encourage the further proliferation of cell-free gene expression technology for new applications.

Rapid, Low-Cost Detection of Water Contaminants Using RegulatedIn VitroTranscription

“…Specifically, the focus on transcriptional RNA outputs allowed us to avoid the complexity of translation, resulting in a simplified set of reaction components that can produce visible signals within tens of minutes. In addition, the defined nature of in vitro reactions removed much of the batch-to-batch variability that can occur when preparing cell extracts [68].…”

Contemporary Tools for Regulating Gene Expression in Bacteria

“…This in vitro prototyping provides a useful method for the rapid exploration and optimisation of the large experimental spaces associated with complex genetic systems ( Figure 2B). Given recent improvement in CFPS lysate production, this tool is likely to see gains in popularity in the future [52][53][54][55]. (A) Introduction of heterologous genes into a production strain leads to competition for resources.…”

Multiplex transcriptional characterizations across diverse and hybrid bacterial cell-free expression systems

“…To further expand upon these advances, we established a streamlined experimental pipeline to construct, test and optimize cell lysates from diverse species (Methods) 18,19 . In short, cells are grown in rich media, lysed by sonication, incubated in a run-off reaction, and dialyzed (Figure 2a) 39 . We used the expression of Broccoli, an RNA fluorescence aptamer, to quantify the transcriptional activity of cell lysates 40 .…”