Deconstructing Cell-Free Extract Preparation for in Vitro Activation 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 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.

Keywords: CFE; CFPS; TX-TL; cell extract; cell-free synthetic biology; endogenous transcription; genetic circuitry; in vitro protein synthesis.

Figure 1.

The impact of different extract preparation procedures on cell-free gene expression (CFE) from different promoter systems. (A) Schematic for extract preparation. Combined transcription and translation activity differs based on extract preparation procedure and the use of endogenous or exogenous (bacteriophage) promoter systems. (B) Extract preparations that did not include postlysis processing steps yielded protein titers of ~ 1250 ng/μL when the sfGFP reporter is expressed from a T7 promoter (P_T7) using exogenously added T7 RNA polymerase, independent of extract preparation steps. (C) Extracts prepared without postlysis processing showed poor yield (~50 ng/μL) when under the control of the consensus E. coli σ⁷⁰ promoter (P_σ⁷⁰) and E. coli RNA polymerase supplied from the lysate. The addition of processing steps to the lysate outlined in green in (A) improved protein expression yields from the bacterial σ⁷⁰ promoter by 5× without impacting yields from the T7 promoter system. Protein yields are from overnight (15-h) in 15 μL reactions carried out in 2.0 mL tubes. Reporter constructs using the consensus E. coli σ⁷⁰ promoter also encoded an RNA stability hairpin before the RBS and downstream coding sequence (Figure S1). Reactions were supplemented with 5 nM of a plasmid-based sfGFP expression cassette in 2.0 mL tubes, and protein yields were background-subtracted (i.e., no plasmid control) and measured by correlation to a known fluorescent standard. Error bars represent the standard deviation of the mean from three technical replicates drawn from a single batch of extract.

Figure 2.

Postlysis processing steps in extract preparation enhance cell-free gene expression yields from E. coli σ⁷⁰ promoters. Extracts prepared with runoff and dialysis (green) increase the yield of an sfGFP reporter across constructs containing a range of synthetic (A) ribosome binding site and (B) E. coli σ⁷⁰ promoter strengths, compared to extracts prepared without these steps (gray). (C) Titration of reporter construct DNA that contains a strong promoter and RBS in both extracts suggests that protein synthesis saturates above 10 nM of reporter template DNA in the processed extract but continues to increase in the nonprocessed extract. (D) Kinetics at the 20 nM reporter template DNA concentration show that despite having equal endpoint sfGFP production levels, protein production is 3× faster for the processed extract. Endpoint data in (A–C) are from 8 h experiments incubated and measured in a plate reader at 30 °C. Error bars represent the standard deviation of the mean across three technical replicates drawn from a single batch of extract.

Figure 3.

Postlysis processing enhances the transcription rate but does not affect RNA degradation rates in CFE systems. (A) In vitro transcription of the malachite green RNA aptamer (MGA) from a strong E. coli σ⁷⁰ promoter in a cell-free gene expression reaction containing the malachite green dye using either processed (green) or nonprocessed (gray) extracts. The construct encoded a 5’ stability hairpin before the MGA DNA sequence to enhance fluorescence observation. (B) In vitro transcription of the MGA construct from a T7 promoter and no 5’ stability hairpin. Kinetic data in (A) and (B) represent the average of three technical replicates, with shaded region representing plus-minus one standard deviation of the mean. (C) Characterization of RNA degradation rates. The malachite green RNA aptamer was purified with the 5’ stability hairpin, mixed with the malachite green dye and 10% extract by volume, and the decay in fluorescence over time was measured to quantify RNA degradation rates (k_degradation)· Inset: the RNA half-life (ln(2)/k_degradation) was estimated by fitting an exponential decay function to the fluorescence kinetics over the first 30 min to fit k_degradation. Errors in half-lives are propagated from three independent measurements and reported as standard deviation.

Figure 4.

Extract activity is sensitive to some protocol variants. (A) Addition of glucose (navy) to the 2× YT + P culture media reduces CFE yields from bacterial promoters under transcriptionally limiting conditions (p = 0.035). (B) Flash freezing the cell pellet before lysis does not significantly impact the extract’s productivity (p = 0.30). (C) Extract clarification by the Kwon et al. protocol for BL21 (gray), with runoff reaction (light blue), and with runoff reaction and dialysis (green) under transcriptionally limiting conditions shows that both the runoff and dialysis steps contribute to the observed increase in transcriptional activity (p = 0.039 for green vs gray and p = 0.031 for green vs light blue). All bars represent the average of six independent cell-free reactions, drawn from three technical replicates of two independently generated extracts made on different days. Error bars represent the standard deviations of the six measurements. In each case, unpaired one-tailed Student’s t tests were performed for N = 2 extracts using Welch’s correction for heteroscedasticity.

Figure 5.

Processed extracts enable cell-free expression of a wide array of genetic circuitry. Postlysis processing enables significantly higher ON states, including activation in some cases, for (A) small RNA transcriptional activators (STARs), (B) RNA transcriptional repressors, (C) LuxR, the ;Y-acyl homoserine lactone (AHL)-inducible σ⁷⁰ transcription factor, (D) CRISPR-Cas9 cleavage, (E) toehold switch RNA translation activators, and (F) translational riboswitches. Each experiment was done in technical triplicate in either a postlysis processed (green) or nonprocessed (gray) extract for 8 h at 30 °C in a plate reader monitoring sfGFP production. The total plasmid DNA concentration was held constant in each reaction between the ON and OFF states of each circuit to remove variation caused by unwanted cell-free transcription and translation of the antibiotic resistance genes encoded on each plasmid. Complete reaction conditions including plasmid and inducer concentrations and optimal magnesium levels are included in Table S1. Kinetics of gene expression trajectories for each circuit are presented in Figure S7. Error bars represent the standard deviation of the mean for three technical replicates drawn from a single batch of each extract.