Cell-free expression is a technology used to synthesize minimal biological cells from natural molecular components. We have developed a versatile and powerful all-E. coli cell-free transcription-translation system energized by a robust metabolism, with the far objective of constructing a synthetic cell capable of self-reproduction. Inorganic phosphate (iP), a byproduct of protein synthesis, is recycled through polysugar catabolism to regenerate ATP (adenosine triphosphate) and thus supports long-lived and highly efficient protein synthesis in vitro. This cell-free TX-TL system is encapsulated into cell-sized unilamellar liposomes to express synthetic DNA programs. In this work, we study the compartmentalization of cell-free TX-TL reactions, one of the aspects of minimal cell module integration. We analyze the signals of various liposome populations by fluorescence microscopy for one and for two reporter genes, and for an inducible genetic circuit. We show that small nutrient molecules and proteins are encapsulated uniformly in the liposomes with small fluctuations. However, cell-free expression displays large fluctuations in signals among the same population, which are due to heterogeneous encapsulation of the DNA template. Consequently, the correlations of gene expression with the compartment dimension are difficult to predict accurately. Larger vesicles can have either low or high protein yields.

We report on and provide a detailed characterization of the performance and properties of a recently developed, all Escherichia coli, cell-free transcription and translation system. Gene expression is entirely based on the endogenous translation components and transcription machinery provided by an E. coli cytoplasmic extract, thus expanding the repertoire of regulatory parts to hundreds of elements. We use a powerful metabolism for ATP regeneration to achieve more than 2 mg/mL of protein synthesis in batch mode reactions, and more than 6 mg/mL in semicontinuous mode. While the strength of cell-free expression is increased by a factor of 3 on average, the output signal of simple gene circuits and the synthesis of entire bacteriophages are increased by orders of magnitude compared to previous results. Messenger RNAs and protein degradation, respectively tuned using E. coli MazF interferase and ClpXP AAA+ proteases, are characterized over a much wider range of rates than the first version of the cell-free toolbox. This system is a highly versatile cell-free platform to construct complex biological systems through the execution of DNA programs composed of synthetic and natural bacterial regulatory parts.

A central goal of synthetic biology is to engineer cellular behavior by engineering synthetic gene networks for a variety of biotechnology and medical applications. The process of engineering gene networks often involves an iterative ‘design–build–test’ cycle, whereby the parts and connections that make up the network are built, characterized and varied until the desired network function is reached. Many advances have been made in the design and build portions of this cycle. However, the slow process of in vivo characterization of network function often limits the timescale of the testing step. Cell-free transcription–translation (TX–TL) systems offer a simple and fast alternative to performing these characterizations in cells. Here we provide an overview of a cell-free TX–TL system that utilizes the native Escherichia coli TX–TL machinery, thereby allowing a large repertoire of parts and networks to be characterized. As a way to demonstrate the utility of cell-free TX–TL, we illustrate the characterization of two genetic networks: an RNA transcriptional cascade and a protein regulated incoherent feed-forward loop. We also provide guidelines for designing TX–TL experiments to characterize new genetic networks. We end with a discussion of current and emerging applications of cell free systems.

RNA regulators are emerging as powerful tools to engineer synthetic genetic networks or rewire existing ones. A potential strength of RNA networks is that they may be able to propagate signals on time scales that are set by the fast degradation rates of RNAs. However, a current bottleneck to verifying this potential is the slow design-build-test cycle of evaluating these networks in vivo. Here, we adapt an Escherichia coli-based cell-free transcription-translation (TX-TL) system for rapidly prototyping RNA networks. We used this system to measure the response time of an RNA transcription cascade to be approximately five minutes per step of the cascade. We also show that this response time can be adjusted with temperature and regulator threshold tuning. Finally, we use TX-TL to prototype a new RNA network, an RNA single input module, and show that this network temporally stages the expression of two genes in vivo.

Toward an “artificial cell” on a chip Cell-free systems that reconstitute biochemical pathways have been critical for unraveling the inner workings of the cell. Karzbrun et al. created a highly miniaturized cell-free system on a silicon chip. A series of tiny linked compartments were fabricated on the chip, in which DNA-driven reactions occurred, with materials flowing into and diffusing between the compartments. The system recreated oscillating protein expression patterns and protein gradients, and provides a stepping stone to creating “artificial cells” on a chip. Science, this issue p. 829 The assembly of artificial cells capable of executing synthetic DNA programs has been an important goal for basic research and biotechnology. We assembled two-dimensional DNA compartments fabricated in silicon as artificial cells capable of metabolism, programmable protein synthesis, and communication. Metabolism is maintained by continuous diffusion of nutrients and products through a thin capillary, connecting protein synthesis in the DNA compartment with the environment. We programmed protein expression cycles, autoregulated protein levels, and a signaling expression gradient, equivalent to a morphogen, in an array of interconnected compartments at the scale of an embryo. Gene expression in the DNA compartment reveals a rich, dynamic system that is controlled by geometry, offering a means for studying biological networks outside a living cell. DNA-driven biochemical reactions on a fabricated silicon chip recreate protein gradients and oscillations. DNA-driven biochemical reactions on a fabricated silicon chip recreate protein gradients and oscillations.

Accelerating the pace of synthetic biology experiments requires new approaches for rapid prototyping of circuits from individual DNA regulatory elements. However, current testing standards require days to weeks due to cloning and in vivo transformation. In this work, we first characterized methods to protect linear DNA strands from exonuclease degradation in an Escherichia coli based transcription-translation cell-free system (TX-TL), as well as mechanisms of degradation. This enabled the use of linear DNA PCR products in TX-TL. We then compared expression levels and binding dynamics of different promoters on linear DNA and plasmid DNA. We also demonstrated assembly technology to rapidly build circuits entirely in vitro from separate parts. Using this strategy, we prototyped a four component genetic switch in under 8 h entirely in vitro. Rapid in vitro assembly has future applications for prototyping multiple component circuits if combined with predictive computational models.

Cell-free protein synthesis is becoming a useful technique for synthetic biology. As more applications are developed, the demand for novel and more powerful in vitro expression systems is increasing. In this work, an all Escherichia coli cell-free system, that uses the endogenous transcription and translation molecular machineries, is optimized to synthesize up to 2.3 mg/ml of a reporter protein in batch mode reactions. A new metabolism based on maltose allows recycling of inorganic phosphate through its incorporation into newly available glucose molecules, which are processed through the glycolytic pathway to produce more ATP. As a result, the ATP regeneration is more efficient and cell-free protein synthesis lasts up to 10 h. Using a commercial E. coli strain, we show for the first time that more than 2 mg/ml of protein can be synthesized in run-off cell-free transcription–translation reactions by optimizing the energy regeneration and waste products recycling. This work suggests that endogenous enzymes present in the cytoplasmic extract can be used to implement new metabolic pathways for increasing protein yields. This system is the new basis of a cell-free gene expression platform used to construct and to characterize complex biochemical processes in vitro such as gene circuits.

There is an increasing interest to express and study membrane proteins in vitro. New techniques to produce and insert functional membrane proteins into planar lipid bilayers have to be developed. In this work, we produce a tethered lipid bilayer membrane (tBLM) to provide sufficient space for the incorporation of the integral membrane protein (IMP) Aquaporin Z (AqpZ) between the tBLM and the surface of the sensor. We use a gold (Au)-coated sensor surface compatible with mechanical sensing using a quartz crystal microbalance with dissipation monitoring (QCM-D) or optical sensing using the surface plasmon resonance (SPR) method. tBLM is produced by vesicle fusion onto a thin gold film, using phospholipid-polyethylene glycol (PEG) as a spacer. Lipid vesicles are composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethyleneglycol)-2000-N-[3-(2-pyridyldithio)propionate], so-called DSPE-PEG-PDP, at different molar ratios (respectively, 99.5/0.5, 97.5/2.5, and 95/5 mol %), and tBLM formation is characterized using QCM-D, SPR, and atomic force technology (AFM). We demonstrate that tBLM can be produced on the gold surface after rupture of the vesicles using an α helical (AH) peptide, derived from hepatitis C virus NS5A protein, to assist the fusion process. A cell-free expression system producing the E. coli integral membrane protein Aquaporin Z (AqpZ) is directly incubated onto the tBLMs for expression and insertion of the IMP at the upper side of tBLMs. The incorporation of AqpZ into bilayers is monitored by QCM-D and compared to a control experiment (without plasmid in the cell-free expression system). We demonstrate that an IMP such as AqpZ, produced by a cell-free expression system without any protein purification, can be incorporated into an engineered tBLM preassembled at the surface of a gold-coated sensor.

The synthesis of living entities in the laboratory is a standing challenge that calls for innovative approaches. Using a cell-free transcription-translation system as a molecular programming platform, we show that the bacteriophage T7, encoded by a 40 kbp DNA program composed of about 60 genes, can be entirely synthesized from its genomic DNA in a test tube reaction. More than a billion infectious bacteriophages T7 per milliliter of reaction are produced after a few hours of incubation. The replication of the genomic DNA occurs concurrently with phage gene expression, protein synthesis, and viral assembly. The demonstration that genome-sized viral DNA can be expressed in a test tube, recapitulating the entire chain of information processing including the replication of the DNA instructions, opens new possibilities to program and to study complex biochemical systems in vitro.

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.