Cell-free Gene Expression Systems Research Articles

Chloroplast Cell-Free Systems from Different Plant Species as a Rapid Prototyping Platform.

Climate change poses a significant threat to global agriculture, necessitating innovative solutions. Plant synthetic biology, particularly chloroplast engineering, holds promise as a viable approach to this challenge. Chloroplasts present a variety of advantageous traits for genetic engineering, but the development of genetic tools and genetic part characterization in these organelles is hindered by the lengthy time scales required to generate transplastomic organisms. To address these challenges, we have established a versatile protocol for generating highly active chloroplast-based cell-free gene expression (CFE) systems derived from a diverse range of plant species, including wheat (monocot), spinach, and poplar trees (dicots). We show that these systems work with conventionally used T7 RNA polymerase as well as the endogenous chloroplast polymerases, allowing for detailed characterization and prototyping of regulatory sequences at both transcription and translation levels. To demonstrate the platform for characterization of promoters and 5' and 3' untranslated regions (UTRs) in higher plant chloroplast gene expression, we analyze a collection of 23 5'UTRs, 10 3'UTRs, and 6 chloroplast promoters, assessed their expression in spinach and wheat extracts, and found consistency in expression patterns, suggesting cross-species compatibility. Looking forward, our chloroplast CFE systems open new avenues for plant synthetic biology, offering prototyping tools for both understanding gene expression and developing engineered plants, which could help meet the demands of a changing global climate.

Inexpensive and colorimetric RNA detection at ambient temperature with a cell-free protein synthesis platform

While many proposals of paper-based diagnostics utilize cell-free gene expression systems, these assays oftentimes suffer from the need for temperature cycling and high operational costs, particularly for developing countries. Here, we explore and report the experimental conditions for the colorimetric detection of viral RNA with an in vitro transcription/translation assay that uses crude E. coli extracts at room temperature where the signal amplification is aided by body heat. Clinically-relevant concentrations of RNA (ca. 600 copies/test) were detected from synthetic RNA samples. The activation of cell-free gene expression was achieved using toehold-switch-mediated riboregulatory elements that are specific to RNA sequences. The colorimetric output was generated by the α-complementation of β-galactosidase ω-fragment (LacZω) with cell-free expressed LacZα, using an X-gal analogue as a substrate. The estimated cost of a single reaction is as low as ~ 0.26 euro/test, which may help to facilitate the accessibility of the diagnostic kit in developing countries. With future optimizations and bacterial strain engineering, production costs can be even further brought down, and the test times can be shortened.Graphical

Cell-Free Translation Quantification via a Fluorescent Minihelix.

Cell-free gene expression systems are used in numerous applications, including medicine making, diagnostics, and educational kits. Accurate quantification of nonfluorescent proteins in these systems remains a challenge. To address this challenge, we report the adaptation and use of an optimized tetra-cysteine minihelix both as a fusion protein and as a standalone reporter with the FlAsH dye. The fluorescent reporter helix is short enough to be encoded on a primer pair to tag any protein of interest via PCR. Both the tagged protein and the standalone reporter can be detected quantitatively in real time or at the end of cell-free expression reactions with standard 96/384-well plate readers, an RT-qPCR system, or gel electrophoresis without the need for staining. The fluorescent signal is stable and correlates linearly with the protein concentration, enabling product quantification. We modified the reporter to study cell-free expression dynamics and engineered ribosome activity. We anticipate that the fluorescent minihelix reporter will facilitate efforts in engineering in vitro transcription and translation systems.

Reversible Control of Gene Expression by Guest-Modified Adenosines in a Cell-Free System via Host-Guest Interaction.

Gene expression technology has become an indispensable tool for elucidating biological processes and developing biotechnology. Cell-free gene expression (CFE) systems offer a fundamental platform for gene expression-based technology, in which the reversible and programmable control of transcription can expand its use in synthetic biology and medicine. This study shows that CFE can be controlled via the host-guest interaction of cucurbit[7]uril (CB[7]) with N6-guest-modified adenosines. These adenosine derivatives were conveniently incorporated into the DNA strand using a post-synthetic approach and formed a selective and stable base pair with complementary thymidine in DNA. Meanwhile, alternate addition of CB[7] and the exchanging guest molecule induced the reversible formation of a duplex structure through the formation and dissociation of a bulky complex on DNA. The kinetics of the reversibility was fine-tuned by changing the size of the modified guest moieties. When incorporated into a specific region of the T7 promoter sequence, the guest-modified adenosines enabled tight and reversible control of in vitro transcription and protein expression in the CFE system. This study marks the first utility of the host-guest interaction for gene expression control in the CFE system, opening new avenues for developing DNA-based technology, particularly for precise gene therapy and DNA nanotechnology.

Cell-free synthesis of the Salmonella specific broad host range bacteriophage, felixO1

Phage-based biocontrol of foodborne Salmonella is limited by the requisite use of Salmonella to propagate the phages. This limitation can be circumvented by producing Salmonella phages using a cell-free gene expression system (CFE) with a non-pathogenic chassis. Here, we produce the Salmonella phage felixO1 using an E. coli-based CFE system.

Reversible Stabilization of Nanofiber-Polyplexes through Introducing Cross-Linkages.

Non-viral gene delivery systems are typically designed vector systems with contradictory properties, namely sufficient stability before cellular uptake and instability to ensure the release of nucleic acid cargoes in the transcription process after being taken up into cells. We reported previously that poly-(L-lysine) terminally bearing a multi-arm PEG (maPEG-PLL) formed nanofiber-polyplexes that suppressed excessive DNA condensation via steric repulsion among maPEGs and exhibited effective transcriptional capability in PCR amplification experiments and a cell-free gene expression system. In this study, the reversible stabilization of a nanofiber-polyplex without impairing the effective transcriptional capability was investigated by introducing cross-links between the PLL side chains within the polyplex using a cross-linking reagent with disulfide (SS) bonds that can be disrupted under reducing conditions. In the presence of dextran sulfate and/or dithiothreitol, the stability of the polyplex and the reactivity of the pDNA were evaluated using agarose gel electrophoresis and real-time PCR. We succeeded in reversibly stabilizing nanofiber-polyplexes using dithiobis (succinimidyl propionate) (DSP) as the cross-linking reagent. The effect of the reversible stabilization was confirmed in experiments using cultured cells, and the DSP-crosslinked polyplexes exhibited gene expression superior to that of polyethyleneimine polyplexes, which are typical polyplexes.

Improving Cell-Free Expression of Model Membrane Proteins by Tuning Ribosome Cotranslational Membrane Association and Nascent Chain Aggregation.

Cell-free gene expression (CFE) systems are powerful tools for transcribing and translating genes outside of a living cell. Synthesis of membrane proteins is of particular interest, but their yield in CFE is substantially lower than that for soluble proteins. In this paper, we study the CFE of membrane proteins and develop a quantitative kinetic model. We identify that ribosome stalling during the translation of membrane proteins is a strong predictor of membrane protein synthesis due to aggregation between the ribosome nascent chains. Synthesis can be improved by the addition of lipid membranes, which incorporate protein nascent chains and, therefore, kinetically compete with aggregation. We show that the balance between peptide-membrane association and peptide aggregation rates determines the yield of the synthesized membrane protein. We define a membrane protein expression score that can be used to rationalize the engineering of lipid composition and the N-terminal domain of a native and computationally designed membrane proteins produced through CFE.

A cell-free strategy for host-specific profiling of intracellular antibiotic sensitivity and resistance

Antimicrobial resistance (AMR) is a pandemic spread across multiple infectious disease-causing microbes. To provide a host-specific tool to study antibiotic susceptibility and resistance, here we develop Klebsiella pneumoniae cell-free gene expression (CFE) systems from laboratory and clinical isolates. Using proteomics, we identify relative differences and unique proteins for these new CFE systems in comparison to an Escherichia coli MG1655 CFE model. Then we profile antimicrobial susceptibility in parallel with whole cells to quantify CFE antibiotic potency. Finally, we apply this native CFE tool to study AMR variants at a proof-of-concept level. Definably we show that RpoB H526L confers a 58-fold increase in CFE resistance to rifampicin—a genotype observed in rifampicin-resistant Mycobacterium tuberculosis clinical isolates. Overall, we provide a cell-free synthetic biology strategy for the profiling of antibiotic sensitivity and resistance from K. pneumoniae. While initial extract processing requires Biosafety Level 2, the CFE system is non-living, suitable for long-term storage and study in a Biosafety Level 1 lab. We anticipate the K. pneumoniae CFE bioassay is advantageous for host-specific antimicrobial testing, the characterisation of intracellular AMR variants and potentially structure-activity relationship studies.

Noise Minimization in Cell-Free Gene Expression.

Biochemical reactions that involve small numbers of molecules are accompanied by a degree of inherent randomness that results in noisy reaction outcomes. In synthetic biology, the ability to minimize noise particularly during the reconstitution of future synthetic protocells is an outstanding challenge to secure robust and reproducible behavior. Here we show that by encapsulation of a bacterial cell-free gene expression system in water-in-oil droplets, in vitro-synthesized MazF reduces cell-free gene expression noise >2-fold. With stochastic simulations we identify that this noise minimization acts through both increased degradation and the autoregulatory feedback of MazF. Specifically, we find that the expression of MazF enhances the degradation rate of mRNA up to 18-fold in a sequence-dependent manner. This sequence specificity of MazF would allow targeted noise control, making it ideal to integrate into synthetic gene networks. Therefore, including MazF production in synthetic biology can significantly minimize gene expression noise, impacting future design principles of more complex cell-free gene circuits.

A Cell-Free Gene Expression Platform for Discovering and Characterizing Stop Codon Suppressing tRNAs.

Non-canonical amino acids (ncAAs) can be incorporated into peptides and proteins to create new properties and functions. Site-specific ncAA incorporation is typically enabled by orthogonal translation systems comprising a stop codon suppressing tRNA (typically UAG), an aminoacyl-tRNA synthetase, and an ncAA of interest. Unfortunately, methods to discover and characterize suppressor tRNAs are limited because of laborious and time-consuming workflows in living cells. In this work, we develop anEscherichia coli crude extract-based cell-free gene expression system to rapidly express and characterize functional suppressor tRNAs. Our approach co-expresses orthogonal tRNAs using endogenous machinery alongside a stop-codon containing superfolder green fluorescent protein (sfGFP) reporter, which can be used as a simple read-out for suppression. As a model, we evaluate the UAG and UAA suppressing activity of several orthogonal tRNAs. Then, we demonstrate that co-transcription of two mutually orthogonal tRNAs can direct the incorporation of two unique ncAAs within a single modified sfGFP. Finally, we show that the cell-free workflow can be used to discover putative UAG-suppressor tRNAs found in metagenomic data, which are nonspecifically recognized by endogenous aminoacyl-tRNA synthetases. We anticipate that our cell-free system will accelerate the development of orthogonal translation systems for synthetic biology.

Activation/Inhibition of Gene Expression Caused by Alcohols: Relationship with the Viscoelastic Property of a DNA Molecule

Alcohols are used in the life sciences because they can condense and precipitate DNA. Alcohol consumption has been linked to many diseases and can alter genetic activity. In the present report, we carried out experiments to make clear how alcohols affect the efficiency of transcription-translation (TX-TL) and translation (TL) by adapting cell-free gene expression systems with plasmid DNA and RNA templates, respectively. In addition, we quantitatively analyzed intrachain fluctuations of single giant DNA molecules based on the fluctuation-dissipation theorem to gain insight into how alcohols affect the dynamical property of a DNA molecule. Ethanol (2-3%) increased gene expression levels four to five times higher than the control in the TX-TL reaction. A similar level of enhancement was observed with 2-propanol, in contrast to the inhibitory effect of 1-propanol. Similar alcohol effects were observed for the TL reaction. Intrachain fluctuation analysis through single DNA observation showed that 1-propanol markedly increased both the spring and damping constants of single DNA in contrast to the weak effects observed with ethanol, whereas 2-propanol exhibits an intermediate effect. This study indicates that the activation/inhibition effects of alcohol isomers on gene expression correlate with the changes in the viscoelastic mechanical properties of DNA molecules.

Phospholipid synthesis inside phospholipid membrane vesicles

Construction of living artificial cells from genes and molecules can expand our understanding of life system and establish a new aspect of bioengineering. However, growth and division of cell membrane that are basis of cell proliferation are still difficult to reconstruct because a high-yielding phospholipid synthesis system has not been established. Here, we developed a cell-free phospholipid synthesis system that combines fatty acid synthesis and cell-free gene expression system synthesizing acyltransferases. The synthesized fatty acids were sequentially converted into phosphatidic acids by the cell-free synthesized acyltransferases. Because the system can avoid the accumulation of intermediates inhibiting lipid synthesis, sub-millimolar phospholipids could be synthesized within a single reaction mixture. We also performed phospholipid synthesis inside phospholipid membrane vesicles, which encapsulated all the components, and showed the phospholipids localized onto the mother membrane. Our approach would be a platform for the construction of self-reproducing artificial cells since the membrane can grow sustainably.

A ubiquitous amino acid source for prokaryotic and eukaryotic cell-free transcription-translation systems.

Cell-free gene expression (CFE) systems are an attractive tool for engineering within synthetic biology and for industrial production of high-value recombinant proteins. CFE reactions require a cell extract, energy system, amino acids, and DNA, to catalyse mRNA transcription and protein synthesis. To provide an amino acid source, CFE systems typically use a commercial standard, which is often proprietary. Herein we show that a range of common microbiology rich media (i.e., tryptone, peptone, yeast extract and casamino acids) unexpectedly provide an effective and low-cost amino acid source. We show that this approach is generalisable, by comparing batch variability and protein production in the following range of CFE systems: Escherichia coli (Rosetta™ 2 (DE3), BL21(DE3)), Streptomyces venezuelae and Pichia pastoris. In all CFE systems, we show equivalent or increased protein synthesis capacity upon replacement of the commercial amino acid source. In conclusion, we suggest rich microbiology media provides a new amino acid source for CFE systems with potential broad use in synthetic biology and industrial biotechnology applications.

Engineering a Synthetic Dopamine-Responsive Riboswitch for In Vitro Biosensing.

The detection of chemicals using natural allosteric transcription factors is a powerful strategy for point-of-use molecular sensing, particularly using fieldable cell-free gene expression (CFE) systems. However, the reliance of detection schemes on characterized protein-based sensors limits the number of measurable analytes. One alternative solution to this issue is to develop new sensors by generating RNA aptamers against the target analyte and then incorporating them directly into a riboswitch scaffold for ligand-inducible genetic control of a reporter protein. However, this strategy has not generated more than a handful of successful portable cell-free molecular sensors. To address this gap, here we convert dopamine-binding aptamers into functional dopamine-sensing riboswitches that regulate gene expression in a freeze-dried CFE reaction. We then develop an assay for direct detection and semi-quantification of dopamine in human urine. We anticipate that this work will be broadly applicable for converting many in vitro-generated RNA aptamers into fieldable molecular diagnostics.

Decentralizing Cell-Free RNA Sensing With the Use of Low-Cost Cell Extracts.

Cell-free gene expression systems have emerged as a promising platform for field-deployed biosensing and diagnostics. When combined with programmable toehold switch-based RNA sensors, these systems can be used to detect arbitrary RNAs and freeze-dried for room temperature transport to the point-of-need. These sensors, however, have been mainly implemented using reconstituted PURE cell-free protein expression systems that are difficult to source in the Global South due to their high commercial cost and cold-chain shipping requirements. Based on preliminary demonstrations of toehold sensors working on lysates, we describe the fast prototyping of RNA toehold switch-based sensors that can be produced locally and reduce the cost of sensors by two orders of magnitude. We demonstrate that these in-house cell lysates provide sensor performance comparable to commercial PURE cell-free systems. We further optimize these lysates with a CRISPRi strategy to enhance the stability of linear DNAs by knocking-down genes responsible for linear DNA degradation. This enables the direct use of PCR products for fast screening of new designs. As a proof-of-concept, we develop novel toehold sensors for the plant pathogen Potato Virus Y (PVY), which dramatically reduces the yield of this important staple crop. The local implementation of low-cost cell-free toehold sensors could enable biosensing capacity at the regional level and lead to more decentralized models for global surveillance of infectious disease.

The all-E. coliTXTL toolbox 3.0: new capabilities of a cell-free synthetic biology platform.

The new generation of cell-free gene expression systems enables the prototyping and engineering of biological systems in vitro over a remarkable scope of applications and physical scales. As the utilization of DNA-directed in vitro protein synthesis expands in scope, developing more powerful cell-free transcription–translation (TXTL) platforms remains a major goal to either execute larger DNA programs or improve cell-free biomanufacturing capabilities. In this work, we report the capabilities of the all-E. coli TXTL toolbox 3.0, a multipurpose cell-free expression system specifically developed for synthetic biology. In non-fed batch-mode reactions, the synthesis of the fluorescent reporter protein eGFP (enhanced green fluorescent protein) reaches 4 mg/ml. In synthetic cells, consisting of liposomes loaded with a TXTL reaction, eGFP is produced at concentrations of >8 mg/ml when the chemical building blocks feeding the reaction diffuse through membrane channels to facilitate exchanges with the outer solution. The bacteriophage T7, encoded by a genome of 40 kb and ∼60 genes, is produced at a concentration of 1013 PFU/ml (plaque forming unit/ml). This TXTL system extends the current cell-free expression capabilities by offering unique strength and properties, for testing regulatory elements and circuits, biomanufacturing biologics or building synthetic cells.

Cell-free gene expression

Cell-free gene expression (CFE) emerged as an alternative approach to living cells for specific applications in protein synthesis and labelling for structural biology and proteomics studies. CFE has since been repurposed as a versatile technology for synthetic biology and bioengineering. However, taking full advantage of this technology requires in-depth understanding of its fundamental workflow beyond existing protocols. This Primer provides new practitioners with a comprehensive, detailed and actionable guide to best practices in CFE, to inform research in the laboratory at the state of the art. We focus on Escherichia coli-based CFE systems, which remain the primary platform for efficient CFE. Producing proteins, biomanufacturing therapeutics, developing sensors and prototyping genetic circuits illustrate the broader utility and opportunities provided by this practical introduction to CFE. With its extensive functionality and portability, CFE is becoming a powerful and enabling research tool for biotechnology. Cell-free gene expression is useful for expressing proteins with post-translational modifications, with special folding requirements and whose expression is difficult in prokaryotic systems. Garenne et al. outline the best practices for the expression of proteins in a cell-free environment.

Longer DNA exhibits greater potential for cell-free gene expression

Cell-free gene expression systems have been valuable tools for understanding how transcription/translation can be regulated in living cells. Many studies have investigated the determining factors that affect gene expression. Here we report the effect of the length of linearized reporter DNAs encoding the firefly luciferase gene so as to exclude the influence of supercoiling. It is found that longer DNA molecules exhibit significantly greater potency in gene expression; for example, the expression level for DNA with 25.7 kbp is 1000-times higher than that for DNA of 1.7 kbp. AFM observation of the DNA conformation indicates that longer DNA takes shrunken conformation with a higher segment density in the reaction mixture for gene expression, in contrast to the stiff conformation of shorter DNA. We propose an underlying mechanism for the favorable effect of longer DNA on gene expression in terms of the enhancement of access of RNA polymerase to the shrunken conformation. It is expected that the enhancement of gene expression efficiency with a shrunken DNA conformation would also be a rather general mechanism in living cellular environments.

In silico Design of Linear DNA for Robust Cell-Free Gene Expression.

Cell-free gene expression systems with linear DNA expression templates (LDETs) have been widely applied in artificial cells, biochips, and high-throughput screening. However, due to the degradation caused by native nucleases in cell extracts, the transcription with linear DNA templates is weak, thereby resulting in low protein expression level, which greatly limits the development of cell-free systems using linear DNA templates. In this study, the protective sequences for stabilizing linear DNA and the transcribed mRNAs were rationally designed according to nucleases’ action mechanism, whose effectiveness was evaluated through computer simulation and cell-free gene expression. The cell-free experiment results indicated that, with the combined protection of designed sequence and GamS protein, the protein expression of LDET-based cell-free systems could reach the same level as plasmid-based cell-free systems. This study would potentially promote the development of the LDET-based cell-free gene expression system for broader applications.

Improving cell-free glycoprotein synthesis by characterizing and enriching native membrane vesicles

Cell-free gene expression (CFE) systems from crude cellular extracts have attracted much attention for biomanufacturing and synthetic biology. However, activating membrane-dependent functionality of cell-derived vesicles in bacterial CFE systems has been limited. Here, we address this limitation by characterizing native membrane vesicles in Escherichia coli-based CFE extracts and describing methods to enrich vesicles with heterologous, membrane-bound machinery. As a model, we focus on bacterial glycoengineering. We first use multiple, orthogonal techniques to characterize vesicles and show how extract processing methods can be used to increase concentrations of membrane vesicles in CFE systems. Then, we show that extracts enriched in vesicle number also display enhanced concentrations of heterologous membrane protein cargo. Finally, we apply our methods to enrich membrane-bound oligosaccharyltransferases and lipid-linked oligosaccharides for improving cell-free N-linked and O-linked glycoprotein synthesis. We anticipate that these methods will facilitate on-demand glycoprotein production and enable new CFE systems with membrane-associated activities.