Synthetic Genes For Dynamic Regulation Of DNA-Based Receptors.

We present a strategy to control dynamically the loading and release of molecular ligands from synthetic nucleic acid receptors using in vitro transcription. We demonstrate this by engineering three model synthetic DNA-based receptors: a triplex-forming DNA complex, an ATP-binding aptamer, and a hairpin strand, whose ability to bind their specific ligands can be cotranscriptionally regulated (activated or inhibited) through specific RNA molecules produced by rationally designed synthetic genes. The kinetics of our DNA sensors and their genetically generated inputs can be captured using differential equation models, corroborating the predictability of the approach used. This approach shows that highly programmable nucleic acid receptors can be controlled with molecular instructions provided by dynamic transcriptional systems, illustrating their promise in the context of coupling DNA nanotechnology with biological signaling.

A dynamic kinetic model captures cell-free metabolism for improved butanol production.

Cell-free systems are useful tools for prototyping metabolic pathways and optimizing the production of various bioproducts. Mechanistically-based kinetic models are uniquely suited to analyze dynamic experimental data collected from cell-free systems and provide vital qualitative insight. However, to date, dynamic kinetic models have not been applied with rigorous biological constraints or trained on adequate experimental data to the degree that they would give high confidence in predictions and broadly demonstrate the potential for widespread use of such kinetic models. In this work, we construct a large-scale dynamic model of cell-free metabolism with the goal of understanding and optimizing butanol production in a cell-free system. Using a combination of parameterization methods, the resultant model captures experimental metabolite measurements across two experimental conditions for nine metabolites at timepoints between 0 and 24 h. We present analysis of the model predictions, provide recommendations for butanol optimization, and identify the aldehyde/alcohol dehydrogenase as the primary bottleneck in butanol production. Sensitivity analysis further reveals the extent to which various parameters are constrained, and our approach for probing valid parameter ranges can be applied to other modeling efforts.

Emerging regulatory challenges of next-generation synthetic biology.

Cell-free synthetic biology is a rapidly developing biotechnology with the potential to solve the world's biggest problems; however, this promise also has implications for global biosecurity and biosafety. Given the current situation of COVID-19 and its economic impact, capitalizing on the potential of cell-free synthetic biology from an economic, biosafety, and biosecurity perspective contributes to our preparedness for the next pandemic, and urges the development of appropriate policies and regulations, together with the necessary mitigation technologies. Proactive involvement from scientists is necessary to avoid misconceptions and assist in the policymaking process.

Total in vitro biosynthesis of the nonribosomal macrolactone peptide valinomycin.

Natural products are important because of their significant pharmaceutical properties such as antiviral, antimicrobial, and anticancer activity. Recent breakthroughs in DNA sequencing reveal that a great number of cryptic natural product biosynthetic gene clusters are encoded in microbial genomes, for example, those of Streptomyces species. However, it is still challenging to access compounds from these clusters because many source organisms are uncultivable or the genes are silent during laboratory cultivation. To address this challenge, we develop an efficient cell-free platform for the rapid, in vitro total biosynthesis of the nonribosomal peptide valinomycin as a model. We achieve this goal in two ways. First, we used a cell-free protein synthesis (CFPS) system to express the entire valinomycin biosynthetic gene cluster (>19 kb) in a single-pot reaction, giving rise to approximately 37 µg/L of valinomycin after optimization. Second, we coupled CFPS with cell-free metabolic engineering system by mixing two enzyme-enriched cell lysates to perform a two-stage biosynthesis. This strategy improved valinomycin production ~5000-fold to nearly 30 mg/L. We expect that cell-free biosynthetic systems will provide a new avenue to express, discover, and characterize natural product gene clusters of interest in vitro.

Branched DNA Architectures Produced by PCR-Based Assembly as Gene Compartments for Cell-Free Gene-Expression Reactions.

The physical distance between genes plays important roles in controlling gene expression reactions in vivo. Herein, we report the design and synthesis of a branched gene architecture in which three transcription units are integrated into one framework through assembly based on the polymerase chain reaction (PCR), together with the exploitation of these constructs as "gene compartments" for cell-free gene expression reactions, probing the impact of this physical environment on gene transcription and translation. We find that the branched gene system enhances gene expression yields, in particular at low concentrations of DNA and RNA polymerase (RNAP); furthermore, in a crowded microenvironment that mimics the intracellular microenvironment, gene expression from branched genes maintains a relatively high level. We propose that the branched gene assembly forms a membrane-free gene compartment that resembles the nucleoid of prokaryotes and enables RNAP to shuttle more efficiently between neighboring transcription units, thus enhancing gene expression efficiency. Our branched DNA architecture provides a valuable platform for studying the influence of "cellular" physical environments on biochemical reactions in simplified cell-free systems.

Integrative biology of native cell extracts: a new era for structural characterization of life processes.

Advances in electron microscopy have provided unprecedented access to the structural characterization of large, flexible and heterogeneous complexes. Until recently, cryo-electron microscopy (cryo-EM) has been applied to understand molecular organization in either highly purified, isolated biomolecules or in situ. An emerging field is developing, bridging the gap between the two approaches, and focuses on studying molecular organization in native cell extracts. This field has demonstrated its potential by resolving the structure of fungal fatty acid synthase (FAS) at 4.7 Å [Fourier shell correlation (FSC) = 0.143]; FAS was not only less than 50% enriched, but also retained higher-order binders, previously unknown. Although controversial in the sense that the lysis step might introduce artifacts, cell extracts preserve aspects of cellular function. In addition, cell extracts are accessible, besides cryo-EM, to modern proteomic methods, chemical cross-linking, network biology and biophysical modeling. We expect that automation in imaging cell extracts, along with the integration of molecular/cell biology approaches, will provide remarkable achievements in the study of closer-to-life biomolecular states of pronounced biotechnological and medical importance. Such steps will, eventually, bring us a step closer to the biophysical description of cellular processes in an integrative, holistic approach.

Characterizing and prototyping genetic networks with cell-free transcription-translation reactions.

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.

In vitro synthesis of the E. coli Sec translocon from DNA.

Difficulties in constructing complex lipid/protein membranes have severely limited the development of functional artificial cells endowed with vital membrane-related functions. The Sec translocon membrane channel, which mediates the insertion of membrane proteins into the plasma membrane, was constructed in the membrane of lipid vesicles through in vitro expression of its component proteins. The components of the Sec translocon were synthesized from their respective genes in the presence of liposomes, thereby bringing about a functional complex. The synthesized E. coli Sec translocon mediated the membrane translocation of single- and multi-span membrane proteins. The successful translocation of a functional peptidase into the liposome lumen further confirmed the proper insertion of the translocon complex. Our results demonstrate the feasible construction of artificial cells, the membranes of which can be functionalized by directly decoding genetic information into membrane functions.

Programmable Synthesis of Biobased Materials Using Cell-Free Systems.

Motivated by the intricate mechanisms underlying biomolecule syntheses in cells that chemistry is currently unable to mimic, researchers have harnessed biological systems for manufacturing novel materials. Cell-free systems (CFSs) utilizing the bioactivity of transcriptional and translational machineries in vitro are excellent tools that allow supplementation of exogenous materials for production of innovative materials beyond the capability of natural biological systems. Herein, recent studies that have advanced the ability to expand the scope of biobased materials using CFS are summarized and approaches enabling the production of high-value materials, prototyping of genetic parts and modules, and biofunctionalization are discussed. By extending the reach of chemical and enzymatic reactions complementary to cellular materials, CFSs provide new opportunities at the interface of materials science and synthetic biology.

Multiscale design of cell-free biologically active architectural structures.

Cell-free protein expression systems are here combined with 3D-printed structures to study the challenges and opportunities as biofabrication enters the spaces of architecture and design. Harnessing large-scale additive manufacturing of biological materials, we examined the addition of cell-free protein expression systems ("TXTL" i.e., biological transcription-translation machinery without the use of living cells) to printed structures. This allowed us to consider programmable, living-like, responsive systems for product design and indoor architectural applications. This emergent, pluripotent technology offers exciting potential in support of health, resource optimization, and reduction of energy use in the built environment, setting a new path to interactivity with mechanical, optical, and (bio) chemical properties throughout structures. We propose a roadmap towards creating healthier, functional and more durable systems by deploying a multiscale platform containing biologically-active components encapsulated within biopolymer lattices operating at three design scales: (i) supporting cell-free protein expression in a biopolymer matrix (microscale), (ii) varying material properties of porosity and strength within two-dimensional lattices to support biological and structural functions (mesoscale), and (iii) obtaining folded indoor surfaces that are structurally sound at the meter scale and biologically active (we label that regime macroscale). We embedded commercially available cell-free protein expression systems within silk fibroin and sodium alginate biopolymer matrices and used green fluorescent protein as the reporter to confirm their compatibility. We demonstrate mechanical attachment of freeze-dried bioactive pellets into printed foldable fibrous biopolymer lattices showing the first steps towards modular multiscale fabrication of large structures with biologically active zones. Our results discuss challenges to experimental setup affecting expression levels and show the potential of robust cell-free protein-expressing biosites within custom-printed structures at scales relevant to everyday consumer products and human habitats.

Plasmid Crosstalk in Cell-Free Expression Systems.

Although cell-free protein expression has been widely used for the synthesis of single proteins, cell-free synthetic biology has rapidly expanded to new, more complex applications. One such application is the prototyping or implementation of complex genetic networks involving the expression of multiple proteins at precise ratios, often from different plasmids. However, expression of multiple proteins from multiple plasmids may inadvertently result in unexpected, off-target changes to the levels of the proteins being expressed, a phenomenon termed plasmid crosstalk. Here, we show that the effects of plasmid crosstalk─even at the qualitative level of increases vs decreases in protein expression─depend on the concentration of plasmids in the reaction and the type of transcriptional machinery involved in the expression. This crosstalk can have a significant impact on genetic circuitry function and even interpretation of simple experimental results and thus should be taken into consideration during the development of cell-free applications.

Biochemistry of Aminoacyl tRNA Synthetase and tRNAs and Their Engineering for Cell-Free and Synthetic Cell Applications.

Cell-free biology is increasingly utilized for engineering biological systems, incorporating novel functionality, and circumventing many of the complications associated with cells. The central dogma describes the information flow in biology consisting of transcription and translation steps to decode genetic information. Aminoacyl tRNA synthetases (AARSs) and tRNAs are key components involved in translation and thus protein synthesis. This review provides information on AARSs and tRNA biochemistry, their role in the translation process, summarizes progress in cell-free engineering of tRNAs and AARSs, and discusses prospects and challenges lying ahead in cell-free engineering.

A combined strategy for the overproduction of complex ergot alkaloid agroclavine.

Microbial cell factories (MCFs) and cell-free systems (CFSs) are generally considered as two unrelated approaches for the biosynthesis of biomolecules. In the current study, two systems were combined together for the overproduction of agroclavine (AC), a structurally complex ergot alkaloid. The whole biosynthetic pathway for AC was split into the early pathway and the late pathway at the point of the FAD-linked oxidoreductase EasE, which was reconstituted in an MCF (Aspergillus nidulans) and a four-enzyme CFS, respectively. The final titer of AC of this combined system is 1209 mg/L, which is the highest one that has been reported so far, to the best of our knowledge. The development of such a combined route could potentially avoid the limitations of both MCF and CFS systems, and boost the production of complex ergot alkaloids with polycyclic ring systems.

Cell-Free Systems Enable the Production of AB5 Toxins for Diagnostic Applications.

Cell-free protein synthesis (CFPS) represents a versatile key technology for the production of toxic proteins. As a cell lysate, rather than viable cells, is used, the toxic effects on the host organism can be circumvented. The open nature of cell-free systems allows for the addition of supplements affecting protein concentration and folding. Here, we present the cell-free synthesis and functional characterization of two AB5 toxins, namely the cholera toxin (Ctx) and the heat-labile enterotoxin (LT), using two eukaryotic cell-free systems based on Chinese hamster ovary (CHO) and Spodoptera frugiperda (Sf21) cells. Through an iterative optimization procedure, the synthesis of the individual AB5 toxins was established, and the formation of multimeric structures could be shown by autoradiography. A functional analysis was performed using cell-based assays, thereby demonstrating that the LT complex induced the characteristic cell elongation of target cells after 24 h. The LT complex induced cell death at higher concentrations, starting at an initial concentration of 5 nM. The initial toxic effects of the Ctx multimer could already be detected at 4 nM. The detection and characterization of such AB5 toxins is of utmost importance, and the monitoring of intracellular trafficking facilitates the further identification of the mechanism of action of these toxins. We showed that the B-subunit of LT (LTB) could be fluorescently labeled using an LTB-Strep fusion protein, which is a proof-of-concept for future Trojan horse applications. Further, we performed a mutational analysis of the CtxA subunit as its template was modified, and an amber stop codon was inserted into CtxA's active site. Subsequently, a non-canonical amino acid was site-specifically incorporated using bio-orthogonal systems. Finally, a fluorescently labeled CtxA protein was produced using copper-catalyzed click reactions as well as a Staudinger ligation. As expected, the modified Ctx multimer no longer induced toxic effects. In our study, we showed that CFPS could be used to study the active centers of toxins by inserting mutations. Additionally, this methodology can be applied for the design of Trojan horses and targeted toxins, as well as enabling the intracellular trafficking of toxins as a prerequisite for the analysis of the toxin's mechanism of action.

DNA Input Classification by a Riboregulator-Based Cell-Free Perceptron.

The ability to recognize molecular patterns is essential for the continued survival of biological organisms, allowing them to sense and respond to their immediate environment. The design of synthetic gene-based classifiers has been explored previously; however, prior strategies have focused primarily on DNA strand-displacement reactions. Here, we present a synthetic in vitro transcription and translation (TXTL)-based perceptron consisting of a weighted sum operation (WSO) coupled to a downstream thresholding function. We demonstrate the application of toehold switch riboregulators to construct a TXTL-based WSO circuit that converts DNA inputs into a GFP output, the concentration of which correlates to the input pattern and the corresponding weights. We exploit the modular nature of the WSO circuit by changing the output protein to the Escherichia coli σ28-factor, facilitating the coupling of the WSO output to a downstream reporter network. The subsequent introduction of a σ28 inhibitor enabled thresholding of the WSO output such that the expression of the downstream reporter protein occurs only when the produced σ28 exceeds this threshold. In this manner, we demonstrate a genetically implemented perceptron capable of binary classification, i.e., the expression of a single output protein only when the desired minimum number of inputs is exceeded.

Ribosomal Protein S1 Improves the Protein Yield of an In Vitro Reconstituted Cell-Free Translation System.

Cell-free expression systems, such as the highly purified in vitro reconstituted PURExpress, hold great promise for engineering biological and life-similar systems by exploiting the ability to perform transcription and translation (TX-TL) outside the constraints of living cells, including for example the expression of recombinant proteins that are difficult or toxic to produce in vivo. Currently, the scope of applications utilizing purified reconstituted TX-TL systems is challenged by poor system performance resulting from limitations in the ribosome and ribosome-associated processes, leading to low protein yields. Because of the transient nature of ribosomal protein S1's interaction with the ribosome, the ribosomes in a reconstituted translation system contain varying amounts of S1, potentially impacting translation initiation and the recruitment of mRNA to the 30S ribosomal subunit. Here we report that by being supplemented with purified recombinant S1 the protein yields can be doubled when using a commercial in vitro reconstituted TX-TL system. We hypothesize that the addition of S1 increases the fraction of functional ribosomes available in the in vitro reaction. Improved yields are shown for different reporter proteins (EYFP, sfGFP, and mRFP) and in different 5'UTR contexts (strong, medium, and weak ribosome binding site), including the expression of a highly structured RNA (PSIV IRES). Overall, fine-tuning the S1 concentration provides a previously overlooked venue to increase protein yield by targeting ribosome composition and translation initiation.

Differentially Optimized Cell-Free Buffer Enables Robust Expression from Unprotected Linear DNA in Exonuclease-Deficient Extracts.

The use of linear DNA templates in cell-free systems promises to accelerate the prototyping and engineering of synthetic gene circuits. A key challenge is that linear templates are rapidly degraded by exonucleases present in cell extracts. Current approaches tackle the problem by adding exonuclease inhibitors and DNA-binding proteins to protect the linear DNA, requiring additional time- and resource-intensive steps. Here, we delete the recBCD exonuclease gene cluster from the Escherichia coli BL21 genome. We show that the resulting cell-free systems, with buffers optimized specifically for linear DNA, enable near-plasmid levels of expression from σ70 promoters in linear DNA templates without employing additional protection strategies. When using linear or plasmid DNA templates at the buffer calibration step, the optimal potassium glutamate concentrations obtained when using linear DNA were consistently lower than those obtained when using plasmid DNA for the same extract. We demonstrate the robustness of the exonuclease deficient extracts across seven different batches and a wide range of experimental conditions across two different laboratories. Finally, we illustrate the use of the ΔrecBCD extracts for two applications: toehold switch characterization and enzyme screening. Our work provides a simple, efficient, and cost-effective solution for using linear DNA templates in cell-free systems and highlights the importance of specifically tailoring buffer composition for the final experimental setup. Our data also suggest that similar exonuclease deletion strategies can be applied to other species suitable for cell-free synthetic biology.

The Potential of Eukaryotic Cell-Free Systems as a Rapid Response to Novel Zoonotic Pathogens: Analysis of SARS-CoV-2 Viral Proteins.

The ongoing pandemic caused by the novel coronavirus (SARS-CoV-2) has led to more than 445 million infections and the underlying disease, COVID-19, resulted in more than 6 million deaths worldwide. The scientific world is already predicting future zoonotic diseases. Hence, rapid response systems are needed to tackle future epidemics and pandemics. Here, we present the use of eukaryotic cell-free systems for the rapid response to novel zoonotic diseases represented by SARS-CoV-2. Non-structural, structural and accessory proteins encoded by SARS-CoV-2 were synthesized by cell-free protein synthesis in a fast and efficient manner. The inhibitory effect of the non-structural protein 1 on protein synthesis could be shown in vitro. Structural proteins were quantitatively detected by commercial antibodies, therefore facilitating cell-free systems for the validation of available antibodies. The cytotoxic envelope protein was characterized in electrophysiological planar lipid bilayer measurements. Hence, our study demonstrates the potential of eukaryotic cell-free systems as a rapid response mechanism for the synthesis, functional characterization and antibody validation against a viral pathogen.

Best Practices for DNA Template Preparation Toward Improved Reproducibility in Cell-Free Protein Production.

Performance variability is a common challenge in cell-free protein production and hinders a wider adoption of these systems for both research and biomanufacturing. While the inherent stochasticity and complexity of biology likely contributes to variability, other systematic factors may also play a role, including the source and preparation of the cell extract, the composition of the supplemental reaction buffer, the facility at which experiments are conducted, and the human operator (Cole et al. ACS Synth Biol 8:2080-2091, 2019). Variability in protein production could also arise from differences in the DNA template-specifically the amount of functional DNA added to a cell-free reaction and the quality of the DNA preparation in terms of contaminants and strand breakage. Here, we present protocols and suggest best practices optimized for DNA template preparation and quantitation for cell-free systems toward reducing variability in cell-free protein production.

Detection of Norovirus Using Paper-Based Cell-Free Systems.

Norovirus infections are the leading cause of foodborne illness and human gastroenteritis, afflicting hundreds of millions of people each year. Molecular assays with the capacity to detect norovirus without expensive equipment and with high sensitivity and specificity represent useful tools to track and contain future outbreaks. Here we describe how norovirus can be detected in low-cost paper-based cell-free reactions. These assays combine freeze-dried, thermostable cell-free transcription-translation reactions with toehold switch riboregulators designed to target the norovirus genome, enabling convenient colorimetric assay readouts. Coupling cell-free reactions with synbody-based viral enrichment and isothermal amplification enables detection of norovirus from clinical samples down to concentrations as low as 270 zM. These diagnostic tests are promising assays for confronting norovirus outbreaks and can be adapted to a variety of other human pathogens.