PACRAT: Pathogen detection with aptamer-detected cascaded recombinase polymerase amplification-in vitro transcription.

The SARS-CoV-2 pandemic underscored the need for early, rapid, and widespread pathogen detection tests that are readily accessible. Many existing rapid isothermal detection methods employ the recombinase polymerase amplification (RPA), which exhibits PCR-like sensitivity, specificity, and even higher speed. However, coupling RPA to other enzymatic reactions has proven difficult. For the first time, we demonstrate that with tuning of buffer conditions and optimization of reagent concentrations, RPA can be cascaded into an in vitro transcription reaction, enabling detection using fluorescent aptamers in a one-pot reaction. We show that this reaction, which we term PACRAT (Pathogen detection with Aptamer-detected Cascaded Recombinase polymerase Amplification-in vitro Transcription) can be used to detect SARS-CoV-2 with single-copy detection limits and 10-minute detection times. Further demonstrating the utility of our one-pot, cascaded amplification system, we show PACRAT can be employed for multiplexed detection of the pathogens SARS-CoV-2 and E. coli, along with multiplexed detection of two variants of SARS-CoV-2.

Building Synthetic Cells─From the Technology Infrastructure to Cellular Entities.

The de novo construction of a living organism is a compelling vision. Despite the astonishing technologies developed to modify living cells, building a functioning cell "from scratch" has yet to be accomplished. The pursuit of this goal alone has─and will─yield scientific insights affecting fields as diverse as cell biology, biotechnology, medicine, and astrobiology. Multiple approaches have aimed to create biochemical systems manifesting common characteristics of life, such as compartmentalization, metabolism, and replication and the derived features, evolution, responsiveness to stimuli, and directed movement. Significant achievements in synthesizing each of these criteria have been made, individually and in limited combinations. Here, we review these efforts, distinguish different approaches, and highlight bottlenecks in the current research. We look ahead at what work remains to be accomplished and propose a "roadmap" with key milestones to achieve the vision of building cells from molecular parts.

Preparing for the future of precision medicine: synthetic cell drug regulation.

Synthetic cells are a novel class of cell-like bioreactors, offering the potential for unique advancements in synthetic biology and biomedicine. To realize the potential of those technologies, synthetic cell-based drugs need to go through the drug approval pipeline. Here, we discussed several regulatory challenges, both unique to synthetic cells, as well as challenges typical for any new biomedical technology. Overcoming those difficulties could bring transformative therapies to the market and will create a path to the development and approval of cutting-edge synthetic biology therapies. Graphical Abstract.

Cell-Free Expressed Membraneless Organelles Inhibit Translation in Synthetic Cells.

Compartments within living cells create specialized microenvironments, allowing multiple reactions to be carried out simultaneously and efficiently. While some organelles are bound by a lipid bilayer, others are formed by liquid-liquid phase separation such as P-granules and nucleoli. Synthetic minimal cells are widely used to study many natural processes, including organelle formation. In this work, synthetic cells expressing artificial membrane-less organelles that inhibit translation are described. RGG-GFP-RGG, a phase-separating protein derived from Caenorhabditis elegans P-granules, is expressed by cell-free transcription and translation, forming artificial membraneless organelles that can sequester RNA and reduce protein expression in synthetic cells. The introduction of artificial membrane-less organelles creates complex microenvironments within the synthetic cell cytoplasm and functions as a tool to inhibit protein expression in synthetic cells. The engineering of compartments within synthetic cells furthers the understanding of the evolution and function of natural organelles and facilitates the creation of more complex and multifaceted synthetic lifelike systems.

Controlled exchange of protein and nucleic acid signals from and between synthetic minimal cells.

Synthetic minimal cells are a class of bioreactors that have some, but not all, functions of live cells. Here, we report a critical step toward the development of a bottom-up minimal cell: cellular export of functional protein and RNA products. We used cell-penetrating peptide tags to translocate payloads across a synthetic cell vesicle membrane. We demonstrated efficient transport of active enzymes and transport of nucleic acid payloads by RNA-binding proteins. We investigated influence of a concentration gradient alongside other factors on the efficiency of the translocation, and we show a method to increase product accumulation in one location. We demonstrate the use of this technology to engineer molecular communication between different populations of synthetic cells, to exchange protein and nucleic acid signals. The synthetic minimal cell production and export of proteins or nucleic acids allows experimental designs that approach the complexity and relevancy of natural biological systems. A record of this paper's transparent peer review process is included in the supplemental information.

Sequential gentle hydration increases encapsulation in model protocells.

Small, spherical vesicles are a widely used chassis for the formation of model protocells and investigating the beginning of compartmentalized evolution. Various methods exist for their preparation, with one of the most common approaches being gentle hydration, where thin layers of lipids are hydrated with aqueous solutions and gently agitated to form vesicles. An important benefit to gentle hydration is that the method produces vesicles without introducing any organic contaminants, such as mineral oil, into the lipid bilayer. However, compared to other methods of liposome formation, gentle hydration is much less efficient at encapsulating aqueous cargo. Improving the encapsulation efficiency of gentle hydration would be of broad use for medicine, biotechnology, and protocell research. Here, we describe a method of sequentially hydrating lipid thin films to increase encapsulation efficiency. We demonstrate that sequential gentle hydration significantly improves encapsulation of water-soluble cargo compared to the traditional method, and that this improved efficiency is dependent on buffer composition. Similarly, we also demonstrate how this method can be used to increase concentrations of oleic acid, a fatty acid commonly used in origins of life research, to improve the formation of vesicles in aqueous buffer.

Cell-Free Expression System Derived from a Near-Minimal Synthetic Bacterium.

Cell-free expression (CFE) systems are fundamental to reconstituting metabolic pathways in vitro toward the construction of a synthetic cell. Although an Escherichia coli-based CFE system is well-established, simpler model organisms are necessary to understand the principles behind life-like behavior. Here, we report the successful creation of a CFE system derived from JCVI-syn3A (Syn3A), the minimal synthetic bacterium. Previously, high ribonuclease activity in Syn3A lysates impeded the establishment of functional CFE systems. Now, we describe how an unusual cell lysis method (nitrogen decompression) yielded Syn3A lysates with reduced ribonuclease activity that supported in vitro expression. To improve the protein yields in the Syn3A CFE system, we optimized the Syn3A CFE reaction mixture using an active machine learning tool. The optimized reaction mixture improved the CFE 3.2-fold compared to the preoptimized condition. This is the first report of a functional CFE system derived from a minimal synthetic bacterium, enabling further advances in bottom-up synthetic biology.

A gene expression control technology for cell-free systems and synthetic cells via targeted gene silencing and transfection.

Synthetic cells, expressing proteins using cell-free transcription-translation (TXTL), is a technology utilized for a variety of applications, such as investigating natural gene pathways, metabolic engineering, drug development or bioinformatics. For all these purposes, the ability to precisely control gene expression is essential. Various strategies to control gene expression in TXTL have been developed; however, further advancements on gene-specific and straightforward regulation methods are still needed. Here, we present a method of control of gene expression in TXTL using a "silencing oligo": a short oligonucleotide, designed with a particular secondary structure, that binds to the target messenger RNA. We demonstrated that silencing oligo inhibits protein expression in TXTL in a sequence-dependent manner. We showed that silencing oligo activity is associated with RNase H activity in bacterial TXTL. To complete the gene expression control toolbox for synthetic cells, we also engineered a first transfection system. We demonstrated the transfection of various payloads, enabling the introduction of RNA and DNA of different lengths to synthetic cell liposomes. Finally, we combined the silencing oligo and the transfection technologies, demonstrating control of gene expression by transfecting silencing oligo into synthetic minimal cells.

New Aequorea Fluorescent Proteins for Cell-Free Bioengineering.

Recently, a new subset of fluorescent proteins has been identified from the Aequorea species of jellyfish. These fluorescent proteins were characterized in vivo; however, there has not been validation of these proteins within cell-free systems. Cell-free systems and technology development is a rapidly expanding field, encompassing foundational research, synthetic cells, bioengineering, biomanufacturing, and drug development. Cell-free systems rely heavily on fluorescent proteins as reporters. Here we characterize and validate this new set of Aequorea proteins for use in a variety of cell-free and synthetic cell expression platforms.

Trumpet is an operating system for simple and robust cell-free biocomputing.

Biological computation is becoming a viable and fast-growing alternative to traditional electronic computing. Here we present a biocomputing technology called Trumpet: Transcriptional RNA Universal Multi-Purpose GatE PlaTform. Trumpet combines the simplicity and robustness of the simplest in vitro biocomputing methods, adding signal amplification and programmability, while avoiding common shortcomings of live cell-based biocomputing solutions. We have demonstrated the use of Trumpet to build all universal Boolean logic gates. We have also built a web-based platform for designing Trumpet gates and created a primitive processor by networking several gates as a proof-of-principle for future development. The Trumpet offers a change of paradigm in biocomputing, providing an efficient and easily programmable biological logic gate operating system.

Cell-free expressed membraneless organelles sequester RNA in synthetic cells.

Compartments within living cells create specialized microenvironments, allowing for multiple reactions to be carried out simultaneously and efficiently. While some organelles are bound by a lipid bilayer, others are formed by liquid-liquid phase separation, such as P-granules and nucleoli. Synthetic minimal cells have been widely used to study many natural processes, including organelle formation. Here we describe a synthetic cell expressing RGG-GFP-RGG, a phase-separating protein derived from LAF-1 RGG domains, to form artificial membraneless organelles that can sequester RNA and reduce protein expression. We create complex microenvironments within synthetic cell cytoplasm and introduce a tool to modulate protein expression in synthetic cells. Engineering of compartments within synthetic cells furthers understanding of evolution and function of natural organelles, as well as it facilitates the creation of more complex and multifaceted synthetic life-like systems.

Parasites, Infections, and Inoculation in Synthetic Minimal Cells.

Synthetic minimal cells provide a controllable and engineerable model for biological processes. While much simpler than any live natural cell, synthetic cells offer a chassis for investigating the chemical foundations of key biological processes. Herein, we show a synthetic cell system with host cells, interacting with parasites and undergoing infections of varying severity. We demonstrate how the host can be engineered to resist infection, we investigate the metabolic cost of carrying resistance, and we show an inoculation that immunizes the host against pathogens. Our work expands the synthetic cell engineering toolbox by demonstrating host-pathogen interactions and mechanisms for acquiring immunity. This brings synthetic cell systems one step closer to providing a comprehensive model of complex, natural life.

T7Max transcription system.

BACKGROUND: Efficient cell-free protein expression from linear DNA templates has remained a challenge primarily due to template degradation. In addition, the yields of transcription in cell-free systems lag behind transcriptional efficiency of live cells. Most commonly used in vitro translation systems utilize T7 RNA polymerase, which is also the enzyme included in many commercial kits. RESULTS: Here we present characterization of a variant of T7 RNA polymerase promoter that acts to significantly increase the yields of gene expression within in vitro systems. We have demonstrated that T7Max increases the yield of translation in many types of commonly used in vitro protein expression systems. We also demonstrated increased protein expression yields from linear templates, allowing the use of T7Max driven expression from linear templates. CONCLUSIONS: The modified promoter, termed T7Max, recruits standard T7 RNA polymerase, so no protein engineering is needed to take advantage of this method. This technique could be used with any T7 RNA polymerase- based in vitro protein expression system.

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 Ribosomes to Alleviate Abiotic Stress in Plants: A Perspective.

As the centerpiece of the biomass production process, ribosome activity is highly coordinated with environmental cues. Findings revealing ribosome subgroups responsive to adverse conditions suggest this tight coordination may be grounded in the induction of variant ribosome compositions and the differential translation outcomes they might produce. In this perspective, we go through the literature linking ribosome heterogeneity to plants' abiotic stress response. Once unraveled, this crosstalk may serve as the foundation of novel strategies to custom cultivars tolerant to challenging environments without the yield penalty.

Traditional protocols and optimization methods lead to absent expression in a mycoplasma cell-free gene expression platform.

Cell-free expression (CFE) systems are one of the main platforms for building synthetic cells. A major drawback is the orthogonality of cell-free systems across species. To generate a CFE system compatible with recently established minimal cell constructs, we attempted to optimize a Mycoplasma bacterium-based CFE system using lysates of the genome-minimized cell JCVI-syn3A (Syn3A) and its close phylogenetic relative Mycoplasma capricolum (Mcap). To produce mycoplasma-derived crude lysates, we systematically tested methods commonly used for bacteria, based on the S30 protocol of Escherichia coli. Unexpectedly, after numerous attempts to optimize lysate production methods or composition of feeding buffer, none of the Mcap or Syn3A lysates supported cell-free gene expression. Only modest levels of in vitro transcription of RNA aptamers were observed. While our experimental systems were intended to perform transcription and translation, our assays focused on RNA. Further investigations identified persistently high ribonuclease (RNase) activity in all lysates, despite removal of recognizable nucleases from the respective genomes and attempts to inhibit nuclease activities in assorted CFE preparations. An alternative method using digitonin to permeabilize the mycoplasma cell membrane produced a lysate with diminished RNase activity yet still was unable to support cell-free gene expression. We found that intact mycoplasma cells poisoned E. coli cell-free extracts by degrading ribosomal RNAs, indicating that the mycoplasma cells, even the minimal cell, have a surface-associated RNase activity. However, it is not clear which gene encodes the RNase. This work summarizes attempts to produce mycoplasma-based CFE and serves as a cautionary tale for researchers entering this field. Graphical Abstract.

Expanding luciferase reporter systems for cell-free protein expression.

Luciferases are often used as a sensitive, versatile reporter in cell-free transcription-translation (TXTL) systems, for research and practical applications such as engineering genetic parts, validating genetic circuits, and biosensor outputs. Currently, only two luciferases (Firefly and Renilla) are commonly used without substrate cross-talk. Here we demonstrate the expansion of the cell-free luciferase reporter system, with two orthogonal luciferase reporters: N. nambi luciferase (Luz) and LuxAB. These luciferases do not have cross-reactivity with the Firefly and Renilla substrates. We also demonstrate a substrate regeneration pathway for one of the new luciferases, enabling long-term time courses of protein expression monitoring in the cell-free system. Furthermore, we reduced the number of genes required in TXTL expression, by engineering a cell extract containing part of the luciferase enzymes. Our findings lead to an expanded platform with multiple orthogonal luminescence translation readouts for in vitro protein expression.

Akaby-Cell-free protein expression system for linear templates.

Cell-free protein expression is increasingly becoming popular for biotechnology, biomedical and research applications. Among cell-free systems, the most popular one is based on Escherichia coli (E. coli). Endogenous nucleases in E. coli cell-free transcription-translation (TXTL) degrade the free ends of DNA, resulting in inefficient protein expression from linear DNA templates. RecBCD is a nuclease complex that plays a major role in nuclease activity in E. coli, with the RecB subunit possessing the actual nuclease activity. We created a RecB knockout of an E. coli strain optimized for cell-free expression. We named this new strain Akaby. We demonstrated that Akaby TXTL successfully reduced linear DNA degradations, rescuing the protein expression efficiency from the linear DNA templates. The practicality of Akaby for TXTL is an efficient, simple alternative for linear template expression in cell-free reactions. We also use this work as a model protocol for modifying the TXTL source E. coli strain, enabling the creation of TXTL systems with other custom modifications.