ConspectusCells are highly advanced microreactors that form the basis of all life. Their fascinating complexity has inspired scientists to create analogs from synthetic and natural components using a bottom-up approach. The ultimate goal here is to assemble a fully man-made cell that displays functionality and adaptivity as advanced as that found in nature, which will not only provide insight into the fundamental processes in natural cells but also pave the way for new applications of such artificial cells.In this Account, we highlight our recent work and that of others on the construction of artificial cells. First, we will introduce the key features that characterize a living system; next, we will discuss how these have been imitated in artificial cells. First, compartmentalization is crucial to separate the inner chemical milieu from the external environment. Current state-of-the-art artificial cells comprise subcompartments to mimic the hierarchical architecture of eukaryotic cells and tissue. Further...

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Artificial cells: synthetic compartments with life-like functionality and adaptivity

Cell-free biology is the activation of biological processes without the use of intact living cells. It has been used for more than 50 years across the life sciences as a foundational research tool, but a recent technical renaissance has facilitated high-yielding (grams of protein per litre), cell-free gene expression systems from model bacteria, the development of cell-free platforms from non-model organisms and multiplexed strategies for rapidly assessing biological design. These advances provide exciting opportunities to profoundly transform synthetic biology by enabling new approaches to the model-driven design of synthetic gene networks, the fast and portable sensing of compounds, on-demand biomanufacturing, building cells from the bottom up, and next-generation educational kits. Cell-free gene expression systems have long been used to address fundamental research questions. Now, owing to technological advances, these systems are finding wider applications in the field of synthetic biology, including in biosensing, biomanufacturing, education and the design of gene networks.

Cell-free gene expression: an expanded repertoire of applications.

Portable, On-Demand Biomolecular Manufacturing

Genetic circuits and reaction cascades are of great importance for synthetic biology, biochemistry and bioengineering. An open question is how to maximize the modularity of their design to enable the integration of different reaction networks and to optimize their scalability and flexibility. One option is encapsulation within liposomes, which enables chemical reactions to proceed in well-isolated environments. Here we adapt liposome encapsulation to enable the modular, controlled compartmentalization of genetic circuits and cascades. We demonstrate that it is possible to engineer genetic circuit-containing synthetic minimal cells (synells) to contain multiple-part genetic cascades, and that these cascades can be controlled by external signals as well as inter-liposomal communication without crosstalk. We also show that liposomes that contain different cascades can be fused in a controlled way so that the products of incompatible reactions can be brought together. Synells thus enable a more modular creation of synthetic biology cascades, an essential step towards their ultimate programmability. Genetic circuits are important for synthetic biology, biochemistry and bioengineering. Now, the encapsulation of genetic circuits into liposomes has been shown to enable a more modular design, the selective isolation of reactions from the environment and from each other, and the hierarchical assembly of reaction products.

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Engineering genetic circuit interactions within and between synthetic minimal cells

INTRODUCTION Embedded controllers enable complex functionality in man-made mechanical and electronic devices. Similarly, intricate regulatory networks within living organisms are at the heart of all cellular processes. However, man-made embedded chemical controllers still cannot be engineered with the ease of their electronic counterparts. Synthetic biology attempts to close the gap by drawing upon billions of years of evolution, borrowing the complex machinery of the central dogma. By contrast, DNA nanotechnology starts with bottom-up understanding of simple molecular components with predictable composition rules. Therefore, it becomes possible—in principle—to recapitulate for molecular systems the hierarchical design strategies that were essential to the development of complex electronic systems. The central concept is to establish a hierarchy of abstractions that permits the programming of high-level dynamical behavior separately from the design of low-level components. RATIONALE Hierarchical molecular programming languages have been previously described, but they have yet to be robustly implemented for molecular dynamical systems. Enzyme-free nucleic acid systems are a promising substrate for molecular design because they obey a predictable class of interactions that arise from just Watson-Crick binding. Beyond static binding, a strand competing with another for the same complement can be displaced (strand displacement), and the displaced strands can, in turn, displace strands in other molecules (cascading). Sequence- and domain-level models (which abstract contiguous sequences that act as a unit) are made possible by predictable thermodynamics and strand displacement rules and are used in easy-to-use tools, such as NUPACK and Visual DSD. At a higher level, formal chemical reaction networks (CRNs)—coupled chemical reaction equations between formal symbols representing different species—have been proposed as a programming language for specifying the dynamics of strand displacement cascades. Extensive theoretical work exploring dynamical systems and algorithms written in the language of CRNs shows the power of reasoning about chemical algorithms independently of the chemical identity of the participating species. RESULTS As a challenging test case for hierarchical design, we set out to implement an oscillator specified by an abstract CRN known as the “rock-paper-scissors” oscillator. This CRN has been proposed and analyzed extensively in theoretical ecology, theoretical physics, theoretical chemistry, and theoretical computer science; yet, until this work, it had no known chemical implementation. To systematically implement each formal chemical reaction, we designed a corresponding set of abstract molecules that mediate the desired interaction pathway according to a domain-level model; subsequent sequence design yielded specific DNA molecules that were expected to satisfy the specifications. Using chemically synthesized oligonucleotides, we experimentally demonstrated a working oscillator after debugging the main sources of design error. We then constructed a mathematical model of individual strand displacement steps and nonidealities that quantitatively captured the observed dynamics. Finally, the critical design principles were codified into a compiler that automates the design process for arbitrary networks. The compiler was tested by successfully designing a new oscillator that worked on the first try. CONCLUSION The programmable nature of base-pairing interactions and the minimal requirements on the chemical environment make DNA a particularly attractive engineering material. Nucleic acids as chemical controllers naturally integrate with the ever-expanding range of molecular structures, machines, and devices developed in DNA nanotechnology and could eventually be embedded within complex synthetic organelles or artificial cells that sense, compute, and respond to their chemical and molecular environment. Besides addressing a technological challenge, we also answer a fundamental scientific question, showing that Watson-Crick base pairing alone suffices for complex temporal dynamics.

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Enzyme-free nucleic acid dynamical systems.

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 t...

The All E. coli TX-TL Toolbox 2.0: A Platform for Cell-Free Synthetic Biology.

The bottom-up construction of biological entities from genetic information provides a broad range of opportunities to better understand fundamental processes within living cells, as well as holding great promise for the development of novel biomedical applications. Cell-free transcription-translation (TXTL) systems have become suitable platforms to tackle such topics because they recapitulate the process of gene expression. TXTL systems have advanced to where the in vitro construction of viable, complex, self-assembling deoxyribonucleic acid-programmed biological entities is now possible. Previously, we demonstrated the cell-free synthesis of three bacteriophages from their genomes: MS2, ΦX174, T7. In this work, we present the complete synthesis of the phage T4 from its 169-kbp genome in one-pot TXTL reactions. This achievement, for one of the largest coliphages, demonstrates the integration of complex gene regulation, metabolism and self-assembly, and brings the bottom-up synthesis of biological systems to a new level.

Cell-free TXTL synthesis of infectious bacteriophage T4 in a single test tube reaction.

A new generation of cell-free transcription-translation (TXTL) systems, engineered to have a greater versatility and modularity, provide novel capabilities to perform basic and applied sciences in test tube reactions. Over the past decade, cell-free TXTL has become a powerful technique for a broad range of novel multidisciplinary research areas related to quantitative and synthetic biology. The new TXTL platforms are particularly useful to construct and interrogate biochemical systems through the execution of synthetic or natural gene circuits. In vitro TXTL has proven convenient to rapidly prototype regulatory elements and biological networks as well as to recapitulate molecular self-assembly mechanisms found in living systems. In this article, we describe how infectious bacteriophages, such as MS2 (RNA), ΦΧ174 (ssDNA), and T7 (dsDNA), are entirely synthesized from their genome in one-pot reactions using an all Escherichia coli, cell-free TXTL system. Synthesis of the three coliphages is quantified using the plaque assay. We show how the yield of synthesized phage depends on the biochemical settings of the reactions. Molecular crowding, emulated through a controlled concentration of PEG 8000, affects the amount of synthesized phages by orders of magnitudes. We also describe how to amplify the phages and how to purify their genomes. The set of protocols and results presented in this work should be of interest to multidisciplinary researchers involved in cell-free synthetic biology and bioengineering.

Mark Rustad

Papers

The All E. coli TX-TL Toolbox 2.0: A Platform for Cell-Free Synthetic Biology.

Cell-free TXTL synthesis of infectious bacteriophage T4 in a single test tube reaction.

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System.

P469: Clinical relevance of mosaic variants detected from exome sequencing data

Clinical relevance of somatic mosaic variants detected from exome sequencing data