Cell free systems for biodesign.

Cell free systems for production of chemicals.

Chapter 24 - Cell-free synthetic biology as an emerging biotechnology

Synthetic biological circuits are the foundation for the ultimate goals of controlling cells and building artificial cells from the ground up. To get closer to these goals in a more efficient way, we utilize a cell-free transcription-translation system to help perfect biological circuits for the simplicity, freedom, and convenience that the system offers. In this thesis, we demonstrate three distinct aspects of biological circuits in a cell-free transcription-translation system: circuit dynamics, phosphorylation, and membrane proteins. We start with a simple feedforward circuit, which shows dynamic responses to the input. We first prototype the feedforward circuit in the cell-free system with the aid of mathematical modeling. Then, based on the knowledge learned from prototyping, we successfully implement the circuit in cells. Not only do we show that a circuit with dynamics can be prototyped in the cell- free system, but we also test a more complicated circuit involving a phosphorylation cycle. The phosphorylation-based insulator circuit is prototyped and then a model created for the circuit is shown to be identifiable in the cell-free system. To further expand the capability of the cell-free system, we demonstrate that biologically active membrane proteins can be generated in the cell-free system with engineering, suggesting that even biological circuits requiring membrane proteins can be prototyped in the system. These results help advance our knowledge of both biological circuits and the cell-free transcription-translation system, and bring us one step closer to our ultimate goals of implementing control theory in synthetic biology.

Cell-free systems are powerful tools in synthetic biology with versatile and wide-ranging applications. However, a significant bottleneck for these systems, particularly the PURE cell-free system, is their limited reaction lifespan and yield. Dialysis offers a promising approach to prolong reaction lifetimes and increase yields, yet most custom dialysis systems require access to sophisticated equipment like 3D printers or microfabrication tools. In this study, we utilized an easy-to-assemble, medium-scale dialysis system for cell-free reactions using commercially available components. By employing dialysis with periodic exchange of the feeding solution, we achieved a protein yield of 1.16 mg/mL GFP in the PURE system and extended protein synthesis for at least 12.5 consecutive days, demonstrating the system's excellent stability.

Cell-free expression (CFE) systems are powerful tools in synthetic biology that allow biomimicry of cellular functions like biosensing and energy regeneration in synthetic cells. Reconstruction of a wide range of cellular processes, however, requires successful reconstitution of membrane proteins into the membrane of synthetic cells. While the expression of soluble proteins is usually successful in common CFE systems, the reconstitution of membrane proteins in lipid bilayers of synthetic cells has proven to be challenging. Here, a method for reconstitution of a model membrane protein, bacterial glutamate receptor (GluR0), in giant unilamellar vesicles (GUVs) as model synthetic cells based on encapsulation and incubation of the CFE reaction inside synthetic cells is demonstrated. Utilizing this platform, the effect of substituting the N-terminal signal peptide of GluR0 with proteorhodopsin signal peptide on successful cotranslational translocation of GluR0 into membranes of hybrid GUVs is demonstrated. This method provides a robust procedure that will allow cell-free reconstitution of various membrane proteins in synthetic cells.

A challenge facing the 21st century is global sustainability. Synthetic biology is an emerging technology, with the potential to be a critical part of the solution. Although it is hard to predict the ultimate applications of synthetic biology, it is imperative to prepare the groundwork required for responsible innovation. This entails raising awareness of the technology and its potential consequences. The Cambridge BioDesign TechEvent presented recent advances in synthetic biology to a wide community in order to promote interactions within and across academia, industry and governance. Our species's global footprint is unsustainable and it is unclear how or whether we will be able to change this. Biological engineering and particularly the emerging field of synthetic biology are considered to be a major part of the solution, promising clean, renewable and better food, energy production, goods manufacturing and healthcare. Synthetic biology introduces rational design rules to the engineering of biological systems, using a common suite of standard workflows and design tools. Technical advancements alone, however, are unlikely to provide solutions without accompanying changes in societal attitudes and behavior. The ultimate consequences of such a technological revolution are difficult, if not impossible, to predict. How can we shape the development of this emerging field to serve the common good? How can we realize its potential, while minimizing the risks? Our species's global footprint is unsustainable and it is unclear how or whether we will change this. ..synthetic biology is considered to be a major part of the solution The Cambridge BioDesign Symposium (25–27 September, 2012) brought together experts in diverse fields to debate synthetic biology's role in global sustainability. It aimed to share technical expertise (TechEvent), as well as to explore potential synthetic biology's applications and their consequences (Forum). While this report focuses mainly on the technological presentations of the TechEvent, it also provides a flavor of broader perspectives addressed at the Forum. New synthetic biology tools: our ability to successfully engineer biology relies heavily on the development of novel design, construction and measurement tools. Central to our construction efforts is the ability to synthesize and assemble bespoke DNA sequences. Ideally, DNA assembly should be fast, accurate, inexpensive, standardized, efficient across diverse assemblies, and amendable to both human and automated operation. Prof. Tom Knight's (MIT, Boston, USA and Ginkgo BioWorks) concept of “Biobricks” laid the foundation for the Registry of Standard Biological Parts – a collection of modular biological parts allowing easy mixing and matching to build synthetic biology devices. In his presentation, Prof. Knight examined different approaches to DNA assembly: overlap-type assemblies such as Gibson assembly are flexible, allowing bespoke part boundaries, but can be less reliable (e.g. where parts contain repetitive sequences) and are less automatable than restriction enzyme-based tools, such as BioBrick™ assembly (e.g. due to lower reliability and the requirement for large numbers of unique oligonucleotides). Restriction enzyme-based assembly remains Prof. Knight's preferred method for high-throughput, automated assembly. He outlined advantages of Ginkgo BioWorks' novel “RAD” assembly method, which is especially well-suited to automated assembly, though he found that its separation of physical and part boundaries makes direct human operation difficult. He reiterated the importance of making biology “work for us, as part of the assembly process”. The ultimate aims of genome engineering include determination of the fundamental features of life by designing minimal genomes, and writing custom genomes for specific applications. In many contexts however, the problem is what to build, rather than how to build it. Dr. Claes Gustafsson (DNA2.0, Palo Alto, USA) presented an approach to efficient and systematic exploration of nucleotide and amino acid sequence space, using what he called “Infologs” – information-rich products of custom DNA synthesis and bioinformatics – provided to the synthetic biology community by his company. Dr. James Brown (Microsoft Research, Cambridge, UK) highlighted bottlenecks in the design-build-test product development cycle as applied to bioengineering and presented tools that could impact the design and testing phases: Microsoft Research's Genetic Engineering of Cells (GEC) software [1] and a novel dual reporter system for standard measurement of gene expression. Genome engineering: whole genome engineering, including the recent in silico synthesis of a Mycoplasma genome, is an area of synthetic biology that resonates with both the scientific community and the general public. The ultimate aims of genome engineering include determination of the fundamental features of life by designing minimal genomes, and writing custom genomes for specific applications [2]. Dr. Tom Ellis (Imperial College London, UK) in his talk: “From parts-based synthetic biology to genome engineering” presented his laboratory's work on design and characterization of biological parts, including a novel class of orthogonal transcription factors called “Talors”, which facilitates genome-level engineering [3]. He highlighted the impact modifying key regulatory switches can have on the expression of whole genetic networks. Furthermore, he outlined his work on the Synthetic Yeast 2.0 project – an international genome engineering initiative, which aims to build the world's first synthetic eukaryotic genome. Rather than direct replacement of the yeast genome, the project entails an iterative redesign: removal of non-essential parts, simplification of telomeres, and introduction of loxP sites to allow large-scale recombination and potentially self-minimization of the genome. ...engineering higher organisms with simple genetic switches might be easier than engineering unicellular organisms... Dr. Philipp Holliger (MRC Laboratory of Molecular Biology, UK) has taken an alternative approach to examining the basic features of life by trying to reconstruct a minimal cell as it might have looked in the early phases of the Earth's development, harboring nothing but two fundamental properties of life: compartmentalization and (RNA-based) replication. His group has built modern-day doppelgangers of the early “RNA world's” replication machinery: RNA replicase ribozymes [4]. The prospect of genuine engineering at the genome level offers exciting challenges. Dr. Jim Haseloff (University of Cambridge, UK) took the discussion one level higher when he described approaches to engineering complex multicellular plant systems – from high-throughput chromosome engineering to the possibility of exploiting the hierarchical organization of natural systems. After highlighting the morphogenetic modularity of multicellular organisms, he concluded that “engineering higher organisms with simple genetic switches might be easier than engineering unicellular organisms, if we can tap into these levels of hierarchy”. Synthetic biology applications on Earth and beyond: generation of novel, clinically or industrially interesting materials (e.g. rebuilding bone for clinical purposes) using the tools of synthetic biology [5] was also discussed at the meeting. Dr. Michelle Oyen (University of Cambridge, UK) presented her research, which utilizes simple robots to automate integration of organic components into inorganic crystals, to form novel biocomposites that resemble natural materials with high stiffness-density ratios, such as bone and eggshell. Dr. David Radford (University of Edinburgh, UK) reported novel industrial and therapeutic uses of Bacillus subtilis, with particular emphasis on optimization of this model bacterium for protein production. Prof. Lynn Rothschild (NASA Ames Research Center, California, USA) presented a plethora of potential synthetic biology applications for space exploration, such as growing food and materials in space, reprograming human cells and endosymbionts for better adaptation to foreign environments, or construction of self-repairable space suits and habitats. Prof. Rothschild described some recent synthetic biology projects from her lab, including engineering microorganisms for biomining (re-extraction of metals from spent electronics) and biocementation (precipitation of calcite for building materials). ...just as the second half of the past century was characterized by the impact of the invention of programing in silicon, this century will be shaped at least as much by the ability to program biology. Public engagement: in a public lecture, Prof. Stephen Emmott (Microsoft Research, Cambridge, UK) argued that solutions to some of the biggest problems of our times, such as climate change and the ability to sustainably feed and power a planet of ten billion people, will require something akin to a scientific revolution, which in turn will require entirely new ways of thinking, new methods, and new kinds of scientists fluent in the “programing languages” of biology. According to Prof. Emmott, to solve these problems we need to think about biology as programmable. He argued that just as the second half of the past century was characterized by the impact of the invention of programing in silicon, this century will be shaped at least as much by the ability to program biology. Venue of the meeting: Centre for Mathematical Sciences, University of Cambridge Perspectives: “If the dominant science in the new 'Age of Wonder' is biology, then the dominant art form should be the design of genomes to create new animals and plants” (Freeman Dyson). The Cambridge BioDesign TechEvent aimed to raise awareness in the wider scientific community of the technical advances of synthetic biology. The TechEvent, as part of the Cambridge BioDesign Symposium, provided a technical focus to a new series of meetings that will develop a multidisciplinary community to engage and inform society on the tough decisions we face regarding the development of synthetic biology. We cordially thank speakers for their contributions to the meeting and for allowing us to cite their unpublished work and apologize to those whose talks were not presented due to space limitation. The meeting was hosted by the University of Cambridge and sponsored by the University of Cambridge, Microsoft Research, the Institution of Engineering and Technology and the Royal College of Art. M.J., P.D. and J.A are supported by EPSRC. Mario Juhas*, Peter W. Davenport*, James R. Brown*, Orr Yarkoni and James W. Ajioka * These authors contributed equally to the work.

Cell-free systems (CFS) have recently evolved into key platforms for synthetic biology applications. Many synthetic biology tools have traditionally relied on cell-based systems, and while their adoption has shown great progress, the constraints inherent to the use of cellular hosts have limited their reach and scope. Cell-free systems, which can be thought of as programmable liquids, have removed many of these complexities and have brought about exciting opportunities for rational design and manipulation of biological systems. Here we review how these simple and accessible enzymatic systems are poised to accelerate the rate of advancement in synthetic biology and, more broadly, biotechnology.

High-throughput screening of biomolecules using cell-free systems.

Applications of Synthetic Biology in Microbial Biotechnology

Advanced approaches that can mimic the structure and function of natural tissue in tissue engineering applications that use multidisciplinary engineering approaches to repair damaged or dysfunctional tissues are fed forward by current engineering applications. Manipulating cells or cell groups in an integrated manner into the scaffold, similar to the native tissue composition, is the main challenge in these approaches. Synthetic biology approaches, originating from genetic engineering, based on the use of advanced tools in the manipulation of cells at the molecular level, are one of the most current issues in tissue engineering that shed light on the programming of cells. Synthetic biology tools allow the reprogramming of cells whose transcriptional, translational, or post-translational molecular mechanisms have been engineered by stimulating them with intrinsic or extrinsic signals. Combining these advanced and excellent tools from synthetic biology with materials engineering applications of tissue engineering is the latest fashion. This chapter discusses going beyond conventional tissue engineering applications, synthetic biological molecular tools, circuit designs that allow the complex behavior of cells to be manipulated with these tools, and approaches that enable the integration of these tools into the material component of tissue engineering

Cell-free synthesis systems can complete the transcription and translation process in vitro to produce complex proteins that are difficult to be expressed in traditional cell-based systems. Such systems also can be used for the assembly of efficient localized multienzyme cascades to synthesize products that are toxic to cells. Cell-free synthesis systems provide a simpler and faster engineering solution than living cells, allowing unprecedented design freedom. This paper reviews the latest progress on the application of cell-free synthesis systems in the field of enzymatic catalysis, including cell-free protein synthesis and cell-free metabolic engineering. In cell-free protein synthesis: complex proteins, toxic proteins, membrane proteins, and artificial proteins containing non-natural amino acids can be easily synthesized by directly controlling the reaction conditions in the cell-free system. In cell-free metabolic engineering, the synthesis of desired products can be made more specific and efficient by designing metabolic pathways and screening biocatalysts based on purified enzymes or crude extracts. Through the combination of cell-free synthesis systems and emerging technologies, such as: synthetic biology, microfluidic control, cofactor regeneration, and artificial scaffolds, we will be able to build increasingly complex biomolecule systems. In the next few years, these technologies are expected to mature and reach industrialization, providing innovative platforms for a wide range of biotechnological applications.

Cell-free systems for low-cost diagnostics.

Purified cell-free systems as standard parts for synthetic biology

Cell-Free Protein Synthesis Containing Disulfide Bonds and Its Application to Protein Engineering

Cell-free systems for development of biosensors.