Synthetic Biology Projects Research Articles

Opportunities in plant synthetic biology

Synthetic biology is an emerging field uniting scientists from all disciplines with the aim of designing or re-designing biological processes. Initially, synthetic biology breakthroughs came from microbiology, chemistry, physics, computer science, materials science, mathematics, and engineering disciplines. A transition to multicellular systems is the next logical step for synthetic biologists and plants will provide an ideal platform for this new phase of research. This meeting report highlights some of the exciting plant synthetic biology projects, and tools and resources, presented and discussed at the 2013 GARNet workshop on plant synthetic biology.

Policy on Synthetic Biology: Deliberation, Probability, and the Precautionary Paradox

Synthetic biology is a cutting-edge area of research that holds the promise of unprecedented health benefits. However, in tandem with these large prospective benefits, synthetic biology projects entail a risk of catastrophic consequences whose severity may exceed that of most ordinary human undertakings. This is due to the peculiar nature of synthetic biology as a 'threshold technology' which opens doors to opportunities and applications that are essentially unpredictable. Fears about these potentially unstoppable consequences have led to declarations from civil society groups calling for the use of a precautionary principle to regulate the field. Moreover, the principle is prevalent in law and international agreements. Despite widespread political recognition of a need for caution, the precautionary principle has been extensively criticized as a guide for regulatory policy. We examine a central objection to the principle: that its application entails crippling inaction and incoherence, since whatever action one takes there is always a chance that some highly improbable cataclysm will occur. In response to this difficulty, which we call the 'precautionary paradox,' we outline a deliberative means for arriving at threshold of probability below which potential dangers can be disregarded. In addition, we describe a Bayesian mechanism with which to assign probabilities to harmful outcomes. We argue that these steps resolve the paradox. The rehabilitated PP can thus provide a viable policy option to confront the uncharted waters of synthetic biology research.

GoldenBraid 2.0: A Comprehensive DNA Assembly Framework for Plant Synthetic Biology

Plant synthetic biology aims to apply engineering principles to plant genetic design. One strategic requirement of plant synthetic biology is the adoption of common standardized technologies that facilitate the construction of increasingly complex multigene structures at the DNA level while enabling the exchange of genetic building blocks among plant bioengineers. Here, we describe GoldenBraid 2.0 (GB2.0), a comprehensive technological framework that aims to foster the exchange of standard DNA parts for plant synthetic biology. GB2.0 relies on the use of type IIS restriction enzymes for DNA assembly and proposes a modular cloning schema with positional notation that resembles the grammar of natural languages. Apart from providing an optimized cloning strategy that generates fully exchangeable genetic elements for multigene engineering, the GB2.0 toolkit offers an evergrowing open collection of DNA parts, including a group of functionally tested, premade genetic modules to build frequently used modules like constitutive and inducible expression cassettes, endogenous gene silencing and protein-protein interaction tools, etc. Use of the GB2.0 framework is facilitated by a number of Web resources that include a publicly available database, tutorials, and a software package that provides in silico simulations and laboratory protocols for GB2.0 part domestication and multigene engineering. In short, GB2.0 provides a framework to exchange both information and physical DNA elements among bioengineers to help implement plant synthetic biology projects.

Meeting report: The Cambridge BioDesign TechEvent – Synthetic Biology, a new “Age of Wonder”?

Meeting report: The Cambridge BioDesign TechEvent – Synthetic Biology, a new “Age of Wonder”?

Building-in biosafety for synthetic biology

As the field of synthetic biology develops, real-world applications are moving from the realms of ideas and laboratory-confined research towards implementation. A pressing concern, particularly with microbial systems, is that self-replicating re-engineered cells may produce undesired consequences if they escape or overwhelm their intended environment. To address this biosafety issue, multiple mechanisms for constraining microbial replication and horizontal gene transfer have been proposed. These include the use of host-construct dependencies such as toxin-antitoxin pairs, conditional plasmid replication or the requirement for a specific metabolite to be present for cellular function. While refactoring of the existing genetic code or tailoring of orthogonal systems, e.g. xeno nucleic acids, offers future promise of more stringent 'firewalls' between natural and synthetic cells, here we focus on what can be achieved using existing technology. The state-of-the-art in designing for biosafety is summarized and general recommendations are made (e.g. short environmental retention times) for current synthetic biology projects to better isolate themselves against potentially negative impacts.

Between the possible and the actual: Philosophical perspectives on the design of synthetic organisms

A number of research projects in synthetic biology funded by public institutions sound like science fiction: for instance DARPA Biodesign project in 2011 aimed at eliminating “the randomness of natural evolutionary advancement, and designing proteins with a 99.5% precision. How should we take such claims? Are they just fantastic visions or hype meant to impress funding agencies and the public or are they symptoms of a specific epistemic attitude? In considering the visions of the future developed in synthetic biology the paper attempts to evaluate the research agendas of synthetic biology from philosophical perspectives. It will first characterize the ambition to re-engineer life and distinguish the visions of the future underlying the various research agendas assembled under the umbrella synthetic biology. Then addressing the question ‘who actually believes in such futures?’ the paper contrasts the complicit belief of ethicists and critical activists with the doubts occasionally formulated by synthetic biologists. Doubts however never generate scepticism within the synthetic biology community, which develops an epistemic opportunism. Finally the third section discusses to what extent, and in which sense, the futuristic visions of synthetic biology belong to the genre of techno-utopia. It concludes that imagined futures are integral part of the techno-epistemic culture of synthetic biology and that the tension between the possible and the actual is a criterion of distinction between the various trends that constitute synthetic biology.

Parallel In Vivo DNA Assembly by Recombination: Experimental Demonstration and Theoretical Approaches

The development of synthetic biology requires rapid batch construction of large gene networks from combinations of smaller units. Despite the availability of computational predictions for well-characterized enzymes, the optimization of most synthetic biology projects requires combinational constructions and tests. A new building-brick-style parallel DNA assembly framework for simple and flexible batch construction is presented here. It is based on robust recombination steps and allows a variety of DNA assembly techniques to be organized for complex constructions (with or without scars). The assembly of five DNA fragments into a host genome was performed as an experimental demonstration.

Self-Assembly of Synthetic Metabolons through Synthetic Protein Scaffolds: One-Step Purification, Co-immobilization, and Substrate Channeling

One-step purification of a multi-enzyme complex was developed based on a mixture of cell extracts containing three dockerin-containing enzymes and one family 3 cellulose-binding module (CBM3)-containing scaffoldin through high-affinity adsorption on low-cost solid regenerated amorphous cellulose (RAC). The three-enzyme complex, called synthetic metabolon, was self-assembled through the high-affinity interaction between the dockerin in each enzyme and three cohesins in the synthetic scaffoldin. The metabolons were either immobilized on the external surface of RAC or free when the scaffoldin contained an intein between the CBM3 and three cohesins. The immobilized and free metabolons containing triosephosphate isomerase, aldolase, and fructose 1,6-biphosphatase exhibited initial reaction rates 48 and 38 times, respectively, that of the non-complexed three-enzyme mixture at the same enzyme loading. Such reaction rate enhancements indicated strong substrate channeling among synthetic metabolons due to the close spatial organization among cascade enzymes. These results suggested that the construction of synthetic metabolons by using cohesins, dockerins, and cellulose-binding modules from cellulosomes not only decreased protein purification labor and cost for in vitro synthetic biology projects but also accelerated reaction rates by 1 order of magnitude compared to non-complexed enzymes. Synthetic metabolons would be an important biocatalytic module for in vitro and in vivo synthetic biology projects.

Thermophilic Thermotoga maritima ribose-5-phosphate isomerase RpiB: Optimized heat treatment purification and basic characterization

The open reading frame TM1080 from Thermotoga maritima encoding ribose-5-phosphate isomerase type B (RpiB) was cloned and over-expressed in Escherichia coli BL21 (DE3). After optimization of cell culture conditions, more than 30% of intracellular proteins were soluble recombinant RpiB. High-purity RpiB was obtained by heat pretreatment through its optimization in buffer choice, buffer pH, as well as temperature and duration of pretreatment. This enzyme had the maximum activity at 70°C and pH 6.5–8.0. Under its suboptimal conditions (60°C and pH 7.0), kcat and Km values were 540s−1 and 7.6mM, respectively; it had a half lifetime of 71h, resulting in its turn-over number of more than 2×108mol of product per mol of enzyme. This study suggests that it is highly feasible to discover thermostable enzymes from exploding genomic DNA database of extremophiles with the desired stability suitable for in vitro synthetic biology projects and produce high-purity thermoenzymes at very low costs.

Modular Modelling in Synthetic Biology: Light-Based Communication in E. coli

Modularity – the notion that individual components can be described as self-contained units and composed in different combinations – is a concept integral to synthetic biology. In vitro, it is embodied by the notion of BioBrickTMparts: standardised DNA sequences of defined structure, function, and common interfaces that can be composed and integrated into living cells. A similar modularity in the modelling of such systems, however, has not been explored until much more recently. In the course of the 2010 International Genetically Engineered Machine (iGEM) competition, the University of Edinburgh team adopted an iterative rule-based approach to developing a BioBrick model of light-based communication in Escherichia coli. The system was modelled in the Kappa stochastic rule-based biological modelling language, allowing for the modular development and analysis of a complicated biological system. It also utilised newly developed spatial extensions to aid in the depiction of intercellular communication via light emitting and light sensing pathways, demonstrating the extensibility of the language as a whole. This paper provides a case study of modelling synthetic biology projects using the rule-based techniques outlined above, documenting the benefits of this modular iterative approach.

The Ribosome Meets Synthetic Biology

A recent advance in custom-designed, cell-free translation systems that makes use of purified and recombinant components opens up new perspectives for synthesizing non-natural translation products. Combining these systems with chemically engineered ribosomes sets the stage for novel synthetic biology projects.

Model annotation for synthetic biology: automating model to nucleotide sequence conversion

Motivation: The need for the automated computational design of genetic circuits is becoming increasingly apparent with the advent of ever more complex and ambitious synthetic biology projects. Currently, most circuits are designed through the assembly of models of individual parts such as promoters, ribosome binding sites and coding sequences. These low level models are combined to produce a dynamic model of a larger device that exhibits a desired behaviour. The larger model then acts as a blueprint for physical implementation at the DNA level. However, the conversion of models of complex genetic circuits into DNA sequences is a non-trivial undertaking due to the complexity of mapping the model parts to their physical manifestation. Automating this process is further hampered by the lack of computationally tractable information in most models.Results: We describe a method for automatically generating DNA sequences from dynamic models implemented in CellML and Systems Biology Markup Language (SBML). We also identify the metadata needed to annotate models to facilitate automated conversion, and propose and demonstrate a method for the markup of these models using RDF. Our algorithm has been implemented in a software tool called MoSeC.Availability: The software is available from the authors' web site http://research.ncl.ac.uk/synthetic_biology/downloads.html.Contact: anil.wipat@ncl.ac.ukSupplementary information: Supplementary data are available at Bioinformatics online.

Toward low-cost biomanufacturing through in vitro synthetic biology: bottom-up design

While most in vitro synthetic biology projects are usually used for the purposes of basic science research or the formation of high-value products, cell-free synthetic pathway biotransformation (SyPaB), which can implement complicated biochemical reactions by the in vitro assembly of numerous enzymes and coenzymes, would be used for low-cost biomanufacturing. In this article, we present bottom-up design principles for SyPaB from basic building blocks (enzymes and/or immobilized enzymes) to basic modules, such as NAD(P)H regeneration, NAD(P)H consumption, ATP regeneration, and extra ATP removal. A combination of thermostable enzymes (called thermoenzymes) with immobilization on solid supports, especially nano-materials and/or electrodes, would greatly prolong enzyme lifetime, enhance mass transfer, and facilitate product/biocatalyst separation. With developments in stable building blocks and modules (called biocatalytic modules), SyPaB has the potential to become a low-cost biomanufacturing platform for biofuels production and even biological CO2 fixation.

CLONEQC: lightweight sequence verification for synthetic biology

Synthetic biology projects aim to produce physical DNA that matches a designed target sequence. Chemically synthesized oligomers are generally used as the starting point for building larger and larger sequences. Due to the error rate of chemical synthesis, these oligomers can have many differences from the target sequence. As oligomers are joined together to make larger and larger synthetic intermediates, it becomes essential to perform quality control to eliminate intermediates with errors and retain only those DNA molecules that are error free with respect to the target. This step is often performed by transforming bacteria with synthetic DNA and sequencing colonies until a clone with a perfect sequence is identified. Here we present CloneQC, a lightweight software pipeline available as a free web server and as source code that performs quality control on sequenced clones. Input to the server is a list of desired sequences and forward and reverse reads for each clone. The server generates summary statistics (error rates and success rates target-by-target) and a detailed report of perfect clones. This software will be useful to laboratories conducting in-house DNA synthesis and is available at http://cloneqc.thruhere.net/ and as Berkeley Software Distribution (BSD) licensed source.

Special issue: societal aspects of synthetic biology

Special issue: societal aspects of synthetic biology

The smallest chemical reaction system with bistability

BackgroundBistability underlies basic biological phenomena, such as cell division, differentiation, cancer onset, and apoptosis. So far biologists identified two necessary conditions for bistability: positive feedback and ultrasensitivity.ResultsBiological systems are based upon elementary mono- and bimolecular chemical reactions. In order to definitely clarify all necessary conditions for bistability we here present the corresponding minimal system. According to our definition, it contains the minimal number of (i) reactants, (ii) reactions, and (iii) terms in the corresponding ordinary differential equations (decreasing importance from i-iii). The minimal bistable system contains two reactants and four irreversible reactions (three bimolecular, one monomolecular).We discuss the roles of the reactions with respect to the necessary conditions for bistability: two reactions comprise the positive feedback loop, a third reaction filters out small stimuli thus enabling a stable 'off' state, and the fourth reaction prevents explosions. We argue that prevention of explosion is a third general necessary condition for bistability, which is so far lacking discussion in the literature.Moreover, in addition to proving that in two-component systems three steady states are necessary for bistability (five for tristability, etc.), we also present a simple general method to design such systems: one just needs one production and three different degradation mechanisms (one production, five degradations for tristability, etc.). This helps modelling multistable systems and it is important for corresponding synthetic biology projects.ConclusionThe presented minimal bistable system finally clarifies the often discussed question for the necessary conditions for bistability. The three necessary conditions are: positive feedback, a mechanism to filter out small stimuli and a mechanism to prevent explosions. This is important for modelling bistability with simple systems and for synthetically designing new bistable systems. Our simple model system is also well suited for corresponding teaching purposes.

A highly active phosphoglucomutase from Clostridium thermocellum: cloning, purification, characterization and enhanced thermostability

Discovery and utilization of highly active and thermostable phosphoglucomutase (PGM) would be vital for biocatalysis mediated by multiple enzymes, for example, high-yield production of enzymatic hydrogen. The thermophilic cellulolytic bacterium Clostridium thermocellum was hypothesized to have a very active PGM because of its key role in microbial cellulose utilization. The Cl. thermocellum ORF Cthe1265 encoding a putative PGM was cloned and expressed in Escherichia coli. The purified enzyme appeared to be a monomer with an estimated molecular weight of 64.9 kDa. This enzyme was found to be a dual-specificity enzyme - PGM/phosphomannomutase (PMM). Mg(2+) and Mn(2+) were activators. Ser144 was identified as an essential catalytic residue through site-directed mutagenesis. The k(cat) and K(m) of PGM were 190 s(-1) and 0.41 mmol l(-1) on glucose-1-phosphate and 59 s(-1) and 0.44 mmol l(-1) on mannose-1-phosphate, respectively, at 60 degrees C. Thermostability of PGM at a low concentration (2 nmol l(-1), 100 U l(-1)) was enhanced by 12-fold (i.e. t(1/2) = 72 h) at 60 degrees C with addition of bovine serum albumin, Triton X-100, Mg(2+)and Mn(2+). The ORF Cthe1265 was confirmed to encode a PGM with PMM activity. This enzyme was the most active PGM reported. This highly active PGM with enhanced thermostability would be an important building block for in vitro synthetic biology projects (complicated biotransformation mediated by multiple enzymes in one pot).

An integrated cell‐free metabolic platform for protein production and synthetic biology

Cell-free systems offer a unique platform for expanding the capabilities of natural biological systems for useful purposes, i.e. synthetic biology. They reduce complexity, remove structural barriers, and do not require the maintenance of cell viability. Cell-free systems, however, have been limited by their inability to co-activate multiple biochemical networks in a single integrated platform. Here, we report the assessment of biochemical reactions in an Escherichia coli cell-free platform designed to activate natural metabolism, the Cytomim system. We reveal that central catabolism, oxidative phosphorylation, and protein synthesis can be co-activated in a single reaction system. Never before have these complex systems been shown to be simultaneously activated without living cells. The Cytomim system therefore promises to provide the metabolic foundation for diverse ab initio cell-free synthetic biology projects. In addition, we describe an improved Cytomim system with enhanced protein synthesis yields (up to 1200 mg/l in 2 h) and lower costs to facilitate production of protein therapeutics and biochemicals that are difficult to make in vivo because of their toxicity, complexity, or unusual cofactor requirements.

Vanillin cell sensor

Our project for iGEM 2006 consisted of designing a cellular vanillin biosensor. We used an EnvZ–E. coli strain as a chassis, and constructed two different devices: a sensor and an actuator, assembled using OmpR-P as a standardised mediator. The sensor device contained a computationally designed vanillin receptor and a synthetic two-component signal transduction protein (Trz). The receptor protein was based on a ribose-binding protein as scaffold. The Trz was built by fusion of the periplasmic and transmembrane domains of a Trg protein with an EnvZ kinase domain. When the receptor complex binds Trg, an allosteric motion is propagated to the cytoplasmic EnvZ kinase domain, resulting in autophosphorylation and subsequent phosphate transfer to the OmpR transcription factor, which finally induces transcription of the ompC promoter. As actuator, we used a synthetic transcriptional circuit, which implements an OmpR-P band detector having GFP and RFP as an output. We designed this circuit using a synthetic promoter working as an AND gate, which is synergistically activated by cI and CRP. Our constructed Trg-EnvZ fusion and AND promoter will be very useful to future synthetic biology projects.

Enzymatic Synthesis of Phosphonomethyl Oligonucleotides by Therminator Polymerase

Projects in synthetic biology imply the study of the substrate specificity of natural and non-natural biopolymers with enzymatic activity and the identification of the products that are obtained. Herein we describe the enzymatic synthesis of 3’–2’ phosphonomethyl–threosyl and 5’–3’ phosphonomethyl–deoxyribosyl oligonucleotides by Therminator polymerase (Scheme 1). We demonstrate that phosphonate nucleotides can be polymerized by this enzyme with the formation of oligonucleotides with more than sixinternucleotide bonds. The enzymes that catalyze the formation of the 5’–3’ phosphodiester bond in DNA and RNA are polymerases and use nucleoside triphosphates as substrates. Therminator polymerase is a mutant variant of the 98 Ne x o polymerase (Thermococcus species 98N-7), in which the Ala 485 residue has been replaced with a Leu residue. With this mutation in an a helixthat is oriented away from the nucleotide-binding site, Therminator polymerase possesses an enhanced ability to incorporate modified substrates, such as dideoxynucleosides, ribonucleosides, and acyclic nucleosides, by using their triphosphate moiety as a substrate. [1–3] We envisaged the synthesis of 3’–2’ phosphonomethyl–