The emerging age of cell-free synthetic biology

The engineering of and mastery over biological parts has catalyzed the emergence of synthetic biology. This field has grown exponentially in the past decade. As increasingly more applications of synthetic biology are pursued, more challenges are encountered, such as delivering genetic material into cells and optimizing genetic circuits in vivo. An in vitro or cell-free approach to synthetic biology simplifies and avoids many of the pitfalls of in vivo synthetic biology. In this review, we describe some of the innate features that make cell-free systems compelling platforms for synthetic biology and discuss emerging improvements of cell-free technologies. We also select and highlight recent and emerging applications of cell-free synthetic biology.     

从DNA合成技术角度阐述合成生物学

合成生物学是一个拥有巨大潜力的新兴学科,合成生物学技术的发展将会对未来生物、医药、农业、能源、材料和环保等方面产生巨大的推进作用。基因合成是合成生物学中最基本和使用最多的一种技术手段,合成生物学的快速发展对基因合成能力提出了空前需求。综述基因合成技术的发展历史、现状和未来趋势,探讨基因合成技术存合成生物学以及整个生命科学研究中的应用和重要意义。     

合成生物学中的DNA组装技术

合成生物学旨在应用工程学的研究思路及手段去设计或改造生物系统,是一个综合了科学与工程的拥有发展潜力的新兴学科,在生物医药、农业、能源、环保等方面发挥着巨大作用。DNA组装技术是合成生物学中的关键技术,也是合成生物学快速发展的限制性技术。综述了众多DNA组装技术的发展及其在合成生物学研究中的意义和应用。     

合成生物学发展现状与前景

合成生物学是综合了科学与工程的一个崭新的生物学研究领域,为生命现象及其运动规律的解析提供了一种采用“白下而上”合成策略的正向工程学的研究思路和方法手段,在经济和社会发展中具有巨大的应用开发潜力。近年来,DNA合成与系统生物学技术的发展使生命系统复杂基因回路的设计、合成与组装逐步成为可能,并应用于生物基化学品、生物燃料、医药中间体、保健产品的生产和环境保护等领域。但是,合成生物学的研究仍然面临科学、技术和伦理的挑战,只有积极地应对这些问题,在加大研究开发支持力度的同时,做好必要的风险监管,才能真正把握合成生物学发展带来的历史机遇。     

The imminent role of protein engineering in synthetic biology

Protein engineering has for decades been a powerful tool in biotechnology for generating vast numbers of useful enzymes for industrial applications. Today, protein engineering has a crucial role in advancing the emerging field of synthetic biology, where metabolic engineering efforts alone are insufficient to maximize the full potential of synthetic biology. This article reviews the advancements in protein engineering techniques for improving biocatalytic properties to optimize engineered pathways in host systems, which are instrumental to achieve high titer production of target molecules. We also discuss the specific means by which protein engineering has improved metabolic engineering efforts and provide our assessment on its potential to continue to advance biology engineering as a whole.     

The promise of behavioural biology

The studies of behaviour that were strongly rooted in biology have had a long tradition of bringing together the ‘how’ and the ‘why’ questions. This integrative approach will serve the subject well in the postgenomic era as the long trend towards analysis at lower and lower levels starts to reverse. The new studies will make use of the resources uncovered by molecular biology and the neurosciences but will use the behaviour of the whole animal to measure outcomes and the context in which behaviour occurs to frame analytical questions. Two examples are given of how movement between levels of analysis is being used with increasing power and promise. The first is the study of filial imprinting in birds where many of the molecular and neural mechanisms involved have been uncovered and are now being integrated to explain the behaviour of the whole animal. The second is the triggering during sensitive periods in early life by environmental events of one of several alternative modes of development leading to different phenotypes. Copyright 2003 The Association for the Study of Animal Behaviour. Published by Elsevier Science Ltd. All rights reserved.     

The simulation approach in synthetic biology

Synthetic biology and systems biology are often highlighted as antagonistic strategies for dealing with the overwhelming complexity of biology (engineering versus understanding; tinkering in the lab versus modelling in the computer). However, a closer view of contemporary engineering methods (inextricably interwoven with mathematical modelling and simulation) and of the situation in biology (inextricably confronted with the intrinsic complexity of biomolecular environments) demonstrates that tinkering in the lab is increasingly supported by rational design methods. In other words: Synthetic biology and systems biology are merged by the use of computational techniques. These computational techniques are needed because the intrinsic complexity of biomolecular environments (stochasticity, non-linearities, system-level organization, evolution, independence, etc.) require advanced concepts of bio bricks and devices. A philosophical investigation of the history and nature of bio parts and devices reveals that these objects are imitating generic objects of engineering (switches, gates, oscillators, sensors, etc.), but the well-known design principles of generic objects are not sufficient for complex environments like cells. Therefore, the rational design methods have to be used to create more advanced generic objects, which are not only generic in their use, but also adaptive in their behavior. Case studies will show how simulation-based rational design methods are used to identify adequate parameters for synthesized designs (stability analyses), to improve lab experiments by ‘looking through noise’ (estimation of hidden variables and parameters), and to replace laborious and time-consuming post hoc tweaking in the lab by in-silico guidance (in-silico variation of bio brick properties). The overall aim of these developments, as will be argued in the discussion, is to achieve adaptive-generic instrumentality for bio parts and devices and thus increasingly merging systems and synthetic biology.     

The synthesis of artificial genome as the basis of synthetic biology

Recent achievements in the whole-genome sequencing especially viral and bacterial ones together with the development of methods of bioinformatics and molecular biology, have created preconditions for transition from synthesis of genes to assembly of the whole genomes based on chemically synthesized blocks, oligonucleotides. The creation of artificial genomes and artificial cells will undoubtedly render huge influence on a deepening of knowledge on mechanisms of functioning of living systems at a cellular level, on a way of origin and evolution of life, and also on biotechnology of the future, and will generate preconditions for the further development of synthetic biology and nanobiotechnology.     

Interactive learning and action: realizing the promise of synthetic biology for global health

The emerging field of synthetic biology has the potential to improve global health. For example, synthetic biology could contribute to efforts at vaccine development in a context in which vaccines and immunization have been identified by the international community as being crucial to international development efforts and, in particular, the millennium development goals. However, past experience with innovations shows that realizing a technology’s potential can be difficult and complex. To achieve better societal embedding of synthetic biology and to make sure it reaches its potential, science and technology development should be made more inclusive and interactive. Responsible research and innovation is based on the premise that a broad range of stakeholders with different views, needs and ideas should have a voice in the technological development and deployment process. The interactive learning and action (ILA) approach has been developed as a methodology to bring societal stakeholders into a science and technology development process. This paper proposes an ILA in five phases for an international effort, with national case studies, to develop socially robust applications of synthetic biology for global health, based on the example of vaccine development. The design is based on results of a recently initiated ILA project on synthetic biology; results from other interactive initiatives described in the literature; and examples of possible applications of synthetic biology for global health that are currently being developed.     

The logic of the Membrane, Magnesium, Mitosis (MMM) model for the regulation of animal cell proliferation

The addition of animal serum or specific protein growth factors to quiescent, serum-deprived vertebrate cells in culture activates a wide variety of biochemical responses within minutes, which are followed in 5-10h by the initiation of DNA synthesis and then by mitosis. The quintessential early and continuing activation step for the increase in DNA synthesis is the increased initiation rate of protein synthesis, which must be continuously maintained throughout the G1 phase for advancement into S. The aggregate of biochemical reactions to growth factors is called the coordinate response, to indicate that many related and unrelated processes are orchestrated to repetitively reproduce cells. It is, however, crucial to recognize that the coordinate response can be induced for one or more rounds of replication by a variety of non-specific and quasi-specific membrane effectors. The logic of considering this framework of events in growth control implied that a single multi-target second messenger plays a central role in coordinating the events of the overall response. The same reasoning suggested that free Mg(2+) is the unifying regulatory element in that response which includes protein kinase pathways, and that the cytoplasmic activity of Mg(2+) increases with the binding of growth factors to their receptors in the cell membrane, or of less specific perturbations of the membrane. Experimental support of this conclusion is presented here and is represented in the MMM model of cell proliferation control.     

The making of the minibody: An engineered β-protein for the display of conformationally constrained peptides

Conformationally constraining selectable peptides onto a suitable scaffold that enables their conformation to be predicted or readily determined by experimental techniques would considerably boost drug discovery process by reducing the gap between the discovery of a peptide lead and the design of a peptidomimetic with a more desirable pharmacological profile. With this in mind, we designed the minibody, a 61-residue β-protein aimed at retaining some desirable features of immunogloblin variable domains, such as tolerance to sequence variability in selected regions of the protein and predictability of main chain conformation of the same regions, based on the ‘canonical structures’ model. To test the ability of the minibody scaffold to support functional sites we also designed a metal binding version of the protein by suitably choosing the sequences of its loops. The minibody was produced both by chemical syntyhesis and expression in E. coli and charactgerized by size exclusion chromatography, UV CD (circular dichroism) spectroscopy and metal binding activity. All our data supported the model, but a more detailed structural characterization of the molecule was impaired by its low soubility. We were able to overcome this problem both by further; mutagenesis of the framework and by addition of a solublizing motif. The minibody is being used to select constrained human IL-6 peptidic ligands from a library displayed on the surface of the f1 bacteriophage.     

The 'atom-splitting' moment of synthetic biology. Nuclear physics and synthetic biology share common features

Synthetic biology and nuclear physics share many commonalities in terms of public perception and funding. Synthetic biologists could learn valuable lessons from the history of the atomic bomb and nuclear power.On 16 July 1945, in the desert of New Mexico, the first nuclear bomb was exploded. It was a crucial moment in the history of the physical sciences—proof positive of the immense forces at work in the heart of atoms—and inevitably changed the world. In 2010, a team at the J. Craig Venter Research Institute in the USA first created artificial life by inserting a synthetic 1.08 megabase pair genome into a mycoplasma cell that lacked its own. They demonstrated that this new cell with its man-made genome was capable of surviving and reproducing [1]. It was a colossal achievement for biology, and its significance might well rank alongside the detonation of the first atomic bomb in terms of scientific advance.…as with post-war physics, synthetic biology''s promises of a brighter future might not all materialize and could have far-reaching effects on society, science and politicsThere are several similarities between twentieth century physics, and twentieth and twenty-first century biology. The nuclear explosion in New Mexico was the result of decades of research and the first splitting of an atom in Otto Hahn''s laboratory in 1938. It ushered in an era of new ideas and hopes for a brighter future built on the power of the atom, but the terrible potential of nuclear weapons and the threat of nuclear warfare ultimately overshadowed these hopes and changed the course of science and politics. The crucial achievement of synthetic life is a strikingly similar event; the culmination of decades of research that started with its own atom-splitting moment: recombinant DNA technology. It promises to bring forth a new era for biology and enable a huge variety of applications for industry, medicine and the military. However, as with post-war physics, synthetic biology''s promises of a brighter future might not all materialize and could have far-reaching effects on society, science and politics. Biology should therefore take note of the consequences of nuclear physics'' iconic event in 1945 for science, politics and society.To appreciate the similarities of these breakthroughs and their consequences for society, it is necessary to understand the historical perspective. The pivotal discoveries for both disciplines were related to fundamental elements of nature. The rise of nuclear physics can be traced back to the discovery of neutrons by James Chadwick in 1932 [2]. Neutrons are essential to the stability of atoms as they insulate the nucleus against the repulsive forces of its positively charged protons. However, the addition of an extra neutron can destabilize the nucleus and cause it to split, releasing more neutrons and a tremendous amount of energy. This nuclear fission reaction was first described by Otto Hahn and Fritz Strassmann in 1938. Leo Szilard realized the possibility of using the neutrons released from the fission of heavy atoms to trigger a nuclear chain reaction to release huge quantities of energy. The first successful chain reactions took place in 1942 in Germany at Leipzig University in the laboratory of Robert Döpel, and in the USA at the University of Chicago in the so-called Chicago Pile-1 reactor, developed by Enrico Fermi. These first nuclear reactors provided the proof of concept for using a nuclear chain reaction as a source of energy. However, even before that, Albert Einstein and Leo Szilard wrote to US President Franklin D. Roosevelt in 1939, suggesting that the US government should develop a new powerful bomb based on nuclear fission. President Roosevelt created the Manhattan Project, which developed the first atomic bomb in 1945.Similarly to nuclear physics, the advent of rDNA technology has concerned the public…The Cold War and the mutually assured nuclear destruction between the USA and the USSR fanned widespread fears about a nuclear Third World War that could wipe out human civilization; Robert Oppenheimer, one of the physicists who developed the atomic bomb, was actually among the first to warn of the spectre of nuclear war. By contrast, the civilian use of nuclear physics, mainly in the form of nuclear reactors, promised a brave new future based on harnessing the power of the atom, but it also generated increasing concerns about the harmful effects of radioactivity, the festering problems of nuclear waste and the safety of nuclear power plants. The nuclear disasters at the Chernobyl reactor in 1986 and the Fukushima power plant in 2011 heightened these concerns to the point that several nations might now abandon nuclear energy altogether.The fundamental discovery in biology, crucial to the creation of synthetic organisms was the double helix structure of DNA in 1953 by Francis Crick and James Watson [3]. The realization that DNA molecules have a universal chemical structure to store and pass on genetic information was the intellectual basis for the development of recombinant DNA (rDNA) technology and genetic engineering. Twenty years after this discovery, Stanley Cohen and Herbert Boyer first transferred DNA from one organism into another by using endonucleases and DNA ligases [4]. This early toolkit was later expanded to include DNA sequencing and synthesizing technologies as well as PCR, which culminated in the creation of the first artificial organism in 2010. Craig Venter''s team synthesized a complete bacterial chromosome from scratch and transferred it into a bacterial cell lacking a genome: the resulting cell was able to synthesize a new set of proteins and to replicate. This proof of concept experiment now enables scientists to pursue further challenges, such as creating organisms with fully designed genomes to achieve agro-biotechnological, commercial, medical and military goals.Similarly to nuclear physics, the advent of rDNA technology has concerned the public, as many fear that genetically modified bacteria could escape the laboratory and wreak havoc, or that the technology could be abused to create biological weapons. Unlike with nuclear physics, the scientists working on rDNA technology anticipated these concerns very early on. In 1974, a group of scientists led by Paul Berg decided to suspend research into rDNA technology to discuss possible hazards and regulation. This discussion took place at a meeting in Asilomar, California, in 1975 [5].A pertinent similarity between these two areas of science is the confluence of several disciplines to create a hybrid technoscience, in which the boundaries between science and technology have become transient [6]. This convergence was vital for the success of both nuclear physics and later synthetic biology, which combines biotechnology, nanotechnology, information technologies and other new fields that have been created along the way [7]. In physics, technoscience received massive support from the government when the military potential of nuclear fission was realized. Although the splitting of the atom took place before the Manhattan Project, the Second World War served as a catalyst to combine research in nuclear physics with organized and goal-directed funding. As most of this funding came from the government, it changed the relationship between politics and research, as scientists were employed to meet specific goals. In the wake of the detonation of the first atomic bombs, the post-war period was another watershed moment for politics, technoscience, industry and society as it generated new and more intimate relationships between science and governments. These included the appointment of a scientific advisor to the President of the USA, the creation of funding organizations such as the National Science Foundation, or research organizations such as the National Aeronautics and Space Administration, and large amounts of federal funding for technoscience research at private and public universities. It also led to the formation of international organizations such as the civilian-controlled International Atomic Energy Agency [6].There is no global war to serve as a catalyst for government spending on synthetic biology. Although the research has benefited tremendously from government agencies and research infrastructure, the funding for Venter''s team largely came from the private sector. In this regard, the relationship between biological techno-science and industry might already be more advanced than with the public sector given the enormous potential of synthetic life for industrial, medical and environmental applications.Research and innovation at universities has always played a vital role in the success of industry-based capitalism [8]; technoscience is now the major determinant of a knowledge-based economy or ''technocapitalism'' [9]. At the heart of technocapitalism are private and public organizations, driven by research and innovation, which are in sharp contrast to industrial capitalism, where the factories were production-driven and research was of less importance [10]. Furthermore, synthetic biology might provide valuable resources to the scientific community and thereby generate new research opportunities and directions for many biological fields [11].However, given the far-reaching implications of creating synthetic life and the risk of abuse, it is probable that the future relationship between synthetic biology and government will include issues of national security. In the light of potential misuse of synthetic biology for bioterrorism, and the safety risks involved in commercial applications, synthetic biology will eventually require some government regulation and oversight. In contrast to nuclear physics, in which the International Atomic Energy Commission was established only after the atomic bomb, the synthetic biology community should hold a new Asilomar meeting to address concerns and formulate guidelines and management protocols, rather than waiting for politicians or commercial enterprises to regulate the field.So far, synthetic biology differs from nuclear physics in terms of handling information. The Manhattan Project inevitably created a need for secrecy as it was created at the height of the Second World War, but the research maintained this shroud of secrecy after the war. After the bombing of Hiroshima and Nagasaki in August 1945, the US government released carefully compiled documents to the American public. The existence of useable nuclear power had been secret until then, and the control of information ensured that the public further supported or tolerated the technology of nuclear fission and the subsequent use of atomic bombs [12]. This initially positive view changed in the ensuing decades with the threat of a global nuclear war.…synthetic biology has side-stepped the mistakes of nuclear physics and might well achieve a more balanced public integration of future developmentsInformation management in synthetic biology differs from nuclear physics, in that most of the crucial breakthroughs are immediately published in peer-reviewed journals and covered by the media. The value of early public discourse on science issues is evident from the reaction towards genetically modified crops and stem cell research. In this regard, synthetic biology has side-stepped the mistakes of nuclear physics and might well achieve a more balanced public integration of future developments.The main issues that might threaten to dampen public support for synthetic biology and favourable public perception are ethics and biosecurity concerns. Ethical concerns have already been addressed in several forums between scientists and public interest groups; this early engagement between science and society and their continuing dialogue might help to address the public''s ethical objections. In terms of biosecurity, biology might learn from nuclear physics'' intimate entanglement with politics and the military. Synthetic biologists should maintain control and regulation of their research and avoid the fate of nuclear physicists, who were recruited to fight the Cold War and were not free to pursue their own research. For synthetic biology to stay independent of government, industry and society, it must capitalize on its public engagement and heed the lessons and mistakes of nuclear physics'' atom-splitting moment. It should not just evaluate, discuss and address the risks for human or environmental health or biosafety concerns, but should also evaluate potential risks to synthetic biology research itself that could either come from falling public acceptance or government intrusion.? Open in a separate windowAlex J ValentineOpen in a separate windowAleysia KleinertOpen in a separate windowJerome Verdier     

Design-driven, multi-use research agendas to enable applied synthetic biology for global health

Many of the synthetic biological devices, pathways and systems that can be engineered are multi-use, in the sense that they could be used both for commercially-important applications and to help meet global health needs. The on-going development of models and simulation tools for assembling component parts into functionally-complex devices and systems will enable successful engineering with much less trial-and-error experimentation and laboratory infrastructure. As illustrations, I draw upon recent examples from my own work and the broader Keasling research group at the University of California Berkeley and the Joint BioEnergy Institute, of which I was formerly a part. By combining multi-use synthetic biology research agendas with advanced computer-aided design tool creation, it may be possible to more rapidly engineer safe and effective synthetic biology technologies that help address a wide range of global health problems.     

Overexpression of a synthetic gene encoding human alpha interferon in Escherichia coli

Interferons (IFNs) represent an important defense mechanism in vertebrates. In this work, we describe gene synthesis and assembly using the polymerase chain reaction as a method for single-step synthesis of DNA sequences. The oligonucleotides designed were based on Escherichia coli codon usage and two genes of IFN were synthesized: one containing a DNA sequence already known and the other, a mutated form in which two cysteine amino acid residues were replaced by serines in an attempt to improve the stability of the protein. DNA sequences were cloned into pAE, an E. coli vector that allows heterologous protein expression with or without a histidine tag. Recombinant human interferons (rhIFNs) were identified by Western blotting and ELISA using anti-human interferon polyclonal antibodies. Purification of the recombinant His-tagged proteins was achieved in a single step by Ni(2+)-charged column chromatography while proteins without His-tag were purified by extensively washing the inclusion bodies, the final yields being approximately 210 and 75mg/L, respectively. The rhIFNs expressed within this system were biologically active ( approximately 1,1x10(8)IU/mg) based on antiviral assay. The combined methodologies described here proved to be cost-effective and could be extended to other genes/proteins of interest.     

Evolving cell models for systems and synthetic biology

This paper proposes a new methodology for the automated design of cell models for systems and synthetic biology. Our modelling framework is based on P systems, a discrete, stochastic and modular formal modelling language. The automated design of biological models comprising the optimization of the model structure and its stochastic kinetic constants is performed using an evolutionary algorithm. The evolutionary algorithm evolves model structures by combining different modules taken from a predefined module library and then it fine-tunes the associated stochastic kinetic constants. We investigate four alternative objective functions for the fitness calculation within the evolutionary algorithm: (1) equally weighted sum method, (2) normalization method, (3) randomly weighted sum method, and (4) equally weighted product method. The effectiveness of the methodology is tested on four case studies of increasing complexity including negative and positive autoregulation as well as two gene networks implementing a pulse generator and a bandwidth detector. We provide a systematic analysis of the evolutionary algorithm’s results as well as of the resulting evolved cell models.     

In vivo engineering of proteins with nitrogen-containing tryptophan analogs

Recently, it has become possible to reprogram the protein synthesis machinery such that numerous noncanonical amino acids can be translated into target sequences yielding tailor-made proteins. The canonical amino acid tryptophan (Trp) encoded by a single nucleotide triplet (UGG) is a particularly interesting target for protein engineering and design. Trp-residues can be substituted with a variety of analogs and surrogates generated biosynthetically or by organic chemistry. Among them, nitrogen-containing tryptophan analogs occupy a central position, as they have distinct chemical properties in comparison with aliphatic amines and imines. They resemble purine bases of DNA and share their capacity for pH-sensitive intramolecular charge transfer. These special properties of the analogs can be directly transmitted into related protein structures via in vivo ribosome-mediated translation. Proteins expressed in this way are further endowed with unique properties like new spectral, altered redox and titration features or might serve as useful biomaterials. We present and discuss current works and future developments in protein engineering with nitrogen-containing tryptophan analogs and related compounds as well as their relevance for academic and applicative research.The term noncanonical amino acid refers to an amino acid that does not belong, in contrast to a canonical amino acid, to the genetically encoded, proteinogenic amino acids. The term analog defines a strict isosteric exchange of a canonical/noncanonical amino acid (e.g., tryptophan/azatryptophan), while the term surrogate defines a nonisosteric change (e.g., tryptophan/azulene). Mutant denotes a protein in which the wild-type sequence was changed by site-directed mutagenesis (codon manipulation on the DNA level) within the repertoire of the standard amino acids. Variant denotes a protein in which one or more canonical amino acids derived from a wild-type or a mutant sequence were replaced by a noncanonical one (expanded amino acid repertoire, codon reassignment on the protein translation level).