Synthetic biology

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Synthetic Biology Research at NASA Ames.

Synthetic biology is an interdisciplinary branch of biology and engineering.

The subject combines disciplines from within these domains, such as biotechnology, genetic engineering, molecular biology, molecular engineering, systems biology, membrane science, biophysics, chemical and biological engineering, electrical and computer engineering, control engineering and evolutionary biology. Synthetic biology applies these disciplines to build artificial biological systems for research, engineering and medical applications.

Definition

Synthetic biology is seen differently by biologists and engineers. Originally seen as part of biology, in recent years the role of electrical and chemical engineering has become more important. For example, one description designates synthetic biology as "an emerging discipline that uses engineering principles to design and assemble biological components".[1] Another portrayed it as "a new emerging scientific field where ICT, biotechnology and nanotechnology meet and strengthen each other".[2]

The definition of synthetic biology is also debated in the human sciences, arts and politics.[3] One popular definition:[4]

"designing and constructing biological modules,[5] biological systems, and biological machines or, re-design of existing biological systems for useful purposes".

The functional aspects of this definition are rooted in molecular biology and biotechnology.[6]

As usage of the term has expanded, synthetic biology was recently defined as the artificial design and engineering of biological systems and living organisms for purposes of improving applications for industry or biological research.[7]

Synthetic Biology Open Language (SBOL) standard visual symbols for use with BioBricks Standard

In general its purpose can be described as the design and construction of novel artificial biological pathways, organisms or devices, or the redesign of existing natural biological systems.

Synthetic biology has traditionally been divided into two different approaches. Top down synthetic biology involves using metabolic and genetic engineering techniques to impart new functions to living cells. Bottom up synthetic biology involves creating new biological systems in vitro by bringing together 'non-living' biomolecular components,[8] often with the aim of constructing an artificial cell. Biological systems are thus assembled module-by-module. Cell-free protein expression systems are often employed,[9][10][11] as are membrane-based molecular machinery. There are increasing efforts to bridge the divide between these approaches by forming hybrid living/synthetic cells,[12] and engineering communication between living and synthetic cell populations.[13]

History

The first identifiable use of the term "synthetic biology" was in Stéphane Leduc’s publication of Théorie physico-chimique de la vie et générations spontanées (1910)[14] and his La Biologie Synthétique (1912).[15]

A contemporary interpretation of synthetic biology was given by Polish geneticist Wacław Szybalski in a panel discussion during Eighteenth Annual "OHOLO" Biological Conference on Strategies for the Control of Gene Expression in 1973 Zichron Yaakov, Israel.[16][17]

In 1978, Arber, Nathans and Smith won the Nobel Prize in Physiology or Medicine for the discovery of restriction enzymes, leading Szybalski to offer an editorial comment in the journal Gene:

The work on restriction nucleases not only permits us easily to construct recombinant DNA molecules and to analyze individual genes, but also has led us into the new era of synthetic biology where not only existing genes are described and analyzed but also new gene arrangements can be constructed and evaluated.[18]

A notable advance in synthetic biology occurred in 2000, when two articles in Nature discussed the creation of synthetic biological circuit devices of a genetic toggle switch and a biological clock by combining genes within E. coli cells.[19][20]

Perspectives

Engineers view biology as a technology – a given system's biotechnology or its biological engineering.[21] Synthetic biology includes the broad redefinition and expansion of biotechnology, with the ultimate goals of being able to design and build engineered biological systems that process information, manipulate chemicals, fabricate materials and structures, produce energy, provide food, and maintain and enhance human health (see Biomedical Engineering) and our environment.[22]

Studies in synthetic biology can be subdivided into broad classifications according to the approach they take to the problem at hand: standardization of biological parts, biomolecular engineering, genome engineering. Biomolecular engineering includes approaches that aim to create a toolkit of functional units that can be introduced to present new technological functions in living cells. Genetic engineering includes approaches to construct synthetic chromosomes for whole or minimal organisms. Biomolecular design refers to the general idea of de novo design and additive combination of biomolecular components. Each of these approaches share a similar task: to develop a more synthetic entity at a higher level of complexity by inventively manipulating a simpler part at the preceding level.[23]

On the other hand, "re-writers" are synthetic biologists interested in testing the irreducibility of biological systems. Due to the complexity of natural biological systems, it would be simpler to rebuild the natural systems of interest from the ground up; In order to provide engineered surrogates that are easier to comprehend, control and manipulate.[24] Re-writers draw inspiration from refactoring, a process sometimes used to improve computer software.

Enabling technologies

Several novel enabling technologies were critical to the success of synthetic biology. Concepts include standardization of biological parts and hierarchical abstraction to permit using those parts in synthetic systems.[25] Basic technologies include reading and writing DNA (sequencing and fabrication). Measurements under multiple conditions are needed for accurate modeling and computer-aided-design (CAD).

DNA and gene synthesis

Driven by dramatic decreases in costs of oligonucleotide ("oligos") synthesis, the sizes of DNA constructions from oligos have increased to the genomic level.[26] In 2000, researchers reported synthesis of the 9.6 kbp (kilo bp) Hepatitis C virus genome from chemically synthesized 60 to 80-mers.[27] In 2002 researchers at SUNY Stony Brook succeeded in synthesizing the 7741 bp poliovirus genome from its published sequence, producing the second synthetic genome, spanning two years.[28] In 2003 the 5386 bp genome of the bacteriophage Phi X 174 was assembled in about two weeks.[29] In 2006, the same team, at the J. Craig Venter Institute, constructed and patented a synthetic genome of a novel minimal bacterium, Mycoplasma laboratorium and were working on getting it functioning in a living cell.[30][31][32]

In 2007 it was reported that several companies were offering synthesis of genetic sequences up to 2000 base pairs (bp) long, for a price of about $1 per bp and a turnaround time of less than two weeks.[33] Oligonucleotides harvested from a photolithographic- or inkjet-manufactured DNA chip combined with DNA mismatch error-correction allows inexpensive large-scale changes of codons in genetic systems to improve gene expression or incorporate novel amino-acids (see George M. Church's and Anthony Forster's synthetic cell projects.[34][35] This favors a synthesis-from-scratch approach.

Additionally, the CRISPR/Cas system has emerged as a promising technique for gene editing. It was described as "the most important innovation in the synthetic biology space in nearly 30 years".[36] While other methods take months or years to edit gene sequences, CRISPR speeds that time up to weeks.[36] Due to its ease of use and accessibility, however, it has raised ethical concerns, especially surrounding its use in biohacking.[37][38][39]

Sequencing

DNA sequencing determines the order of nucleotide bases in a DNA molecule. Synthetic biologists use DNA sequencing in their work in several ways. First, large-scale genome sequencing efforts continue to provide information on naturally occurring organisms. This information provides a rich substrate from which synthetic biologists can construct parts and devices. Second, sequencing can verify that the fabricated system is as intended. Third, fast, cheap, and reliable sequencing can facilitate rapid detection and identification of synthetic systems and organisms.[40]

Microfluidics

Microfluidics, in particular droplet microfluidics, is an emerging tool used to construct new components, and to analyse and characterize them.[41][42] It is widely employed in screening assays.[43]

Modularity

The most used[44]:22–23 standardized DNA parts are BioBrick plasmids, invented by Tom Knight in 2003.[45] Biobricks are stored at the Registry of Standard Biological Parts in Cambridge, Massachusetts. The BioBrick standard has been used by thousands of students worldwide in the international Genetically Engineered Machine (iGEM) competition.[44]:22–23

While DNA is most important for information storage, a large fraction of the cell's activities are carried out by proteins. Tools can send proteins to specific regions of the cell and to link different proteins together. The interaction strength between protein partners should be tunable between a lifetime of seconds (desirable for dynamic signaling events) up to an irreversible interaction (desirable for device stability or resilient to harsh conditions). Interactions such as coiled coils,[46] SH3 domain-peptide binding[47] or SpyTag/SpyCatcher[48] offer such control. In addition it is necessary to regulate protein-protein interactions in cells, such as with light (using light-oxygen-voltage-sensing domains) or cell-permeable small molecules by chemically induced dimerization.[49]

In a living cell, molecular motifs are embedded in a bigger network with upstream and downstream components. These components may alter the signalling capability of the modeling module. In the case of ultrasensitive modules, the sensitivity contribution of a module can differ from the sensitivity that the module sustains in isolation.[50][51]

Modeling

Models inform the design of engineered biological systems by better predicting system behavior prior to fabrication. Synthetic biology benefits from better models of how biological molecules bind substrates and catalyze reactions, how DNA encodes the information needed to specify the cell and how multi-component integrated systems behave. Multiscale models of gene regulatory networks focus on synthetic biology applications. Simulations can model all biomolecular interactions in transcription, translation, regulation and induction of gene regulatory networks.[52] [53]

Synthetic transcription factors

Studies have considered the components of the DNA transcription mechanism. One desire of scientists creating synthetic biological circuits is to be able to control the transcription of synthetic DNA in prokaryotes and eukaryotes. One study tested the adjustability of synthetic transcription factors (sTFs) in areas of transcription output and cooperative ability among multiple transcription factor complexes.[54] Researchers were able to mutate zinc fingers, the DNA specific component of sTFs, to decrease their affinity for specific operator DNA sequence sites, and thus decrease the associated site-specific activity of the sTF (usually transcriptional regulation). They further used the zinc fingers as components of complex-forming sTFs, which are the eukaryotic translation mechanisms.[55]

Applications

Biological computers

A biological computer refers to an engineered biological system that can perform computer-like operations, which is a dominant paradigm in synthetic biology. Researchers built and characterized a variety of logic gates in a number of organisms,[56] and demonstrated both analog and digital computation in living cells. They demonstrated that bacteria can be engineered to perform both analog and/or digital computation.[57][58] In human cells research demonstrated a universal logic evaluator that operates in mammalian cells in 2007.[59] Subsequently, researchers utilized this paradigm to demonstrate a proof-of-concept therapy that uses biological digital computation to detect and kill human cancer cells in 2011.[60] Another group of researchers demonstrated in 2016 that principles of computer engineering, can be used to automate digital circuit design in bacterial cells.[61] In 2017, researchers demonstrated the 'Boolean logic and arithmetic through DNA excision' (BLADE) system to engineer digital computation in human cells.[62]

Biosensors

A biosensor refers to an engineered organism, usually a bacterium,that is capable of reporting some ambient phenomenon such as the presence of heavy metals or toxins. One such system is the Lux operon of Aliivibrio fischeri,[63] which codes for the enzyme that is the source of bacterial bioluminescence, and can be placed after a respondent promoter to express the luminescence genes in response to a specific environmental stimulus.[64] One such sensor created consisted of a bioluminescent bacterial coating on a photosensitive computer chip to detect certain petroleum pollutants. When the bacteria sense the pollutant, they luminesce.[65]

Modified organisms can sense environmental signals and send output signals that can be detected and serve diagnostic purposes. Microbe cohorts have been used.[66]

Cell transformation

Cells use interacting genes and proteins, which are called gene circuits, to implement diverse function, such as responding to environmental signals, decision making and communication. Three key components are involved: DNA, RNA and Synthetic biologist designed gene circuits that can control gene expression from several levels including transcriptional, post-transcriptional and translational levels.

Traditional metabolic engineering has been bolstered by the introduction of combinations of foreign genes and optimization by directed evolution. This includes engineering E. coli and yeast for commercial production of a precursor of the antimalarial drug, Artemisinin.[67]

Entire organisms have yet to be created from scratch, although living cells can be transformed with new DNA. Several ways allow constructing synthetic DNA components and even entire synthetic genomes, but once the desired genetic code is obtained, it is integrated into a living cell that is expected to manifest the desired new capabilities or phenotypes while growing and thriving.[68] Cell transformation is used to create biological circuits, which can be manipulated to yield desired outputs.[19][20]

By integrating synthetic biology with materials science, it would be possible to use cells as microscopic molecular foundries to produce materials with properties whose properties were genetically encoded. Re-engineering has produced Curli fibers, the amyloid component of extracellular material of biofilms, as a platform for programmable nanomaterial. These nanofibers were genetically constructed for specific functions, including adhesion to substrates, nanoparticle templating and protein immobilization.[69]

Designed proteins

The Top7 protein was one of the first proteins designed for a fold that had never been seen before in nature[70]

Natural proteins can be engineered, e.g., by directed evolution, novel protein structures that match or improve on the functionality of existing proteins can be produced. One group generated a helix bundle that was capable of binding oxygen with similar properties as hemoglobin, yet did not bind carbon monoxide.[71] A similar protein structure was generated to support a variety of oxidoreductase activities.[72] Another group generated a family of G-protein coupled receptors that could be activated by the inert small molecule clozapine-N-oxide but insensitive to the native ligand, acetylcholine.[73]

Another common investigation is expansion of the natural set of 20 amino acids. Excluding stop codons, 61 codons have been identified, but only 20 amino acids are coded generally in all organisms. Certain codons are engineered to code for alternative amino acids including: nonstandard amino acids such as O-methyl tyrosine; or exogenous amino acids such as 4-fluorophenylalanine. Typically, these projects make use of re-coded nonsense suppressor tRNA-Aminoacyl tRNA synthetase pairs from other organisms, though in most cases substantial engineering is required.[74]

Other researchers investigated protein structure and function by reducing the normal set of 20 amino acids. Limited protein sequence libraries are made by generating proteins where groups of amino acids may be replaced by a single amino acid.[75] For instance, several non-polar amino acids within a protein can all be replaced with a single non-polar amino acid.[76] One project demonstrated that an engineered version of Chorismate mutase still had catalytic activity when only 9 amino acids were used.[77]

Researchers and companies practice synthetic biology to synthesize industrial enzymes with high activity, optimal yields and effectiveness. These synthesized enzymes aim to improve products such as detergents and lactose-free dairy products, as well as make them more cost effective.[78] The improvements of metabolic engineering by synthetic biology is an example of a biotechnological technique utilized in industry to discover pharmaceuticals and fermentive chemicals. Synthetic biology may investigate modular pathway systems in biochemical production and increase yields of metabolic production. Artificial enzymatic activity and subsequent effects on metabolic reaction rates and yields may develop "efficient new strategies for improving cellular properties ... for industrially important biochemical production".[79]

Designed nucleic acid systems

Scientists can encode digital information onto a single strand of synthetic DNA. In 2012, George M. Church encoded one of his books about synthetic biology in DNA. The 5.3 Mb of data was more than 1000 times greater than the previous largest amount of information to be stored in synthesized DNA.[80] A similar project encoded the complete sonnets of William Shakespeare in DNA.[81]

Many technologies have been developed for incorporating unnatural nucleotides and amino acids into nucleic acids and proteins, both in vitro and in vivo. For example, in May 2014, researchers announced that they had successfully introduced two new artificial nucleotides into bacterial DNA. By including individual artificial nucleotides in the culture media, they were able to exchange the bacteria 24 times; they did not generate mRNA or proteins able to use the artificial nucleotides.[82][83][84]

Space exploration

Synthetic biology raised NASA's interest as it could help to produce resources for astronauts from a restricted portfolio of compounds sent from Earth.[85][86][87] On Mars, in particular, synthetic biology could lead to production processes based on local resources, making it a powerful tool in the development of manned outposts with less dependence on Earth.[85]

Synthetic life

Gene functions in the minimal genome of the synthetic organism, Syn 3.[88]

One important topic in synthetic biology is synthetic life, that is concerned with hypothetical organisms created in vitro from biomolecules and their component materials. Synthetic life experiments attempt to either probe the origins of life, study some of the properties of life, or more ambitiously to recreate life from non-living (abiotic) components. Synthetic life biology attempts to create living organisms capable of carrying out important functions, from manufacturing pharmaceuticals to detoxifying polluted land and water.[89] In medicine, it offers prospects of using designer biological parts as a starting point for new classes of therapies and diagnostic tools.[89]

A living "artificial cell" has been defined as a completely synthetic cell that can capture energy, maintain ion gradients, contain macromolecules as well as store information and have the ability to mutate.[90] Nobody has been able to create such a cell.[90]

A completely synthetic bacterial chromosome was produced in 2010 by Craig Venter, and his team introduced it to genomically emptied bacterial host cells.[91] The host cells were able to grow and replicate.[92][93]

The first living organism with 'artificial' expanded DNA code was presented in 2014; the team used E. coli that had its genome extracted and replaced with a chromosome with an expanded genetic code. The nucleosides added are d5SICS and dNaM.[84]

Drug delivery platforms

Engineered bacteria-based platform

Bacteria have long been used in cancer treatment. Bifidobacterium and Clostridium selectively colonize tumors and reduce their size.[94] Recently synthetic biologists reprogrammed bacteria to sense and respond to a particular cancer state. Most often bacteria are used to deliver a therapeutic molecule directly to the tumor to minimize off-target effects. To target the tumor cells, peptides that can specifically recognize a tumor were expressed on the surfaces of bacteria. Peptides used include an affibody molecule that specifically targets human epidermal growth factor receptor 2[95] and a synthetic adhsin.[96] The other way is to allow bacteria to sense the tumor microenvironment, for example hypoxia, by building an AND logic gate into bacteria.[97] The bacteria then only release target therapeutic molecules to the tumor through either lysis[98] or the bacterial secretion system.[99] Lysis has the advantage that it can stimulate the immune system and control growth. Multiple types of secretion systems can be used and other strategies as well. The system is inducible by external signals. Inducers include chemicals, electromagnetic or light waves.

Multiple species and strains are applied in these therapeutics. Most commonly used bacteria are Salmonella typhimurium, Escherichia Coli, Bifidobacteria, Streptococcus, Lactobacillus, Listeria and Bacillus subtilis. Each of these species have their own property and are unique to cancer therapy in terms of tissue colonization, interaction with immune system and ease of application.

Cell-based platform

The immune system plays an important role in cancer and can be harnessed to attack cancer cells. Cell-based therapies focus on immunotherapies, mostly by engineering T cells.

T cell receptors were engineered and ‘trained’ to detect cancer epitopes. Chimeric antigen receptors (CARs) are composed of a fragment of an antibody fused to intracellular T cell signaling domains that can activate and trigger proliferation of the cell. A second generation CAR-based therapy was approved by FDA.[citation needed]

Gene switches were designed to enhance safety of the treatment. Kill switches were developed to terminate the therapy should the patient show severe side effects.[100] Mechanisms can more finely control the system and stop and reactivate it.[101][102] Since the number of T-cells are important for therapy persistence and severity, growth of T-cells is also controlled to dial the effectiveness and safety of therapeutics.[103]

Although several mechanisms can improve safety and control, limitations include the difficulty of inducing large DNA circuits into the cells and risks associated with introducing foreign components, especially proteins, into cells.

Bioethics and security

Synthetic biology raises ethical issues and biosecurity issues. With the exception of regulating DNA synthesis companies,[104][105] however, the issues are not seen as new because they were raised during the earlier recombinant DNA and genetically modified organism (GMO) debates and extensive regulations of genetic engineering and pathogen research were in place in many jurisdictions.[106]

Ethical questions include: Who will have control and access to the products of synthetic biology, and who will gain from these innovations? Does the patent system allow patents on living organisms? Can embryos be engineered?[107]

European Union

The European Union-funded project SYNBIOSAFE[108] has issued reports on how to manage synthetic biology. A 2007 paper identified key issues in safety, security, ethics and the science-society interface, which the project defined as public education and ongoing dialogue among scientists, businesses, government and ethicists.[109][110] The key security issues that SYNBIOSAFE identified involved engaging companies that sell synthetic DNA and the biohacking community of amateur biologists. Key ethical issues concerned the creation of new life forms.

A subsequent report focused on biosecurity, especially the so-called dual-use challenge. For example, while synthetic biology may lead to more efficient production of medical treatments, it may also lead to synthesis or modification of harmful pathogens (e.g., smallpox).[111] The biohacking community remains a source of special concern, as the distributed and diffuse nature of open-source biotechnology makes it difficult to track, regulate or mitigate potential concerns over biosafety and biosecurity.[112]

COSY, another European initiative, focuses on public perception and communication.[113][114][115] To better communicate synthetic biology and its societal ramifications to a broader public, COSY and SYNBIOSAFE published SYNBIOSAFE, a 38-minute documentary film, in October 2009.[116]

The International Association Synthetic Biology has proposed self-regulation.[117] This proposes specific measures that the synthetic biology industry, especially DNA synthesis companies, should implement. In 2007, a group led by scientists from leading DNA-synthesis companies published a "practical plan for developing an effective oversight framework for the DNA-synthesis industry".[104]

United States

In January 2009, the Alfred P. Sloan Foundation funded the Woodrow Wilson Center, the Hastings Center, and the J. Craig Venter Institute to examine the public perception, ethics and policy implications of synthetic biology.[118]

On July 9–10, 2009, the National Academies' Committee of Science, Technology & Law convened a symposium on "Opportunities and Challenges in the Emerging Field of Synthetic Biology".[119]

After the publication of the first synthetic genome and the accompanying media coverage about "life" being created, President Barack Obama established the Presidential Commission for the Study of Bioethical Issues to study synthetic biology.[120] The commission convened a series of meetings, and issued a report in December 2010 titled "New Directions: The Ethics of Synthetic Biology and Emerging Technologies." The commission stated that "while Venter’s achievement marked a significant technical advance in demonstrating that a relatively large genome could be accurately synthesized and substituted for another, it did not amount to the “creation of life”.[121] It noted that synthetic biology is an emerging field, which creates potential risks and rewards. The commission did not recommend policy or oversight changes and called for continued funding of the research and new funding for monitoring, study of emerging ethical issues and public education.[106]

Synthetic biology, as a major tool for biological advances, results in the "potential for developing biological weapons, possible unforeseen negative impacts on human health ... and any potential environmental impact".[122] These security issues may be avoided by regulating industry uses of biotechnology through policy legislation. Federal guidelines on genetic manipulation are being proposed by "the President's Bioethics Commission ... in response to the announced creation of a self-replicating cell from a chemically synthesized genome, put forward 18 recommendations not only for regulating the science ... for educating the public".[122]

Opposition

On March 13, 2012, over 100 environmental and civil society groups, including Friends of the Earth, the International Center for Technology Assessment and the ETC Group issued the manifesto The Principles for the Oversight of Synthetic Biology. This manifesto calls for a worldwide moratorium on the release and commercial use of synthetic organisms until more robust regulations and rigorous biosafety measures are established. The groups specifically call for an outright ban on the use of synthetic biology on the human genome or human microbiome.[123][124] Richard Lewontin wrote that some of the safety tenets for oversight discussed in The Principles for the Oversight of Synthetic Biology are reasonable, but that the main problem with the recommendations in the manifesto is that "the public at large lacks the ability to enforce any meaningful realization of those recommendations".[125]

Health and safety

As research procedures scale up to industrial processes, the number and variety of workers exposed to commercial synthetic biology risk is expected to increase. Exposure to lentiviral vectors is one important health hazard. Hazard controls include increased workplace health surveillance and proactive risk assessment and management, as well as biocontainment methods.[126] Risk assessment may be more difficult for organisms constructed from individual synthetic genetic components since they do not occur in nature. Because synthetic biology engages a wide range of disciplines outside of biology who may be less familiar with microbiological risk assessment.[127]

See also

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Bibliography

  • European commission (2016) Synthetic biology and biodiversity ; Science for Environment Policy (2016) ; Future Brief 15. Produced for the European Commission DG Environment by the Science Communication Unit, UWE, Bristol. <http://ec.europa.eu/science-environment-policy>, PDF, 36 pages

External links

  • Synthetic biology books recent popular science book and textbooks
  • Introductory Summary of Synthetic Biology. Concise overview of synthetic biology concepts, developments and applications
  • Collaborative overview article on Synthetic Biology
  • Controversial DNA startup wants to let customers create creatures (2015-01-03), San Francisco Chronicle
  • It's Alive, But Is It Life: Synthetic Biology and the Future of Creation (28 Sept 2016), World Science Festival
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