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1. IntroductionSynthetic biology (SynBio) aims to: a) design and engineer biologically based parts, novel devices and systems not found in nature and b) re-design existing natural biological systems for useful purposes
SynBio strives to make the engineering of biology easier and more predictable
and is influenced by a wide variety of fields such as genetic engineering, biochemistry, bioinformatics, microbiology and nanotechnology .
Erwin Schr?dinger, a Nobel Laureate for his work on quantum physics, was intrigued by how life seemed to create order in a molecular level while the entropy of the universe continued increasing. Moreover, living things could pass on that order from one generation to the next. His article 'What Is Life?' inspired James Watson and Francis Crick and the subsequent discovery of DNA. Though there was no unanimous definition of DNA, life scientists decided that it was a self-sustaining chemical system capable of undergoing Darwinian evolution . Scientists began to understand life as a system, consisting of building blocks which again arose from molecular complexes. Hence, the conventional approach to biological research has been to isolate a few genes or proteins in order to understand their structure and function. The understanding that biological systems are multi-level and multi-scale has led to a realization that biological systems can no longer be studied using a reductionist approach (assuming that single biochemical events result in single effects). In fact, there is a complex network of interactions between biological components (e.g. genes, proteins), with positive and negative feedback loops that regulate their operation. This systems approach led to the emergence of systems biology, as well as synthetic biology . SynBio has the potential to produce clean fuel in an efficient and sustainable manner , to fabricate practical organisms that could clean hazardous waste in inaccessible places , to recognize and destroy tumors , to produce newer analogues of existing drugs with more specificity and less side-effects , to use plants to sense chemicals and respond accordingly
and a wide range of other applications. In the field of energy, SynBio is being used to develop much more efficient biofuels, which have the potential to alleviate current problems like competition for land use between energy and food crops . The actual process of deriving biofuels from crops such as sugar cane or palm oil wastes around 90% of the biomass. SynBio derived biofuels are being developed in order to use a much higher percentage of the biomass, leading to a significant increase in yields and carbon savings .In health, the synthetic anti-malarial drug artemisinin - which is being developed using SynBio techniques - could be produced in large scale and have a major impact on the treatment of malaria in the developing world. Also, the cost of treatment should be low as the development of the drug is being funded by the Gates Foundation
. Whether addressing an existing problem or creating new capabilities, efficient solutions can be inspired by, but need not necessarily mimic, natural biological processes. Our new designs could even be more robust or efficient than systems that have been fashioned by evolution .SynBio will revolutionize how we conceptualize and approach the engineering of biological systems. The vision and applications of this emerging field will influence a lot of scientific and engineering disciplines and affect various aspects of daily life and society .2. HistoryIn 1980, the term 'synthetic biology' came out in the literature when Barbara Hobom described genetically engineered bacteria, using recombinant DNA technology. Bacteria are living systems (hence biological) that have been modified by human intervention (that is, synthetically). In this respect, synthetic biology was mainly synonymous with 'bioengineering' . In 2000, the title 'synthetic biology' was presented again by Eric Kool and other speakers at the annual meeting of the American Chemical Society in San Francisco. Here, the term was related to the synthesis of unnatural organic molecules that operate in living systems. More generally in this sense, the term has been used with reference to attempts to 'redesign life'
. In this sense, the term is an expansion of the concept of 'biomimetic chemistry', where organic synthesis is used to produce artificial molecules that summarize the behavior of biological components ( e.g. enzymes). However, synthetic biology has a wider scope because it aims to recreate in unnatural chemical systems the emergent properties of living systems (e.g. inheritance, genetics, evolution) .In 2004, a community of engineers and scientists gave further meaning to the term. As it is described in section '3.1.3 Bioparts', and according to Benner & Sismour, 2005 "this community seeks to extract from living systems interchangeable parts that might be tested, validated as construction units, and reassembled to create devices that might (or might not) have analogues in living systems. The parts come from natural living systems (that is, they are biological); their assembly is, however, unnatural. Therefore, one engineering goal might be to assemble biological components (such as proteins that bind DNA and the DNA sequences that they bind) to create, for example, outputs analogous to those of a computer.”3. Basic Concepts3.1 Bioinformatics and systems biology for synthetic biologyAs synthetic biology develops and matures as a field, it has to develop its own ontology and tool box. For large scale synthetic biology applications, bioinformatics and systems biology models will be harnessed using engineering principles of computer aided synthetic modules and parts design. Trend for this has already started. Here we review developments in this interface discipline variously christened as synthetic bioinformatics or synthetic systems biology. We also enlist the direction and trends for this emerging field.Bioinformatics uses biological information and computational methods to discover knowledge. The application of its methods results in a collective construction that is constantly being deposited in distributed information systems in which knowledge about interesting biomolecules is organized. It ultimately encodes relationships between composition (sequence), structure, functions, binding, abundance, etc. Bioinformatics products are, therefore, of prime importance for SynBio. Likewise, Bioinformatics methodologies, often resulting from the combination of others, are highly inspirational for SynBio. Bioinformatics is keen on evaluating similarities and screening results against defined discriminating criteria. It is also keen on integrating heterogenous data resources, often originated in vast data set, and distilling the most valuable results. It is expected that bioinformatics will provide contributions to SynBio on a wide variety of levels, that can range from the selection and characterization of components (parts or modules), to the derivation of naturally inspired design rules, and to the creation of protocols for testing its products. Conversely, SynBio is constantly creating new challenges for bioinformatics practitioners - both developers and users, especially but not exclusively in the area of confluence with systems biology.SynBio will open new possibilities of validating models and predictions that emerge from bioinformatics studies in the laboratory or even in organisms. The validity of in-silico predictions is often poorly tested because it involves inducing difficult changes in biological systems that aim at reproducing the changes that are easily induced in models. BioParts can play a very valuable role there, by making such changes possible by direct replacement of parts, such as a in the case of the activation/inactivation of a sector of a pathway. 3.1.1 The relationship between systems biology and synthetic biologySystems biology aims to study natural biological systems in their entirety, frequently with a biomedical focus, and uses simulation and modeling tools in comparisons with experimental information. Synthetic biology aims to construct novel and artificial biological parts, devices and systems. These two disciplines share a lot of methods, so there is a close relationship between them. But in SynBio, the methods are used as the basis for engineering applications. The basis of quantitative systems biology resides in the application of signal theory, concerning analysis or operations on signals over discret and engineering systems to the analysis of biological systems, allowing the description of systems in terms of mathematical equations. Once a system, or part of a system, has been defined in this manner, then synthetic biology permits the reduction of the system to biological parts (bioparts) whose function is represented in terms of input/output characteristics. These characteristics are then presented on a standard specification sheet, quite like that of electronic components in the form of datasheets, so that a system designer can comprehend the functional characteristics of the part. The parts are then entered into an inventory. The parts defined in an inventory (registry) can then be assembled into devices and, finally, into systems. The design of any engineering part, device or system has tolerances to compensate for defects in the manufacturing. Bioparts tend to have broader tolerances than standard engineering parts, so biologically-based devices are designed to fit better for such features. Therefore, synthetic biology includes the classic reductionist method through which complex systems or processes are built from defined parts and devices
whereas systems biology is typically 'anti-reductionistic' or holistic. 3.1.2 The Engineering design cycle and rational designA key aspect of SynBio is the application to biology of methods that are ordinarily used in engineering design and development. The essence of this approach is to describe the specification of the part, device or system that is needed and to develop a design which fits these specifications. Therefore, in engineering, systems are usually built from standard devices, which in turn are built from standard parts. These standard parts and devices are all adequately characterized and may be used in the design of multiple systems. This general approach is part of what is famous as the engineering cycle .We encourage you to take a look at the figure 2 of “Synthetic Biology: scope, applications and implications” by The Royal Academy of Engineering . It can be seen that the specification step is followed by an elaborated design step. One of the main features of the modern design is the ability to undertake detailed computer modeling. This is valid to synthetic biology as well. Nowadays it is possible to carry out detailed computer modeling due to advances in technology and therefore simulate in detail the expected behavior of the part, device or system under development. The next stage is implementation (in SynBio it means e.g. to modify synthetic DNA and insert it into an E. coli cell or some other chassis). The following stage, testing and validation, is especially meaningful in SynBio because the response to the insertion of modified bacterial DNA determines whether or not the specification and the design have been realized the right way .The elaboration of a part, device or system can involve many iterations of the cycle, occurring a refinement of the design and its implementation. The field of electronics is sometimes used as a conceptual model for SynBio. For instance, a simple audio amplifier is designed using standard resistors, capacitors and transistors. There is a set of specifications for the amplifier that the designer has to follow, and he has to find component parts which meet the exact specifications. It is essential to note that once built, tested and validated, the audio amplifier becomes a standard device built from standard parts – with its own specification sheet - the same is true for standard parts and devices in synthetic biology . 3.1.3 BiopartsA biopart is a modular biological part designed to be easily combined with other parts. Ultimately, the intention is to produce a range of standard devices (built from standard parts) to be used in standard systems. The biopart standard gives a framework where parts can be re-used in a lot of applications to achieve a specific function. The behavior of any biopart component is characterized on a data sheet comprising a set of parameters and performance descriptions. A particular combination of parts (a device) is then modeled prior to physical assembly of parts, to guarantee correct functionality The BioBricks Foundation is a nonprofit organization founded by engineers and scientists from MIT, Harvard, and UCSF. The Registry of Standard Biological Parts, run by MIT, contains information about the bioparts or BioBricks(TM). "What makes us truly unique is our focus on standardizing BioBrick(TM) parts. This has never been done before. We’re also the only organization actively developing industry standards for biotechnology. And we’re the first to stage the world." - BioBricks Foundation . 4. Methods and Fundamental Techniques in Synthetic BiologyThere are three key technological enablers that have facilitated the rise and rapid progress of SynBio: computational modeling, DNA synthesis and DNA sequencing.4.1 Computational modelingSynthetic biology, just like systems biology, depends on computer modeling of biological processes. Lately, multi-scale models of gene regulatory networks have been developed to model the entire set of bio-molecular interactions in gene regulatory networks, (i.e. transcription, translation and regulation by induction or repression). There are commercially available software tools for systems biologists, but there is a demand for an integrated design environment (IDE) for synthetic biologists, like the computer aided design (CAD) systems developed for other areas of engineering . High Throughput Computing has helped validate drug discovery using 'parallel computing'
and 'cloud computing' . For the exact specification, design, modeling and validation of devices and systems, the quantitative measurement of biological parameters is essential. A reason for that is because discrepancies between the behavior predicted by a model and real measurements may identify defects in actual biological control hypotheses and shed light on the malfunction of synthetic systems. In the future, technologies that allow a lot of parallel, even single cell, and time-dependent measurements, will be particularly powerful for synthetic biology . (A list of bioinformatics tools used in SynBio and a description of their application can be found here: 8.2 )
4.2 DNA synthesisChemical synthesis of DNA or oligonucleotide synthesis is an essential component of synthetic biology. Thanks to the advancements in automated DNA synthesizer, it is now possible to synthesize and assemble complete genes, regulatory elements, gene circuits or even entire genomes of micro organisms. Khorana and co-workers pioneered the chemical synthesis of DNA from oligonucleotides and demonstrated the first chemical synthesis of a yeast tRNA gene . This process is also known as artificial synthesis of gene because no initial DNA template was used. Somatostatin was the first peptide and Leukocyte Interferon was the first protein-coding gene to be chemically synthesized and expressed in bacteria . These studies demonstrated the potential applications of synthetic blology. Chemical synthesis of DNA is often more straight forward and economical than recombinant DNA cloning and is routinely employed in biotechnology.4.3 DNA sequencing First of all, DNA has to be extracted from the cell. This is achieved by mechanical or chemical means, followed by purification and separation of the DNA strands from binding proteins. Then they are incorporated into plasmid vectors, and transferred to bacteria or virus, which divides exponentially to form a high yield of DNA clones. The strands are separated by heating, followed by 'primer annealing'. Next, 'extension' of the strand is done using deoxynucleotides (dNTP), with the help of DNA Polymerase. In the 'termination' step, fluorescent-labeled dideoxynucleotides (ddNTP) are attached leading to termination of further chain growth as ddNTPs lack 3' hydroxyl group without which DNA Polymerase can not work. Each four ddNTPs are coded with a differently colored fluorescent dye. The ratio of dNTPs to ddNTPs are carefully chosen to obtain different chain lengths. In the next step, these mixtures are poured into a slender capillary tube which is subjected to an electrical field (capillary electrophoresis). The smallest pass through first and in this order the largest exits last. As they exit, a laser excites the fluorescent-labeled base and the event is read by a photocell which ultimately passes the signal onto a computer which decodes the 'terminator' base by showing a peak characteristic of that particular base (Fig. ). Sequencing is done in this (smallest first etc.) sequence. The sequencing of whole genomes of several organisms has provided a wealth of information regarding the chassis within which synthetic biologists try to construct functional devices in vitro. (Chassis are the environments or scaffolds into which synthetic DNA is placed). Also, sequencing is used to assure that engineered sections of DNA or even entire organisms have been correctly fabricated (i.e. proofread). The rapid speed of sequencing of DNA by DNA chip [see
the animation] and the inexpensive costs will help to detect and identify novel systems and organisms. The decade since the Human Genome Project finished has witnessed a remarkable sequencing technology explosion that has allowed numerous questions about the genome to be asked and answered, at unprecedented speed and resolution
. Sequencing technology seems poised to another landmark shift as Life Technologies announced single day human genome sequencing at the cost of an ipad with its revolutionary ion proton technology . Oxford nanopore has reportedly develop a nanopore based disruptive DNA strand sequencing technology . These advances will provide a synergistic effect for advances in synthetic biology. 4.4 DNA Parts AssemblyDNA assembly challenge is to take a set of double-stranded DNA sequence fragments, and stitch them together in the user-defined order and orientation to yield a single, potentially circular, assembled DNA sequence.The following is the development progress of DNA parts assembly, from which it is inferred that parts standardization, one-step (cut-ligation), and large-scale are the future. 1. Traditional MCS (Multiple Cloning Site) Approach3. SLIC, Gibson, CPEC and USER assembly methods.4. Golden Gate assembly method (and MoClo and GoldenBraid)Self-explaining details for public are available in J5's webpage . In addition, more professional guides and protocols are available from the journal The Methods in Enzymology. Volume 497, Pages 2-662 (2011), is dedicated to Synthetic Biology . 4.5 DNA Part Analysis and Description Standards
4.6 DNA arraysGene expression in eukaryotes is a dynamic process and involves complex interactions of genetic regulatory networks (or GRN) . We need to understand the design principles of these networks in order to advance synthetic biology. Micro arrays or gene chips are mainly made up of known cDNA (complimentary DNA) or oligonucleotide sequences of thousands of genes on a chip and extensively used for gene expression analysis; see animation . This method is based on hybridization of labeled cDNA species derived from biological samples to the gene chips. This step is followed by fluorescent detection and quantitative analysis of data to reveal differential expression. Micro arrays enable simultaneous, quantitative monitoring of expression of thousands of genes in response to multiple experimental conditions on a single platform. The data sets of gene expression are subjected to a clustering algorithm to reveal genes that are co-expressed (simultaneously increased or decreased) and are regulated as discrete networks . 5.Biological networks and circuits5.1 The concept of regulatory and metabolic circuits 5.2 Oscillators5.2.1 Types of oscillators5.2.1.1 The Elowitz and Leiber Represillator5.2.1.2 The Atkinson Oscillator5.2.1.3 The Metabolator5.2.1.4 Circadian circuits 5.2.2 Designing oscillators (with examples) 5.3 Inverters 5.3.1 Designing inverters (with examples) 5.4 Chemotaxis and quorum sensing 5..4.1 Cell - cell communication systems
5.5 Design and characterization of synthetic gene networks - coordinated expression- stability of expression- transcriptional cascades- toggles- logic gates- signal amplifiers- pulse generators
6. Applications of Synthetic Biology 6.1 Synthetic biology and health Artemesinin, an antimalarial drug, is one of the prototypical drug that is being synthesized at a low cost using SynBio technique. Although the Chinese have been using this herbal medication (qinghaosu) containing the active principle for over 2000 years as an antipyretic, the pure form was prepared only recently. Now it is being synthesized in E.coli or as a precursor, amorphadiene in yeast which can be chemically converted to artemisinin
. Sulfonamides (antimicrobials), many antimetabolite anticancer drugs, monoclonal antibodies like Trastuzumab (used in the treatment of breast cancer), Rituximab (used in the treatment of non-Hodgkin's lymphoma) are some of the many designer drugs synthesized using SynBio approach . 6.2 Synthetic biology and environment 6.3 Synthetic biology and new sources of energy A microbial platform for the direct production of bioethanol from alginate, a polysaccharide constituent of seaweed was reported
. The key advance in this platform is the bioengineering of enzymes involved in alginate metabolism and transport from Vibrio splendidus into E.coli for industrial applications. 6.4 Synthetic biology and new biomaterials In a very recent development scientists at Wyss Institute created a DNA nanorobot that could harmlessly deliver anticancer drugs on target tumor cell surfaces with almost no collateral damage (i.e. without harming healthy cells)
(see video below). The barrel shaped molecule identifies specific target cells by latching onto the cell surface with the help of two aptamers present on the barrel surface, in much the same way a key fits to a lock. This (binding) causes unfolding of the barrel and consequent delivery of the drug which is kept inside the barrel to prevent the drug from interacting with other cells which do not express the 'key'. This bioengineering concept known as DNA origami may also be used in diagnostics apart from therapeutic purposes. 6.5 Nanobiotechnology 6.6 Synthetic genomics6.6.1 Genome re-writing and refactoring6.6.2 Genome transplantation 6.6.3 Synthetic genomes and 'synthetic' organisms
6.7 Other applications
7. Discussion Given the power that SynB it is no wonder that manipulating microorganisms, therapeutic or otherwise administration on animal or human subjects are certain to raise bioethical and religious concerns. Properly educating the public is very necessary to gain trust and obviate any hurdle that might arise. Moreover, the fear of bioterrorism will always be lurking around. Protection from such biohacking attempts should be anticipated and methodically countered, if not prevented. Emergence of highly virulent strains as a consequence of therapeutic misadventure is also possible. The future of SynBio looks very bright. With the discovery of green fluorescent protein (GFP) and the subsequent synthesis of its analogs which fluoresce with many different colors of the electromagnetic spectrum , have empowered us with diagnostics, e.g. detecting urinary tract infection (UTI); to map and trace the neuronal circuitry etc. GECIs (genetically-encoded calcium indicators) are SynBio derived molecules which respond to calcium ions, a reliable indicator of Action Potential in excitable tissues such as neurons. We can picture them in 'real time', in action, thus gaining valuable insight to their functional aspects. Optogenetics , at present, allows us to manipulate genetically modified rats using light shone into their brains using very slender fiber-optic probes. It has the potential to generate courage, erase fear memories and many more .Bionic prostheses e.g. cochlear implants , articular prostheses where tissue-engineered chondrocytes would adhere to in a more biologically friendly manner
are not far beyond. Functioning cardiomyocytes (cardiac muscle cells) has been created from bone
then cultured and repositioned into the damaged portions of the heart of the same rat the bone marrow cells came from (autologous transplantation) . This improved myocardial functions of these rats when compared to the control population. This is a milestone in transplantation research since it steers clear of the 'stem cell controversy'. Nowadays scientists are attempting at 3D sculpting of animal including human tissues (bioparts) by 'ink-jet printing' using individual cells, which promises potential benefits . A real case in point is that of Luke Massella who received an engineered bladder 10 years ago 'printed' by Dr. Anthony Atala, a surgeon . Liposomal drug delivery systems have made lipid-insoluble drugs to manoeuvre past the lipid bilayer to reach their target location possible . Biosensors capable of detecting heavy metals, TNT have also been developed. Construction of logic gates such as AND, NAND gates from the four bases of DNA and then using these parts to construct a biological DNA computer is also bound to happen. Though slow, they will be biologically compatible. Biosensors that sense blood glucose level may be incorporated in a positive feedback driven insulin pump and then implanted in a patient of Insulin Dependent Diabetes Mellitus (IDDM) patients. SynBio is also ready to modify gene expression by
the modification of DNA methylation or acetylation status of DNA. This is poised to alleviate or cure cancer. 7.1 Biosafety and biosecurity 7.2 Bioethics in synthetic biology
7.3 New approaches towards synthetic lifeProtocells, non-DNA genetic information, non-protein enzymes etc. 7.4 Future of synthetic biology
8. Resources There are many excellent sources of synthetic biology on the Internet. A comprehensive source of Internet links was developed by Dana Antonucci-Durgan of the Stony Brook University Library (USA) .8.1 DNA synthesis, sequencing- INTERPRETING DNA SEQUENCE - DNA - Protein Sequencing & Synthesis Facilities - Tech Summary: Illumina's Solexa Sequencing Technology (Forum)
8.2 (A list and description of the different tools can be found by clicking on the heading. see also 4.1 Computational modeling)
8.3 Educational materials: videos, reviews, journals, mashups, news, comics8.3.1 Videos:- YouTube Education
It contains lectures about synbio from top universities.- Synthetic Biology Explained - Andrew Hessel?s Introduction to Synthetic Biology - Synthetic Biology on the BBC - Decoding Synthetic Biology - KQED QUEST - J. Craig Venter on Synthetic Biology at NASA Ames - Drew Endy on Synthetic Biology - Drew Endy on Engineering Biology - The New Biology - Anthony Atala: Printing a human kidney - DNA nanorobot 8.3.2 Reviews:- Five hard truths for synthetic biology - Science/AAAS | Special Issue: Synthetic Biology - Nature Biotechnology December 2009, Volume 27 | Focus on Synthetic Biology - Journal of the Royal Society Interface | Synthetic Biology Focus Issue - EMBO reports special issue
8.3.3 Journals:- ACS SyntheticBiology - Journal of Biological Engineering - Journal of Computer-Aided Molecular Design - Journal of Synthetic Biology - Molecular Systems Biology - Systems and Synthetic Biology 8.3.4 Mashup: - The SynBioLogist - SynBioFromLeukipposInstitute 8.3.5 News:-Synthetic Biology on GEN 8.3.6 Comics:- Adventures in Synthetic Biology
8.3.7 iGEM Foundation and Competition :The International Genetically Engineered Machine foundation
is dedicated to the advancement of synthetic biology by creating summer undergraduate competition to build biological systems in living cells from a kit of biological parts (BioBricks from the registry of standard biological parts). This competition began as a month long course at M.I.T in 2003 and grew to a summer competition in 2004 and became very popular with 165 international teams participating in 2011.8.3.8: This section includes influential people, institutions, companies, social and professional networks such as Facebook, LinkedIn, Mendely and Twitter. (For details please click on the heading.)
8.4 Graduate Studies in Synthetic Biology A list of universities with graduate programs in Synthetic Biology.
8.5 Please click on the heading and find some of the most cited and influential papers in SynBio.
References Andrianantoandro, E., Basu, S., Karig, D.K., Weiss, R. Mol. Syst. Biol. (2006) [] Collins, J. Nature. (2012) [] Benner, S.A., Sismour, A.M. Nat. Rev. Genet. (2005) [] O'Malley, M.A., Powell, A., Davies, J.F., Calvert, J. Bioessays. (2008) [] Deplazes, A. EMBO. Rep. (2009) [] Purnick, P.E., Weiss, R. Nat. Rev. Mol. Cell. Biol. (2009) [] Savage, D.F., Way, J., Silver, P.A. ACS. Chem. Biol. (2008) [] Cases, I., de Lorenzo, V. Int. Microbiol. (2005) [] Anderson, J.C., Clarke, E.J., Arkin, A.P., Voigt, C.A. J. Mol. Biol. (2006) [] Neumann, H., Neumann-Staubitz, P. Appl. Microbiol. Biotechnol. (2010) [] Antunes, M.S., Ha, S.B., Tewari-Singh, N., Morey, K.J., Trofka, A.M., Kugrens, P., Deyholos, M., Medford, J.I. Plant. Biotechnol. J. (2006) [] Bowen, T.A., Zdunek, J.K., Medford, J.I. New. Phytol. (2008) [] Ro, D.K., Paradise, E.M., Ouellet, M., Fisher, K.J., Newman, K.L., Ndungu, J.M., Ho, K.A., Eachus, R.A., Ham, T.S., Kirby, J., Chang, M.C., Withers, S.T., Shiba, Y., Sarpong, R., Keasling, J.D. Nature. (2006) [] Szostak, J.W., Bartel, D.P., Luisi, P.L. Nature. (2001) [] Khorana, H.G., Agarwal, K.L., Büchi, H., Caruthers, M.H., Gupta, N.K., Kleppe, K., Kumar, A., Otsuka, E., RajBhandary, U.L., Van de Sande, J.H., Sgaramella, V., Terao, T., Weber, H., Yamada, T. J. Mol. Biol. (1972) [] Itakura, K., Hirose, T., Crea, R., Riggs, A.D., Heyneker, H.L., Bolivar, F., Boyer, H.W. Science. (1977) [] Edge, M.D., Green, A.R., Heathcliffe, G.R., Meacock, P.A., Schuch, W., Scanlon, D.B., Atkinson, T.C., Newton, C.R., Markham, A.F. Nature. (1981) [] Davidson, E.H. Nature. (2010) [] Amit, I., Garber, M., Chevrier, N., Leite, A.P., Donner, Y., Eisenhaure, T., Guttman, M., Grenier, J.K., Li, W., Zuk, O., Schubert, L.A., Birditt, B., Shay, T., Goren, A., Zhang, X., Smith, Z., Deering, R., McDonald, R.C., Cabili, M., Bernstein, B.E., Rinn, J.L., Meissner, A., Root, D.E., Hacohen, N., Regev, A. Science. (2009) [] Chevrier, N., Mertins, P., Artyomov, M.N., Shalek, A.K., Iannacone, M., Ciaccio, M.F., Gat-Viks, I., Tonti, E., DeGrace, M.M., Clauser, K.R., Garber, M., Eisenhaure, T.M., Yosef, N., Robinson, J., Sutton, A., Andersen, M.S., Root, D.E., von Andrian, U., Jones, R.B., Park, H., Carr, S.A., Regev, A., Amit, I., Hacohen, N. Cell. (2011) [] Martin, V.J., Pitera, D.J., Withers, S.T., Newman, J.D., Keasling, J.D. Nat. Biotechnol. (2003) [] Westfall, P.J., Pitera, D.J., Lenihan, J.R., Eng, D., Woolard, F.X., Regentin, R., Horning, T., Tsuruta, H., Melis, D.J., Owens, A., Fickes, S., Diola, D., Benjamin, K.R., Keasling, J.D., Leavell, M.D., McPhee, D.J., Renninger, N.S., Newman, J.D., Paddon, C.J. Proc. Natl. Acad. Sci. U. S. A. (2012) [] Wargacki, A.J., Leonard, E., Win, M.N., Regitsky, D.D., Santos, C.N., Kim, P.B., Cooper, S.R., Raisner, R.M., Herman, A., Sivitz, A.B., Lakshmanaswamy, A., Kashiyama, Y., Baker, D., Yoshikuni, Y. Science. (2012) []}