CiteULike: macowell's watchlist

Targeted development of registries of biological parts.

J Peccoud — 2008-07-17T21:39:45-00:00

PLoS ONE, Vol. 3, No. 7. (2008)

BACKGROUND: The design and construction of novel biological systems by combining basic building blocks represents a dominant paradigm in synthetic biology. Creating and maintaining a database of these building blocks is a way to streamline the fabrication of complex constructs. The Registry of Standard Biological Parts (Registry) is the most advanced implementation of this idea. METHODS/PRINCIPAL FINDINGS: By analyzing inclusion relationships between the sequences of the Registry entries, we build a network that can be related to the Registry abstraction hierarchy. The distribution of entry reuse and complexity was extracted from this network. The collection of clones associated with the database entries was also analyzed. The plasmid inserts were amplified and sequenced. The sequences of 162 inserts could be confirmed experimentally but unexpected discrepancies have also been identified. CONCLUSIONS/SIGNIFICANCE: Organizational guidelines are proposed to help design and manage this new type of scientific resources. In particular, it appears necessary to compare the cost of ensuring the integrity of database entries and associated biological samples with their value to the users. The initial strategy that permits including any combination of parts irrespective of its potential value leads to an exponential and economically unsustainable growth that may be detrimental to the quality and long-term value of the resource to its users.

Protein fabrication automation.

JC Cox — 2007-10-31T16:19:39-00:00

Protein Sci, Vol. 16, No. 3. (March 2007), pp. 379-390.

Facile "writing" of DNA fragments that encode entire gene sequences potentially has widespread applications in biological analysis and engineering. Rapid writing of open reading frames (ORFs) for expressed proteins could transform protein engineering and production for protein design, synthetic biology, and structural analysis. Here we present a process, protein fabrication automation (PFA), which facilitates the rapid de novo construction of any desired ORF from oligonucleotides with low effort, high speed, and little human interaction. PFA comprises software for sequence design, data management, and the generation of instruction sets for liquid-handling robotics, a liquid-handling robot, a robust PCR scheme for gene assembly from synthetic oligonucleotides, and a genetic selection system to enrich correctly assembled full-length synthetic ORFs. The process is robust and scalable.

Engineering BioBrick vectors from BioBrick parts.

RP Shetty — 2008-05-22T04:30:41-00:00

Journal of biological engineering, Vol. 2, No. 1. (2008)

ABSTRACT: BACKGROUND: The underlying goal of synthetic biology is to make the process of engineering biological systems easier. Recent work has focused on defining and developing standard biological parts. The technical standard that has gained the most traction in the synthetic biology community is the BioBrick standard for physical composition of genetic parts. Parts that conform to the BioBrick assembly standard are BioBrick standard biological parts. To date, over 2,000 BioBrick parts have been contributed to, and are available from, the Registry of Standard Biological Parts. RESULTS: Here we extended the same advantages of BioBrick standard biological parts to the plasmid-based vectors that are used to provide and propagate BioBrick parts. We developed a process for engineering BioBrick vectors from BioBrick parts. We designed a new set of BioBrick parts that encode many useful vector functions. We combined the new parts to make a BioBrick base vector that facilitates BioBrick vector construction. We demonstrated the utility of the process by constructing seven new BioBrick vectors. We also successfully used the resulting vectors to assemble and propagate other BioBrick standard biological parts. CONCLUSION: We extended the principles of part reuse and standardization to BioBrick vectors. As a result, myriad new BioBrick vectors can be readily produced from all existing and newly designed BioBrick parts. We invite the synthetic biology community to (1) use the process to make and share new BioBrick vectors; (2) expand the current collection of BioBrick vector parts; and (3) characterize and improve the available collection of BioBrick vector parts.

Synthetic Biology: Drawing a Line in Darwin's Sand

Preston — 2008-01-11T12:10:27-00:00

Environmental Values, Vol. 17, No. 1. (February 2008), pp. 23-39.

DNA polymerization on the inner surface of a giant liposome for synthesizing an artificial cell model

Koh-Ichiroh Shohda — 2008-07-14T13:59:23-00:00

Soft Matter, Vol. 2, No. 5. (2006), pp. 402-408.

We have designed a new artificial cell model consisting of a giant liposome, an enzyme, and DNA conjugated with a cholesterol tag by a poly(ethylene glycol) spacer to characterize the model system. The cholesterol tag of the conjugated molecule was anchored to the inner surface of the giant liposome and the single-stranded DNA unit hybridized with a 100-mer template DNA that was added to the water pool inside the liposome. We found that the DNA unit acted as a primer, DNA polymerization proceeded on the inner surface of the liposome. This reaction was a key step of our cell model. Production of a full-length strand was proved by selective cleavage of the polymerized DNA by a restriction enzyme.

Miniaturising the laboratory in emulsion droplets

Andrew Griffiths — 2006-08-18T11:22:00-00:00

Trends in Biotechnology, Vol. 24, No. 9. (September 2006), pp. 395-402.

Biochemical and genetic assays can be both miniaturized and parallelized by compartmentalization in living cells. In vitro compartmentalization (IVC) offers an alternative strategy based on partitioning reactions in water droplets dispersed to form microscopic compartments in water-in-oil emulsions. The cell-like volumes of these compartments (as low as one femtolitre), the ability to freely determine and regulate their content and the large number of compartments (>1010 per millilitre emulsion) have provided the basis for a range of new, ultra-high-throughput, cell-free technologies. This review describes the scope and potential of IVC in areas such as in vitro evolution of proteins and RNAs, cell-free cloning and sequencing, genetics, genomics, and proteomics.

Engineering modular protein interaction switches by sequence overlap.

NA Sallee — 2008-03-14T00:37:01-00:00

J Am Chem Soc, Vol. 129, No. 15. (18 April 2007), pp. 4606-4611.

Many cellular signaling pathways contain proteins whose interactions change in response to upstream inputs, allowing for conditional activation or repression of the interaction based on the presence of the input molecule. The ability to engineer similar regulation into protein interaction elements would provide us with powerful tools for controlling cell signaling. Here we describe an approach for engineering diverse synthetic protein interaction switches. Specifically, by overlapping the sequences of pairs of protein interaction domains and peptides, we have been able to generate mutually exclusive regulation over their interactions. Thus, the hybrid protein (which is composed of the two overlapped interaction modules) can bind to either of the two respective ligands for those modules, but not to both simultaneously. We show that these synthetic switch proteins can be used to regulate specific protein-protein interactions in vivo. These switches allow us to disrupt an interaction with the addition or activation of a protein input that has no natural connection to the interaction in question. Therefore, they give us the ability to make novel connections between normally unrelated signaling pathways and to rewire the input/output relationships of cellular behaviors. Our experiments also suggest a possible mechanism by which complex regulatory proteins might have evolved from simpler components.

Neutral lipid biosynthesis in engineered Escherichia coli: jojoba oil-like wax esters and fatty acid butyl esters.

R Kalscheuer — 2008-04-10T14:32:23-00:00

Applied and environmental microbiology, Vol. 72, No. 2. (February 2006), pp. 1373-1379.

Wax esters are esters of long-chain fatty acids and long-chain fatty alcohols which are of considerable commercial importance and are produced on a scale of 3 million tons per year. The oil from the jojoba plant (Simmondsia chinensis) is the main biological source of wax esters. Although it has a multitude of potential applications, the use of jojoba oil is restricted, due to its high price. In this study, we describe the establishment of heterologous wax ester biosynthesis in a recombinant Escherichia coli strain by coexpression of a fatty alcohol-producing bifunctional acyl-coenzyme A reductase from the jojoba plant and a bacterial wax ester synthase from Acinetobacter baylyi strain ADP1, catalyzing the esterification of fatty alcohols and coenzyme A thioesters of fatty acids. In the presence of oleate, jojoba oil-like wax esters such as palmityl oleate, palmityl palmitoleate, and oleyl oleate were produced, amounting to up to ca. 1% of the cellular dry weight. In addition to wax esters, fatty acid butyl esters were unexpectedly observed in the presence of oleate. The latter could be attributed to solvent residues of 1-butanol present in the medium component, Bacto tryptone. Neutral lipids produced in recombinant E. coli were accumulated as intracytoplasmic inclusions, demonstrating that the formation and structural integrity of bacterial lipid bodies do not require specific structural proteins. This is the first report on substantial biosynthesis and accumulation of neutral lipids in E. coli, which might open new perspectives for the biotechnological production of cheap jojoba oil equivalents from inexpensive resources employing recombinant microorganisms.

Refinement and standardization of synthetic biological parts and devices

Barry Canton — 2008-07-09T01:14:41-00:00

Nat Biotech, Vol. 26, No. 7. (July 2008), pp. 787-793.

Life cycle of a minimal protocell--a dissipative particle dynamics study.

H Fellermann — 2008-03-14T13:26:06-00:00

Artif Life, Vol. 13, No. 4. (2007), pp. 319-345.

Cross-reactions and other systematic difficulties generated by the coupling of functional chemical subsystems pose the largest challenge for assembling a viable protocell in the laboratory. Our current work seeks to identify and clarify such key issues as we represent and analyze in simulation a full implementation of a minimal protocell. Using a 3D dissipative particle dynamics simulation method, we are able to address the coupled diffusion, self-assembly, and chemical reaction processes required to model a full life cycle of a protocell composed of coupled genetic, metabolic, and container subsystems. Utilizing this minimal structural and functional representation of the constituent molecules, their interactions, and their reactions, we identify and explore the nature of the many linked processes for the full protocellular system. Obviously the simplicity of this simulation method combined with the inherent system complexity prevents us from expecting quantitative simulation predictions from these investigations. However, we report important findings on systemic processes, some previously predicted and some newly discovered, as we couple the protocellular self-assembly processes and chemical reactions.

Building a Parallel Metabolism within the Cell

Aleksandra Filipovska — 2008-01-14T16:58:47-00:00

ACS Chem. Biol. (4 January 2008)

One of the key aims of synthetic biology is to engineer artificial processes inside living cells. This requires components that interact in a predictable manner, both with each other and with existing cellular systems. However, the activity of many components is constrained by their interactions with other cellular molecules and often their roles in maintaining cell health. To escape this limitation, researchers are pursuing an orthogonal approach, building a parallel metabolism within the cell. Components of this parallel metabolism can be sourced from evolutionarily distant species or reengineered from existing cellular molecules by using rational design and directed evolution. These approaches allow the study of basic principles in cell biology and the engineering of cells that can function as environmental sensors, simple computers, and drug factories.

Expanding the metabolic engineering toolbox: more options to engineer cells.

KE Tyo — 2007-04-23T17:23:53-00:00

Trends Biotechnol, Vol. 25, No. 3. (March 2007), pp. 132-137.

Metabolic engineering exploits an integrated, systems-level approach for optimizing a desired cellular property or phenotype; and great strides have been made within this scope and context during the past fifteen years. However, due to limitations in the concepts and techniques, these have relied on a focused, pathway-oriented view. Recent advances in 'omics' technologies and computational systems biology have brought the foundational systems approach of metabolic engineering into focus. At the same time, protein engineering and synthetic biology have expanded the breadth and precision of the methods available to metabolic engineers to improve strain properties. Examples are presented that illustrate this broader perspective of tools and concepts, including a recent approach for global transcriptional machinery engineering (gTME), which has demonstrated the ability to elicit multigenic transcriptional changes that have improved phenotypes compared with single-gene perturbations.

Synthetic biology projects in vitro

Anthony Forster — 2008-06-09T18:25:05-00:00

Genome Res., Vol. 17, No. 1. (1 January 2007), pp. 1-6.

Advances in the in vitro synthesis and evolution of DNA, RNA, and polypeptides are accelerating the construction of biopolymers, pathways, and organisms with novel functions. Known functions are being integrated and debugged with the aim of synthesizing life-like systems. The goals are knowledge, tools, smart materials, and therapies. 10.1101/gr.5776007

From Never Born Proteins to Minimal Living Cells: Two Projects in Synthetic Biology

Luisi — 2007-01-24T08:18:26-00:00

Origins of Life and Evolution of the Biosphere, Vol. 36, No. 5-6. (December 2006), pp. 605-616.

Synthetic Biology: Caught between Property Rights, the Public Domain, and the Commons.

Arti Rai — 2007-03-21T11:03:35-00:00

PLoS Biol, Vol. 5, No. 3. (13 March 2007)

Synthetic biology: enormous possibility, exaggerated perils.

Zachary Russ — 2008-04-30T08:04:25-00:00

Journal of biological engineering, Vol. 2, No. 1. (25 April 2008)

ABSTRACT: The following essay was written by a freshman undergraduate student majoring in Bioengineering at the University of Maryland, Mr. Zachary Russ. Mr. Russ was one of 94 students who submitted a 1000 to 1200 word essay to the 3rd Annual Bioethics Essay Contest sponsored by the Institute of Biological Engineering (IBE). A group of professionals in Biological Engineering assessed and ranked the essays in a blinded process. Five semi-finalists were invited to present their essays at a session at the annual meeting of IBE in Chapel Hill, NC on March 8, 2008. Five judges scored the presentations at the annual meeting and selected Mr. Russ's contribution as the overall winner (1st Place). Below is his essay.

Setting the standard in synthetic biology

Adam Arkin — 2008-07-09T01:15:20-00:00

Nat Biotech, Vol. 26, No. 7. (July 2008), pp. 771-774.

RNA and RNP as new molecular parts in synthetic biology

Hirohide Saito — 2007-12-26T03:48:30-00:00

Journal of Biotechnology, Vol. 132, No. 1. (15 October 2007), pp. 1-7.

Synthetic biology has a promising outlook in biotechnology and for understanding the self-organizing principle of biological molecules in life. However, synthetic biologists have been looking for new molecular "parts" that function as modular units required in designing and constructing new "devices" and "systems" for regulating cell function because the number of such parts is strictly limited at present. In this review, we focus on RNA/ribonucleoprotein (RNP) architectures that hold promise as new "parts" for synthetic biology. They are constructed with molecular design and an experimental evolution technique. So far, designed self-folding RNAs, RNA (RNP) enzymes, and nanoscale RNA architectures have been successfully constructed by utilizing Watson-Crick base-pairs together with specific RNA-RNA or RNA-protein binding motifs of known defined 3D structures. Riboregulators for regulating targeted gene expression have also been designed and produced in vitro as well as in vivo. Lately, RNA and ribonucleoprotein complexes have been strongly attracting the attention of molecular biologists because a variety of noncoding RNAs discovered in nature perform spatiotemporal gene expressions. Thus we hope that newly accumulating knowledge on naturally occurring RNAs and RNP complexes will provide a variety of new parts, devices and systems for synthetic biology.

Ribozymes, riboswitches and beyond: regulation of gene expression without proteins

Alexander Serganov — 2007-09-19T03:51:54-00:00

Nature Reviews Genetics, Vol. 8, No. 10. (11 September 2007), pp. 776-790.

Rapid and sensitive pollutant detection by induction of heat shock gene-bioluminescence gene fusions.

TK Van Dyk — 2006-06-04T11:00:58-00:00

Appl Environ Microbiol, Vol. 60, No. 5. (May 1994), pp. 1414-1420.

Heat shock gene expression is induced by a variety of environmental stresses, including the presence of many chemicals. To address the utility of this response for pollutant detection, two Escherichia coli heat shock promoters, dnaK and grpE, were fused to the lux genes of Vibrio fischeri. Metals, solvents, crop protection chemicals, and other organic molecules rapidly induced light production from E. coli strains containing these plasmid-borne fusions. Introduction of an outer membrane mutation, tolC, enhanced detection of a hydrophobic molecule, pentachlorophenol. The maximal response to pentachlorophenol in the tolC+ strain was at 38 ppm, while the maximal response in an otherwise isogenic tolC mutant was at 1.2 ppm. Stress responses were observed in both batch and chemostat cultures. It is suggested that biosensors constructed in this manner may have potential for environmental monitoring.

Robust, Tunable Biological Oscillations from Interlinked Positive and Negative Feedback Loops

Tony Tsai — 2008-07-03T22:13:28-00:00

Science, Vol. 321, No. 5885. (4 July 2008), pp. 126-129.

A simple negative feedback loop of interacting genes or proteins has the potential to generate sustained oscillations. However, many biological oscillators also have a positive feedback loop, raising the question of what advantages the extra loop imparts. Through computational studies, we show that it is generally difficult to adjust a negative feedback oscillator's frequency without compromising its amplitude, whereas with positive-plus-negative feedback, one can achieve a widely tunable frequency and near-constant amplitude. This tunability makes the latter design suitable for biological rhythms like heartbeats and cell cycles that need to provide a constant output over a range of frequencies. Positive-plus-negative oscillators also appear to be more robust and easier to evolve, rationalizing why they are found in contexts where an adjustable frequency is unimportant. 10.1126/science.1156951

An Introduction to Systems Biology: Design Principles of Biological Circuits (Chapman & Hall/Crc Mathematical and Computational Biology Series)

Uri Alon — 2007-05-21T00:56:08-00:00

(07 July 2006)

Cross-species de novo identification of cis-regulatory modules with GibbsModule: application to gene regulation in embryonic stem cells

Dan Xie — 2008-05-27T08:52:20-00:00

Genome Res. (15 May 2008), gr.072769.107.

We introduce the GibbsModule algorithm for de novo detection of cis-regulatory motifs and modules in eukaryote genomes. GibbsModule models the co-expressed genes within one species as sharing a core cis-regulatory motif and each homologous gene group as sharing a homologous cis-regulatory module (CRM), characterized by a similar composition of motifs. Without using a pre-determined alignment result, GibbsModule iteratively updates the core motif shared by co-expressed genes and traces the homologous CRMs that contain the core motif. GibbsModule achieved substantial improvements in both precision and recall as compared to peer algorithms on a number of synthetic and real datasets. Applying GibbsModule to analyze the binding regions of the Kruppel-like factor (Klf) transcription factor in embryonic stem cells (ESCs), we discovered a motif that differs from a previously published Klf motif identified by a SELEX experiment, but the new motif is consistent with mutagenesis analysis. Sox2 motif was found to be a collaborating motif to the Klf motif in ESCs. We used quantitative chromatin immunoprecipitation (ChIP) analysis to test whether GibbsModule could distinguish functional and non-functional binding sites. All 7 tested binding sites in GibbsModule predicted CRMs had higher ChIP signals as compared to the other 7 tested binding sites located outside of predicted CRMs. GibbsModule is available at http://biocomp.bioen.uiuc.edu/GibbsModule. 10.1101/gr.072769.107

Engineering stochasticity in gene expression.

JJ Tabor — 2008-06-22T00:08:33-00:00

Molecular bioSystems, Vol. 4, No. 7. (July 2008), pp. 754-761.

Stochastic fluctuations (noise) in gene expression can cause members of otherwise genetically identical populations to display drastically different phenotypes. An understanding of the sources of noise and the strategies cells employ to function reliably despite noise is proving to be increasingly important in describing the behavior of natural organisms and will be essential for the engineering of synthetic biological systems. Here we describe the design of synthetic constructs, termed ribosome competing RNAs (rcRNAs), as a means to rationally perturb noise in cellular gene expression. We find that noise in gene expression increases in a manner proportional to the ability of an rcRNA to compete for the cellular ribosome pool. We then demonstrate that operons significantly buffer noise between coexpressed genes in a natural cellular background and can even reduce the level of rcRNA enhanced noise. These results demonstrate that synthetic genetic constructs can significantly affect the noise profile of a living cell and, importantly, that operons are a facile genetic strategy for buffering against noise.

Protein Design by Directed Evolution

Christian Jackel — 2008-05-08T08:48:35-00:00

Annual Review of Biophysics, Vol. 37, No. 1. (2008), pp. 153-173.

While nature evolved polypeptides over billions of years, protein design by evolutionary mimicry is progressing at a far more rapid pace. The mutation, selection, and amplification steps of the evolutionary cycle may be imitated in the laboratory using existing proteins, or molecules created de novo from random sequence space, as starting templates. However, the astronomically large number of possible polypeptide sequences remains an obstacle to identifying and isolating functionally interesting variants. Intelligently designed libraries and improved search techniques are consequently important for future advances. In this regard, combining experimental and computational methods holds particular promise for the creation of tailored protein receptors and catalysts for tasks unimagined by nature.

Design of Multi-Specificity in Protein Interfaces

Elisabeth Humphris — 2007-08-24T23:40:23-00:00

PLoS Computational Biology, Vol. 3, No. 8. (1 August 2007), e164.

Interactions in protein networks may place constraints on protein interface sequences to maintain correct and avoid unwanted interactions. Here we describe a “multi-constraint” protein design protocol to predict sequences optimized for multiple criteria, such as maintaining sets of interactions, and apply it to characterize the mechanism and extent to which 20 multi-specific proteins are constrained by binding to multiple partners. We find that multi-specific binding is accommodated by at least two distinct patterns. In the simplest case, all partners share key interactions, and sequences optimized for binding to either single or multiple partners recover only a subset of native amino acid residues as optimal. More interestingly, for signaling interfaces functioning as network “hubs,” we identify a different, “multi-faceted” mode, where each binding partner prefers its own subset of wild-type residues within the promiscuous binding site. Here, integration of preferences across all partners results in sequences much more “native-like” than seen in optimization for any single binding partner alone, suggesting these interfaces are substantially optimized for multi-specificity. The two strategies make distinct predictions for interface evolution and design. Shared interfaces may be better small molecule targets, whereas multi-faceted interactions may be more “designable” for altered specificity patterns. The computational methodology presented here is generalizable for examining how naturally occurring protein sequences have been selected to satisfy a variety of positive and negative constraints, as well as for rationally designing proteins to have desired patterns of altered specificity.

Redesigning enzymes based on adaptive evolution for optimal function in synthetic metabolic pathways.

Y Yoshikuni — 2008-06-20T09:52:00-00:00

Chemistry & biology, Vol. 15, No. 6. (June 2008), pp. 607-618.

Nature has balanced most metabolic pathways such that no one enzyme in the pathway controls the flux through that pathway. However, unnatural or nonnative, constructed metabolic pathways may have limited product flux due to unfavorable in vivo properties of one or more enzymes in the pathway. One such example is the mevalonate-based isoprenoid biosynthetic pathway that we previously reconstructed in Escherichia coli. We have used a probable mechanism of adaptive evolution to engineer the in vivo properties of two enzymes (3-hydroxy-3-methylglutaryl-CoA reductase [tHMGR] and many terpene synthases) in this pathway and thereby eliminate or minimize the bottleneck created by these inefficient or nonfunctional enzymes. Here, we demonstrate how we significantly improved the productivity (by approximately 1000 fold) of this reconstructed biosynthetic pathway using this strategy. We anticipate that this strategy will find broad applicability in the functional construction (or reconstruction) of biological pathways in heterologous hosts.

Implementing arithmetic and other analytic operations by transcriptional regulation.

SM Cory — 2008-04-28T17:06:57-00:00

PLoS computational biology, Vol. 4, No. 4. (April 2008)

The transcriptional regulatory machinery of a gene can be viewed as a computational device, with transcription factor concentrations as inputs and expression level as the output. This view begs the question: what kinds of computations are possible? We show that different parameterizations of a simple chemical kinetic model of transcriptional regulation are able to approximate all four standard arithmetic operations: addition, subtraction, multiplication, and division, as well as various equality and inequality operations. This contrasts with other studies that emphasize logical or digital notions of computation in biological networks. We analyze the accuracy and precision of these approximations, showing that they depend on different sets of parameters, and are thus independently tunable. We demonstrate that networks of these "arithmetic" genes can be combined to accomplish yet more complicated computations by designing and simulating a network that detects statistically significant elevations in a time-varying signal. We also consider the much more general problem of approximating analytic functions, showing that this can be achieved by allowing multiple transcription factor binding sites on the promoter. These observations are important for the interpretation of naturally occurring networks and imply new possibilities for the design of synthetic networks.

Rational design of memory in eukaryotic cells.

CM Ajo-Franklin — 2007-11-05T07:46:12-00:00

Genes Dev, Vol. 21, No. 18. (15 September 2007), pp. 2271-2276.

The ability to logically engineer novel cellular functions promises a deeper understanding of biological systems. Here we demonstrate the rational design of cellular memory in yeast that employs autoregulatory transcriptional positive feedback. We built a set of transcriptional activators and quantitatively characterized their effects on gene expression in living cells. Modeling in conjunction with the quantitative characterization of the activator-promoter pairs accurately predicts the behavior of the memory network. This study demonstrates the power of taking advantage of components with measured quantitative parameters to specify eukaryotic regulatory networks with desired properties.

Enhancement of Cell Type Specificity by Quantitative Modulation of a Chimeric Ligand

Pablo Cironi — 2008-06-02T09:16:44-00:00

J. Biol. Chem., Vol. 283, No. 13. (28 March 2008), pp. 8469-8476.

Evolution modulates the quantitative characteristics of protein interactions and often uses combinations of weak interactions to achieve a particular specificity. We addressed how quantitative optimization might be used in the design of multidomain proteins, using a chimera containing epidermal growth factor (EGF) as a cell targeting element and interferon-alpha-2a (IFNalpha-2a) to initiate signal transduction. We first connected EGF and IFNalpha-2a via a linker that allows both ligands to bind to their receptors on a cell surface and then incorporated a series of mutations into the IFNalpha-2a portion that progressively decrease both the on rate and the dissociation constant of the IFNalpha-2a-IFNalpha receptor 2 (IFNAR2) interaction. Using this strategy, we designed chimeric proteins in which the activation of the IFNalpha receptor in HeLa, A431, and engineered Daudi cells depends on the presence of EGF receptor on the same cell. The mutant chimeric proteins also inhibited proliferation of IFNalpha-sensitive cells in an EGF receptor-dependent manner. These results provide insights into the quantitative requirements for specific binding to multisubunit receptors and illustrate the value of a quantitative approach in the design of synthetic-biological constructs. 10.1074/jbc.M708502200

Defossiling Fuel: How Synthetic Biology Can Transform Biofuel Production

David Savage — 2008-03-05T18:25:56-00:00

ACS Chem. Biol., Vol. 3, No. 1. (18 January 2008), pp. 13-16.

Lauren Nicole, Photodisc, Getty Images Although crude oil production is predicted to peak soon, it is reasonable to assume that unconventional fossil fuel sources can continue to meet societys increasing energy demands for many decades to come (1). The real challenge is sustainability: stabilizing and reversing global climate change, minimizing political and economic energy volatility, and smoothing the transition from fossil fuels in the distant future. In response to this challenge, many are looking to biotechnology to develop biofuels, such as ethanol, butanol, biodiesel, and hydrogen (H2), in which the energy ultimately derives from photosynthetic capture of sunlight. A fundamental issue with biofuels is efficiency. The pathway from sunlight through natural intermediates to final molecule is long, and biofuel production is perhaps the ultimate metabolic engineering problem (2). This challenge is made even greater by its inherent systems complexity, because any solution must be implemented in the context of an energy infrastructure with challenging engineering, economic, political, and environmental realities.

Customized signaling with reconfigurable protein scaffolds

Patrick Guye — 2008-05-09T21:16:16-00:00

Nature Biotechnology, Vol. 26, No. 5., pp. 526-528.

[The importance of synthetic biology.]

M Morange — 2008-05-13T08:15:58-00:00

Medecine sciences : M/S, Vol. 24, No. 5. (May 2008), pp. 447-448.

[First French team success during iGEM Synthetic biology competition.]

David Bikard — 2008-05-13T08:15:14-00:00

Medecine sciences : M/S, Vol. 24, No. 5. (May 2008), pp. 541-544.

Recursive construction of perfect DNA molecules from imperfect oligonucleotides.

G Linshiz — 2008-05-13T08:13:38-00:00

Molecular systems biology, Vol. 4 (2008)

Making faultless complex objects from potentially faulty building blocks is a fundamental challenge in computer engineering, nanotechnology and synthetic biology. Here, we show for the first time how recursion can be used to address this challenge and demonstrate a recursive procedure that constructs error-free DNA molecules and their libraries from error-prone oligonucleotides. Divide and Conquer (D&C), the quintessential recursive problem-solving technique, is applied in silico to divide the target DNA sequence into overlapping oligonucleotides short enough to be synthesized directly, albeit with errors; error-prone oligonucleotides are recursively combined in vitro, forming error-prone DNA molecules; error-free fragments of these molecules are then identified, extracted and used as new, typically longer and more accurate, inputs to another iteration of the recursive construction procedure; the entire process repeats until an error-free target molecule is formed. Our recursive construction procedure surpasses existing methods for de novo DNA synthesis in speed, precision, amenability to automation, ease of combining synthetic and natural DNA fragments, and ability to construct designer DNA libraries. It thus provides a novel and robust foundation for the design and construction of synthetic biological molecules and organisms.

The crucial role of CS in systems and synthetic biology

Jacques Cohen — 2008-05-11T15:34:40-00:00

Commun. ACM, Vol. 51, No. 5. (May 2008), pp. 15-18.

Synthetic gene brushes: a structure-function relationship

Amnon Buxboim — 2008-04-21T02:14:26-00:00

Mol Syst Biol, Vol. 4 (15 April 2008)

Genome Transplantation in Bacteria: Changing One Species to Another

Carole Lartigue — 2007-06-30T05:37:35-00:00

Science (28 June 2007), 1144622.

As a step toward propagation of synthetic genomes, we completely replaced the genome of a bacterial cell with one from another species by transplanting a whole genome as naked DNA. Intact genomic DNA from Mycoplasma mycoides large colony (LC), virtually free of protein, was transplanted into Mycoplasma capricolum cells by polyethylene glycol-mediated transformation. Cells selected for tetracycline resistance, carried by the M. mycoides LC chromosome, contain the complete donor genome and are free of detectable recipient genomic sequences. These cells that result from genome transplantation are phenotypically identical to the M. mycoides LC donor strain as judged by several criteria. 10.1126/science.1144622

Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasma genitalium Genome

Daniel Gibson — 2008-01-25T23:30:03-00:00

Science (24 January 2008), 1151721.

We have synthesized a 582,970 bp Mycoplasma genitalium genome. This synthetic genome, named M. genitalium JCVI-1.0, contains all the genes of wild-type M. genitalium G37 except MG408, which was disrupted by an antibiotic marker to block pathogenicity and to allow for selection. To identify the genome as synthetic, we inserted "watermarks" at intergenic sites known to tolerate transposon insertions. Overlapping "cassettes" of 5 to 7 kb, assembled from chemically synthesized oligonucleotides, were joined by in vitro recombination to produce intermediate assemblies of approximately 24 kb, 72 kb ("1/8 genome"), and 144 kb ("1/4 genome"), which were all cloned as bacterial artificial chromosomes (BACs) in Escherichia coli. Most of these intermediate clones were sequenced, and clones of all four 1/4 genomes with the correct sequence were identified. The complete synthetic genome was assembled by transformation-associated recombination (TAR) cloning in the yeast Saccharomyces cerevisiae, then isolated and sequenced. A clone with the correct sequence was identified. The methods described here will be generally useful for constructing large DNA molecules from chemically synthesized pieces and also from combinations of natural and synthetic DNA segments. 10.1126/science.1151721

Synthetic genomes brought closer to life

Robert Holt — 2008-03-09T04:13:09-00:00

Nature Biotechnology, Vol. 26, No. 3., pp. 296-297.

Computational identification of obligatorily autocatalytic replicators embedded in metabolic networks

Adam Kun — 2008-03-10T16:10:55-00:00

Genome Biology, Vol. 9, No. 3. (2008)

BACKGROUND:If chemical A is necessary for the synthesis of more chemical A then A has the power of replication (such systems are known as autocatalytic systems). We provide the first systems-level analysis to search for small-molecular autocatalytic components in metabolisms of diverse organisms, including an inferred minimal metabolism.RESULTS:We find that intermediary metabolism is invariably autocatalytic for ATP. Furthermore, we provide evidence for the existence of additional, organism-specific autocatalytic seeds in the forms of coenzymes (NAD, CoA, tetrahydrofolate, quinones) and sugars. Although the enzymatic reactions of a number of autocatalytic cycles are present in most of the studied organisms, they display obligatorily autocatalytic behaviour in a few networks only, hence demonstrating the need for a systems-level approach to identify metabolic replicators embedded in large networks.CONCLUSIONS:Metabolic replicators are hence apparently common and potentially both universal and ancestral: without their presence, kick-start of metabolic networks is impossible, even if all enzymes and genes are present in the same cell. Identification of metabolic replicators is also important for attempts to create synthetic cells, as some of these autocatalytic molecules will presumably be needed to be added to the system as by definition the system cannot synthesize them without their initial presence.

Terpenoids: Opportunities for Biosynthesis of Natural Product Drugs Using Engineered Microorganisms.

Parayil Ajikumar — 2008-03-25T09:18:13-00:00

Mol Pharm (21 March 2008)

Terpenoids represent a diverse class of molecules that provide a wealth of opportunities to address many human health and societal issues. The expansive array of structures and functionalities that have been evolved in nature provide an excellent pool of molecules for use in human therapeutics. While this class of molecules has members with therapeutic properties including anticancer, antiparasitic, antimicrobial, antiallergenic, antispasmodic, antihyperglycemic, anti-inflammatory, and immunomodulatory properties, supply limitations prevent the large scale use of some molecules. Many of these molecules are only found in ppm levels in nature thus requiring massive harvesting to obtain sufficient amounts of the drug. Synthetic biology and metabolic engineering provide innovative approaches to increase the production of the desired molecule in the native organism, and most importantly, transfer the biosynthetic pathways to other hosts. Microbial systems are well studied, and genetic manipulations allow the optimization of microbial metabolisms for the production of common terpenoid precursors. Using a host of tools, unprecedented advancements in the large scale production of terpenoids have been achieved in recent years. Identification of limiting steps and pathway regulation, coupled with design strategies to minimize terpenoid byproducts wih a high flux to the desired biosynthetic pathways, have yielded greater than 100-fold improvements in the production of a range of terpenoids. This review focuses on the biodiversity of terpenoids, the biosynthetic pathways involved, and engineering efforts to maximize the production through these pathways.

Hierarchical self-assembly of DNA into symmetric supramolecular polyhedra

Yu He — 2008-03-13T00:31:06-00:00

Nature, Vol. 452, No. 7184. (13 March 2008), pp. 198-201.

Designing biological systems.

DA Drubin — 2007-03-13T01:41:46-00:00

Genes Dev, Vol. 21, No. 3. (1 February 2007), pp. 242-254.

The design of artificial biological systems and the understanding of their natural counterparts are key objectives of the emerging discipline of synthetic biology. Toward both ends, research in synthetic biology has primarily focused on the construction of simple devices, such as transcription-based oscillators and switches. Construction of such devices should provide us with insight on the design of natural systems, indicating whether our understanding is complete or whether there are still gaps in our knowledge. Construction of simple biological systems may also lay the groundwork for the construction of more complex systems that have practical utility. To realize its full potential, biological systems design borrows from the allied fields of protein design and metabolic engineering. In this review, we describe the scientific accomplishments in this field, as well as its forays into biological part standardization and education of future biological designers.

Simplicity in biology

Uri Alon — 2007-03-29T09:58:35-00:00

Nature, Vol. 446, No. 7135. (28 March 2007), pp. 497-497.

Network motifs: theory and experimental approaches

Uri Alon — 2007-05-19T03:15:41-00:00

Nature Reviews Genetics, Vol. 8, No. 6., pp. 450-461.

Enhancement of cellular memory by reducing stochastic transitions

Murat Acar — 2005-05-13T07:43:36-00:00

Nature, Vol. 435, No. 7039., pp. 228-232.

Cost–benefit theory and optimal design of gene regulation functions

Tomer Kalisky — 2007-11-07T20:44:58-00:00

Phys. Biol., Vol. 4, No. 4. (December 2007), 229.

Computational design of a biologically active enzyme.

MA Dwyer — 2005-11-09T14:17:29-00:00

Science, Vol. 304, No. 5679. (25 June 2004), pp. 1967-1971.

Rational design of enzymes is a stringent test of our understanding of protein chemistry and has numerous potential applications. Here, we present and experimentally validate the computational design of enzyme activity in proteins of known structure. We have predicted mutations that introduce triose phosphate isomerase activity into ribose-binding protein, a receptor that normally lacks enzyme activity. The resulting designs contain 18 to 22 mutations, exhibit 10(5)- to 10(6)-fold rate enhancements over the uncatalyzed reaction, and are biologically active, in that they support the growth of Escherichia coli under gluconeogenic conditions. The inherent generality of the design method suggests that many enzymes can be designed by this approach.

Programming gene expression with combinatorial promoters

Robert Cox — 2007-11-13T20:47:10-00:00

Mol Syst Biol, Vol. 3 (13 November 2007)