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Diverse High Throughput Technologies in Cancer Research and Synthetic Biology

Zohar Yakhini, Agilent Technologies and Technion Computational Cancer Biology https://simons.berkeley.edu/talks/zohar-yakhini-02-02-2016...

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Diverse High Throughput Technologies in Cancer Research and Synthetic Biology
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Editorial: Transforming biotechnology with synthetic biology.

Synthetic biology has advanced to contain not only whole cell systems but also cell-free systems. Combined with minimized genome and promoter engineering, synthetic biology can be used to engineer living systems or biomolecular components for production of chemicals, materials or pharmaceutics. The future looks bright!
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Design guidelines for a virtual synthetic biology lab

Many currently developing scientific fields do not end up in secondary school laboratories, for, among others, equipment to perform research in these areas is expensive. Therefore, students are unable to experiment with new fields. One such fields is synthetic biology, in which scientist use elements of genetic information to develop whole new systems. The European Union funded SYNENERGENE project is currently developing a virtual lab to enable upper secondary students to experiment with synthetic biology. However, it is unknown what such virtual labs should look like. This study aims to find design guidelines for a virtual lab promoting conceptual and procedural knowledge on synthetic biology for upper secondary students. To do so, literature on the topic is reviewed, and two biology teacher trainers are interviewed. It becomes clear that among the most important guidelines is authenticity. The lab should focus on real scientific processes, and students should use real world equipment. Additionally, learning aims should be defined clearly, and the abstract and complex nature of synthetic biology should be dealt with using visualisations. When incorporating wishes of teachers, like low energy investment and coverage of curricular aims, the virtual synthetic biology lab has the potential to reconnect the secondary school curriculum with current scientific practice
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A statistical approach reveals designs for the most robust stochastic gene oscillators.

The engineering of transcriptional networks presents many challenges due to the inherent uncertainty in the system structure, changing cellular context and stochasticity in the governing dynamics. One approach to address these problems is to design and build systems that can function across a range of conditions; that is they are robust to uncertainty in their constituent components. Here we examine the robustness landscape of transcriptional oscillators, which underlie many important processes such as circadian rhythms and the cell cycle, plus also serve as a model for the engineering of complex and emergent phenomena. The central questions that we address are: Can we build genetic oscillators that are more robust than those already constructed? Can we make genetic oscillators arbitrarily robust? These questions are technically challenging due to the large model and parameter spaces that must be efficiently explored. Here we use a measure of robustness that coincides with the Bayesian model evidence combined with an efficient Monte Carlo method to traverse model space and concentrate on regions of high robustness, which enables the accurate evaluation of the relative structural robustness of gene network models governed by stochastic dynamics. We report the most robust two and three gene oscillator systems, plus examine how the number of interactions, the presence of auto-regulation, and degradation of mRNA and protein affects the frequency, amplitude and robustness of transcriptional oscillators. We also find that there is a limit to parametric robustness, beyond which there is nothing to be gained by adding additional feedback. Importantly, we provide predictions on new oscillator systems that can be constructed to verify the theory and advance design and modelling approaches to systems and synthetic biology.
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The era of synthetic biology and genome engineering: Where no man has gone before

The era of synthetic biology and genome engineering: Where no man has gone before
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Towards enabling engineered microbial-electronic systems: RK2-based conjugal transfer system for Shewanella synthetic biology

Synthetic biology has been traditionally associated with electronics through the application of circuit design concepts towards the genetic engineering of microbes. Due to recent advances in the understanding of extracellular electron transfer in the bacterium Shewanella oneidensis (Shewanella), synthetic biology advances now have the potential of being used towards electronics applications. Towards this end, there is a need for tools that enable the systematic optimisation of genetic circuits in Shewanella. With the introduction of an RK2 origin of transfer cassette, we show that a modular plasmid system constructed prior for synthetic biology efforts in the bacterium Escherichia coli (E. coli) can be ported to Shewanella. In the process, it is also shown that different replication origins can be maintained in Shewanella and that multiple-plasmid strains can be realised in the bacterium. The results suggest that parts accumulated from E. coli synthetic biology efforts over the past decade and a half may be able to be ported to Shewanella, enabling the future engineering of systems where microbes interface with electronics (e.g. biosensors).
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Fully integrated wearable sensor arrays for multiplexed in situ perspiration analysis

Fully integrated wearable sensor arrays for multiplexed in situ perspiration analysis | SynBioFromLeukipposInstitute | Scoop.it
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Wei Gao, Sam Emaminejad, Hnin Yin Yin Nyein, Samyuktha Challa, Kevin Chen, Austin Peck, Hossain M. Fahad, Hiroki Ota, Hiroshi Shiraki, Daisuke Kiriya, Der-Hsien Lien, George A. Brooks, Ronald W. Davis & Ali Javey

"Wearable sensor technologies are essential to the realization of personalized medicine through continuously monitoring an individual’s state of health1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12. Sampling human sweat, which is rich in physiological information13, could enable non-invasive monitoring. Previously reported sweat-based and other non-invasive biosensors either can only monitor a single analyte at a time or lack on-site signal processing circuitry and sensor calibration mechanisms for accurate analysis of the physiological state14, 15, 16, 17, 18. Given the complexity of sweat secretion, simultaneous and multiplexed screening of target biomarkers is critical and requires full system integration to ensure the accuracy of measurements. Here we present a mechanically flexible and fully integrated (that is, no external analysis is needed) sensor array for multiplexed in situ perspiration analysis, which simultaneously and selectively measures sweat metabolites (such as glucose and lactate) and electrolytes (such as sodium and potassium ions), as well as the skin temperature (to calibrate the response of the sensors). Our work bridges the technological gap between signal transduction, conditioning (amplification and filtering), processing and wireless transmission in wearable biosensors by merging plastic-based sensors that interface with the skin with silicon integrated circuits consolidated on a flexible circuit board for complex signal processing. This application could not have been realized using either of these technologies alone owing to their respective inherent limitations. The wearable system is used to measure the detailed sweat profile of human subjects engaged in prolonged indoor and outdoor physical activities, and to make a real-time assessment of the physiological state of the subjects. This platform enables a wide range of personalized diagnostic and physiological monitoring applications."

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SynBio Future 2016 Conference: Future Axioms in Synthetic Biology

SynBio Future 2016 Conference: Future Axioms in Synthetic Biology | SynBioFromLeukipposInstitute | Scoop.it

Eventbrite - IndieBio EU, UCC School of Microbiology, UCC School of Biochemistry and Cell Biology presents SynBio Future 2016 Conference: Future Axioms in Synthetic Biology - Monday, February 22, 2016 at Devere Hall.

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Introducing a class of standardized and interchangeable parts utilizing programmed ribosomal frameshifts for synthetic biology applications.

Synthetic biology and the rational design of biological devices depend on the availability of standardized and interchangeable biological parts with diverse range of functions. Reliable access to different reading frames during translation has largely been overlooked as functionality for bioengineering applications. Here we report the construction and initial characterization of the first member of such a class of biological parts that conforms to the BioBrick Standard (RFC25), allowing its interchangeable use in biological devices. Using our standardized frameshifting signal consisting of a UUUAAAG slippery sequence, a 6 nt spacer and an engineered pseudoknot based on the infectious bronchitis virus pseudoknot PK401 embedded in a dual reporter construct, we confirm that the frameshifting activity is comparable to the previously published frequency despite the introduced sequence changes. The frameshifting activity is demonstrated using SDS-PAGE and fluorescence spectroscopy. Standardized programmable ribosomal frameshift parts with specific frameshifting frequencies will be of utility for applications such as double coding DNA sequences by expanding the codable space into the -1 frame. Programmed shifting into the -1 frame to bypass a stop codon allows labeling of a protein pool with a fixed stoichiometry of fusion protein, as well as the construction of multi-enzyme expression constructs with specific expression ratios. A detailed understanding of the structural basis of programmed frameshifting will provide the opportunities to rationally design frameshifting elements with a wide range of applications in synthetic biology, including signals that are regulated by small ligands.
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Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9

Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9 | SynBioFromLeukipposInstitute | Scoop.it
CRISPR-Cas9–based genetic screens are a powerful new tool in biology. By simply altering the sequence of the single-guide RNA (sgRNA), one can reprogram Cas9 to target different sites in the genome with relative ease, but the on-target activity and off-target effects of individual sgRNAs can vary widely. Here, we use recently devised sgRNA design rules to create human and mouse genome-wide libraries, perform positive and negative selection screens and observe that the use of these rules produced improved results. Additionally, we profile the off-target activity of thousands of sgRNAs and develop a metric to predict off-target sites. We incorporate these findings from large-scale, empirical data to improve our computational design rules and create optimized sgRNA libraries that maximize on-target activity and minimize off-target effects to enable more effective and efficient genetic screens and genome engineering.
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The all E. coli TX-TL Toolbox 2.0: a platform for cell-free synthetic biology.

We report on and provide a detailed characterization of the performance and properties of a recently developed, all E. coli, cell-free transcription and translation system. Gene expression is entirely based on the endogenous translation components and transcription machinery provided by an E. coli cytoplasmic extract, thus expanding the repertoire of regulatory parts to hundreds of elements. We use a powerful metabolism for ATP regeneration to achieve more than 2 mg/ml of protein synthesis in batch mode reactions, and more than 6 mg/ml in semi-continuous mode. While the strength of cell-free expression is increased by a factor of three on average, the output signal of simple gene circuits and the synthesis of entire bacteriophages are increased by orders of magnitude compared to previous results. Messenger RNAs and protein degradation, respectively tuned using E. coli MazF interferase and ClpXP AAA+ proteases, are characterized over a much wider range of rates than the first version of the cell-free toolbox. This system is a highly versatile cell-free platform to construct complex biological systems through the execution of DNA programs composed of synthetic and natural bacterial regulatory parts.
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High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects

High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects | SynBioFromLeukipposInstitute | Scoop.it
CRISPR–Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations. Existing strategies for reducing genome-wide off-target effects of the widely used Streptococcus pyogenes Cas9 (SpCas9) are imperfect, possessing only partial or unproven efficacies and other limitations that constrain their use. Here we describe SpCas9-HF1, a high-fidelity variant harbouring alterations designed to reduce non-specific DNA contacts. SpCas9-HF1 retains on-target activities comparable to wild-type SpCas9 with >85% of single-guide RNAs (sgRNAs) tested in human cells. Notably, with sgRNAs targeted to standard non-repetitive sequences, SpCas9-HF1 rendered all or nearly all off-target events undetectable by genome-wide break capture and targeted sequencing methods. Even for atypical, repetitive target sites, the vast majority of off-target mutations induced by wild-type SpCas9 were not detected with SpCas9-HF1. With its exceptional precision, SpCas9-HF1 provides an alternative to wild-type SpCas9 for research and therapeutic applications. More broadly, our results suggest a general strategy for optimizing genome-wide specificities of other CRISPR-RNA-guided nucleases.
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Orthogonal intercellular signaling for programmed spatial behavior.

Bidirectional intercellular signaling is an essential feature of multicellular organisms, and the engineering of complex biological systems will require multiple pathways for intercellular signaling with minimal crosstalk. Natural quorum-sensing systems provide components for cell communication, but their use is often constrained by signal crosstalk. We have established new orthogonal systems for cell-cell communication using acyl homoserine lactone signaling systems. Quantitative measurements in contexts of differing receiver protein expression allowed us to separate different types of crosstalk between 3-oxo-C6- and 3-oxo-C12-homoserine lactones, cognate receiver proteins, and DNA promoters. Mutating promoter sequences minimized interactions with heterologous receiver proteins. We used experimental data to parameterize a computational model for signal crosstalk and to estimate the effect of receiver protein levels on signal crosstalk. We used this model to predict optimal expression levels for receiver proteins, to create an effective two-channel cell communication device. Establishment of a novel spatial assay allowed measurement of interactions between geometrically constrained cell populations via these diffusible signals. We built relay devices capable of long-range signal propagation mediated by cycles of signal induction, communication and response by discrete cell populations. This work demonstrates the ability to systematically reduce crosstalk within intercellular signaling systems and to use these systems to engineer complex spatiotemporal patterning in cell populations.
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Structure and Function of a Bacterial Microcompartment Shell Protein Engineered to Bind a [4Fe-4S] Cluster

Structure and Function of a Bacterial Microcompartment Shell Protein Engineered to Bind a [4Fe-4S] Cluster | SynBioFromLeukipposInstitute | Scoop.it
Bacterial microcompartments (BMCs) are self-assembling organelles composed of a selectively permeable protein shell and encapsulated enzymes. They are considered promising templates for the engineering of designed bionanoreactors for biotechnology. In particular, encapsulation of oxidoreductive reactions requiring electron transfer between the lumen of the BMC and the cytosol relies on the ability to conduct electrons across the shell. We determined the crystal structure of a component protein of a synthetic BMC shell, which informed the rational design of a [4Fe-4S] cluster-binding site in its pore. We also solved the structure of the [4Fe-4S] cluster-bound, engineered protein to 1.8 Å resolution, providing the first structure of a BMC shell protein containing a metal center. The [4Fe-4S] cluster was characterized by optical and EPR spectroscopies; it has a reduction potential of −370 mV vs the standard hydrogen electrode (SHE) and is stable through redox cycling. This remarkable stability may be attributable to the hydrogen-bonding network provided by the main chain of the protein scaffold. The properties of the [4Fe-4S] cluster resemble those in low-potential bacterial ferredoxins, while its ligation to three cysteine residues is reminiscent of enzymes such as aconitase and radical S-adenosymethionine (SAM) enzymes. This engineered shell protein provides the foundation for conferring electron-transfer functionality to BMC shells.
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Synthetic Biology and the Moral Significance of Artificial Life: A Reply to Douglas, Powell and Savulescu.

I discuss the moral significance of artificial life within synthetic biology via a discussion of Douglas, Powell and Savulescu's paper 'Is the creation of artificial life morally significant'. I argue that the definitions of 'artificial life' and of 'moral significance' are too narrow. Douglas, Powell and Savulescu's definition of artificial life does not capture all core projects of synthetic biology or the ethical concerns that have been voiced, and their definition of moral significance fails to take into account the possibility that creating artificial life is conditionally acceptable. Finally, I show how several important objections to synthetic biology are plausibly understood as arguing that creating artificial life in a wide sense is only conditionally acceptable.
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End-to-end automated microfluidic platform for synthetic biology: from design to functional analysis.

Synthetic biology aims to engineer biological systems for desired behaviors. The construction of these systems can be complex, often requiring genetic reprogramming, extensive de novo DNA synthesis, and functional screening.
RESULTS:
Herein, we present a programmable, multipurpose microfluidic platform and associated software and apply the platform to major steps of the synthetic biology research cycle: design, construction, testing, and analysis. We show the platform's capabilities for multiple automated DNA assembly methods, including a new method for Isothermal Hierarchical DNA Construction, and for Escherichia coli and Saccharomyces cerevisiae transformation. The platform enables the automated control of cellular growth, gene expression induction, and proteogenic and metabolic output analysis.
CONCLUSIONS:
Taken together, we demonstrate the microfluidic platform's potential to provide end-to-end solutions for synthetic biology research, from design to functional analysis.
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Kernel Architecture of the Genetic Circuitry of the Arabidopsis Circadian System

Kernel Architecture of the Genetic Circuitry of the  Arabidopsis  Circadian System | SynBioFromLeukipposInstitute | Scoop.it
A wide range of organisms features molecular machines, circadian clocks, which generate endogenous oscillations with ~24 h periodicity and thereby synchronize biological processes to diurnal environmental fluctuations. Recently, it has become clear that plants harbor more complex gene regulatory circuits within the core circadian clocks than other organisms, inspiring a fundamental question: are all these regulatory interactions between clock genes equally crucial for the establishment and maintenance of circadian rhythms? Our mechanistic simulation for Arabidopsis thaliana demonstrates that at least half of the total regulatory interactions must be present to express the circadian molecular profiles observed in wild-type plants. A set of those essential interactions is called herein a kernel of the circadian system. The kernel structure unbiasedly reveals four interlocked negative feedback loops contributing to circadian rhythms, and three feedback loops among them drive the autonomous oscillation itself. Strikingly, the kernel structure, as well as the whole clock circuitry, is overwhelmingly composed of inhibitory, rather than activating, interactions between genes. We found that this tendency underlies plant circadian molecular profiles which often exhibit sharply-shaped, cuspidate waveforms. Through the generation of these cuspidate profiles, inhibitory interactions may facilitate the global coordination of temporally-distant clock events that are markedly peaked at very specific times of day. Our systematic approach resulting in experimentally-testable predictions provides insights into a design principle of biological clockwork, with implications for synthetic biology.
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Hyper-production of large proteins of spider dragline silk MaSp2 by Escherichia coli via synthetic biology approach

Hyper-production of large proteins of spider dragline silk MaSp2 by Escherichia coli via synthetic biology approach | SynBioFromLeukipposInstitute | Scoop.it
Spider dragline silk exhibits excellent mechanical properties that make it a promising protein polymer for industrial and biomedical applications. Since farming spiders is not feasible due to their highly territorial nature, recombinant production of dragline silk proteins in a foreign host has received great attention. However, their production titer remains low, because efficient expression of very large, highly repetitive, glycine-rich silk proteins is a challenge. This work demonstrates the design and high-level production of large dragline silk proteins of major ampullate spidroin 2 (MaSp2) in Escherichia coli by synthetic biology approach. The expression levels of MaSp2 with molecular weight of 28.3–256.5 kDa were significantly elevated by down-shifting the induction temperature. The beneficial effect was found to be at least partially attributed to the improved plasmid maintenance in the recombinant cells. Combination of induction temperature downshift with the glycyl-tRNA pool increase in E. coli led to enhanced biosynthesis of glycine-rich silk proteins. A high production titer of about 3.6 g l−1 of a 201.6-kDa MaSp2 protein was achieved in a 3-L fed-batch bioreactor, which was the highest as reported. The developed approach may be useful to cost-effective large-scale production of silk proteins.
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Neri Oxman predicts 3D printing will usher a new age for humanity

Neri Oxman predicts 3D printing will usher a new age for humanity | SynBioFromLeukipposInstitute | Scoop.it
During this year's World Economic Forum in Davos, Switzerland, 3D printing is heavily featured in Neri Oxman's essay. By using modern technology like 3D printing in our digital age, she believes a fourth industrial revolution, what she calls the biological age, is fast approaching.
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BIOFABRICATE: There’s a bio-revolution on the horizon! | Make: DIY Projects, How-Tos, Electronics, Crafts and Ideas for Makers

BIOFABRICATE: There’s a bio-revolution on the horizon! | Make: DIY Projects, How-Tos, Electronics, Crafts and Ideas for Makers | SynBioFromLeukipposInstitute | Scoop.it
If you missed BIOFABRICATE you missed out! Researchers, artists, designers and entrepreneurs are growing building materials, packaging, and lighting products from mushrooms. They're painting with bacteria, dying cloth with microalgae and growing clothing from kombucha. Plus, get the scoop the programmable materials library being built with Autodesk's Project Cyborg and learn to GIY!
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Pathways In Science | PLOS Synthetic Biology Community

Pathways In Science | PLOS Synthetic Biology Community | SynBioFromLeukipposInstitute | Scoop.it
I have walked along many paths, even pathways, on my journey through life. Recently, I have come across new pathways, indeed two different types of pathways, which have made me curious and thoughtful.

I was sitting in a meeting on Responsible Research and Innovation (RRI) and synthetic biology the other day and heard a lot about ‘innovation pathways’, ‘commercial pathways’, ‘translational pathways’, ‘pathways to innovation’ and, of course, ‘pathways to impact’. Some weeks beforehand I had been sitting in various synthetic biology meetings surrounded by people who study ‘metabolic pathways’ (or as some call them, ‘paths of life’). In both cases I was puzzled, but in different ways. What are these pathways we are talking about? How do we journey along them or make others embark on them? How do we map what they are and where they go? How are they made and changed?

Of course, these pathways are very different, but as we shall see, they cross over (might even come into conflict) in the life of natural scientists (who explore metabolic pathways and are supposed to find pathways to [industrial] growth) and in the life of social scientists (who are tasked with embedding RRI into this process and “help avoid lock-in to innovation pathways that do not serve individual patient or public benefit”).
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How to combine biology, chemistry and synthetic biology to add synthetic amino acids to a protein, and why creativity matters.

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Biotechnology, synthetic biology keys to humans colonizing other planets

The lack of technology for such sustainable life support systems is a major factor underlying criticism of human space exploration. If long-term human presence on other worlds is the goal, than genetically altered organisms are the key.

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