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Transcription Activator-like Effectors: A Toolkit for Synthetic Biology

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by
Richard Moore , Anita Chandrahas , and Leonidas Bleris

"Transcription activator-like effectors (TALEs) are proteins secreted by Xanthomonas bacteria to aid the infection of plant species. TALEs assist infections by binding to specific DNA sequences and activating the expression of host genes. Recent results show that TALE proteins consist of a central repeat domain which determines the DNA targeting specificity and can be rapidly synthesized de novo. Considering the highly modular nature of TALEs, their versatility, and the ease of constructing these proteins, this technology can have important implications for synthetic biology applications. Here, we review developments in the area with a particular focus on modifications for custom and controllable gene regulation."



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Synthetic biology in droplet-based microfluidics

Droplet microfluidics is an active multidisciplinary area of research that evolved out of the larger field of microfluidics. It enables the user to handle, process and manipulate micrometer-sized emulsion droplets on a micro- fabricated platform. The capability to carry out a large number of individual experiments per unit time makes the droplet microfluidic technology an ideal high-throughput platform for analysis of biological and biochemical samples. The objective of this thesis was to use such a technology for designing systems with novel implications in the newly emerging field of synthetic biology. Chapter 4, the first results chapter, introduces a novel method of droplet coalescence using a flow-focusing capillary device. In Chapter 5, the development of a microfluidic platform for the fabrication of a cell-free micro-environment for site-specific gene manipulation and protein expression is described. Furthermore, a novel fluorescent reporter system which functions both in vivo and in vitro is introduced in this chapter. Chapter 6 covers the microfluidic fabrication of polymeric vesicles from poly(2-methyloxazoline-b-dimethylsiloxane-b-2-methyloxazoline) tri-block copolymer. The polymersome made from this polymer was used in the next Chapter for the study of a chimeric membrane protein called mRFP1-EstA∗. In Chapter 7, the application of microfluidics for the fabrication of synthetic biological membranes to recreate artificial cell- like chassis structures for reconstitution of a membrane-anchored protein is described.
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Process-based design of dynamical biological systems

The computational design of dynamical systems is an important emerging task in synthetic biology. Given desired properties of the behaviour of a dynamical system, the task of design is to build an in-silico model of a system whose simulated be- haviour meets these properties. We introduce a new, process-based, design methodology for addressing this task. The new methodology combines a flexible process-based formalism for specifying the space of candidate designs with multi-objective optimization approaches for selecting the most appropriate among these candidates. We demonstrate that the methodology is general enough to both formulate and solve tasks of designing deterministic and stochastic systems, successfully reproducing plausible designs reported in previous studies and proposing new designs that meet the design criteria, but have not been previously considered.
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10 Things to See and Do at Vienna Design Week | Artinfo

10 Things to See and Do at Vienna Design Week | Artinfo | SynBioFromLeukipposInstitute | Scoop.it
The Future of Product Design: POSSIBLE TOMORROWS
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The beginner’s guide to biohacking

The beginner’s guide to biohacking | SynBioFromLeukipposInstitute | Scoop.it
Bulletproof founder and biohacking guru Dave Asprey recommends five ways you can change your environment for more energy, better sleep, and weight loss.
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Could your future hard-drive be made of DNA 

Could your future hard-drive be made of DNA  | SynBioFromLeukipposInstitute | Scoop.it
Nick Glass meets a scientist pioneering the use of DNA as a way to store data.
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A Superfast DNA Sequencer Based on Motion Detection

A Superfast DNA Sequencer Based on Motion Detection | SynBioFromLeukipposInstitute | Scoop.it
For more than 20 years, the practice of using a low-intensity electric current to pull long strands of DNA through nanometer-scale pores in a membrane and measure the electric field variations of the four nucleic acids—A, C, G, T—has been growing as the main approach for DNA sequencers. 

We’ve seen the development of this technology reach the point where U.K.-based Oxford Nanopore has been offering portable DNA sequencers based on this fundamental measurement principle for more than a year. Meanwhile, in the research labs, scientists have been tinkering with better materials for the membrane and have started to work with the “wonder material” graphene to see what benefits it might provide in these types of devices.

Now researchers at the National Institute of Standards and Technology (NIST) may have changed the technology paradigm for DNA sequencers in their proposal for an entirely new material architecture that would represent the first DNA sequencer based on sensing motion in the membrane as the DNA thread passes through it.

In research described in the journal ACS Nano, the NIST researchers proposed a device in which a nanoscale ribbon of molybdenum disulfide is suspended over a metal electrode immersed in water. In this arrangement, the molybdenum disulfide acts as a kind of capacitor, storing an electrical charge. When a single strand of DNA is passed through a pore in the membrane, the membrane only flexes when a DNA base pairs up with and then separates from a complementary base affixed to the hole. It is this flexing that the motion sensor detects as an electrical signal.

In the paper, the NIST researchers performed numerical simulations of how fast and accurate this DNA sequencer could be, and they concluded that the membrane would be 79 to 86 percent accurate in identifying DNA bases in a single measurement at speeds up to about 70 million bases per second. It is this speed and accuracy that the NIST researchers see as a game changer.

“It is the promise of true single-base resolution and the ability to reliably detect repeated DNA motifs at the rates of millions of bases per second,” said Alex Smolyanitsky, a NIST researcher and lead author, in an email interview with IEEE Spectrum. “An array of sensors described in our paper has the potential to accurately sequence DNA at speeds far greater than anything on the current market, while the device itself is envisioned to be portable and low-power.”

In a head-to-head comparison with research darling du jour graphene, the benefits are clear.

“The molybdenum disulfide is much less prone to ‘sticking’ to DNA, compared to graphene,” said Smolyanitsky. “Also, it is expected to be electrically conductive at room temperature.”

Before a complete prototype is built, the NIST researchers will be working on chemical functionalization of the material. But there does seem to be an urgency to the research with a patent already being sought on the design.

“We have immediate plans and expertise to work on the experimental aspects of this technology,” said Smolyanitsky. “In addition, we are open to forming early-stage partnerships with the industry.”

DNA sequencing may be just be a starting application for this design with a wide variety of nanoelectromechanical system and device applications on the NIST researchers’ horizon.

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Engineering the Microbiome: Using Synthetic Biology as the Interface Between Ourselves and our Ecology

Engineering the Microbiome: Using Synthetic Biology as the Interface Between Ourselves and our Ecology | SynBioFromLeukipposInstitute | Scoop.it
Engineering the Microbiome: Using Synthetic Biology as the Interface Between Ourselves and our Ecology
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Unintended Consequences of Expanding the Genetic Alphabet

Unintended Consequences of Expanding the Genetic Alphabet | SynBioFromLeukipposInstitute | Scoop.it
The base pair d5SICS·dNaM was recently reported to incorporate and replicate in the DNA of a modified strain of Escherichia coli, thus making the world’s first stable semisynthetic organism. This newly expanded genetic alphabet may allow organisms to store considerably more information in order to translate proteins with unprecedented enzymatic activities. Importantly, however, there is currently no knowledge of the photochemical properties of d5SICS or dNaM—properties that are central to the chemical integrity of cellular DNA. In this contribution, it is shown that excitation of d5SICS or dNaM with near-visible light leads to efficient trapping of population in the nucleoside’s excited triplet state in high yield. Photoactivation of these long-lived, reactive states is shown to photosensitize cells, leading to the generation of reactive oxygen species and to a marked decrease in cell proliferation, thus warning scientists of the potential phototoxic side effects of expanding the genetic alphabet.
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New research center is dedicated to engineering cells into living machines

New research center is dedicated to engineering cells into living machines | SynBioFromLeukipposInstitute | Scoop.it
The Golden State will soon house its own “blue-sky” bioengineering center thanks to a healthy grant from the National Science Foundation.
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Materials Ecology: design that works with and is inspired by nature.

Materials Ecology: design that works with and is inspired by nature. | SynBioFromLeukipposInstitute | Scoop.it

Really cool concept of "materials ecology" in design/architecture 

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The Next Generation of Synthetic Biology Chassis: Moving Synthetic Biology from the Laboratory to the Field 

The Next Generation of Synthetic Biology Chassis: Moving Synthetic Biology from the Laboratory to the Field  | SynBioFromLeukipposInstitute | Scoop.it
Escherichia coli (E. coli) has played a pivotal role in the development of genetics and molecular biology as scientific fields. It is therefore not surprising that synthetic biology (SB) was built upon E. coli and continues to dominate the field. However, scientific capabilities have advanced from simple gene mutations to the insertion of rationally designed, complex synthetic circuits and creation of entirely synthetic genomes. The point is rapidly approaching where E. coli is no longer an adequate host for the increasingly sophisticated genetic designs of SB. It is time to develop the next generation of SB chassis; robust organisms that can provide the advanced physiology novel synthetic circuits will require to move SB from the laboratory into fieldable technologies. This can be accomplished by developing chassis-specific genetic toolkits that are as extensive as those for E. coli. However, the holy grail of SB would be the development of a universal toolkit that can be ported into any chassis. This viewpoint article underscores the need for new bacterial chassis, as well as discusses some of the important considerations in their selection. It also highlights a few examples of robust, tractable bacterial species that can meet the demands of tomorrow’s state-of-the-art in SB. Significant advances have been made in the first 15 years since this field has emerged. However, the advances over the next 15 years will occur not in laboratory organisms, but in fieldable species where the potential of SB can be fully realized in game changing technology.
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Alternative Watson-Crick Synthetic Genetic Systems

In its "grand challenge" format in chemistry, "synthesis" as an activity sets out a goal that is substantially beyond current theoretical and technological capabilities. In pursuit of this goal, scientists are forced across uncharted territory, where they must answer unscripted questions and solve unscripted problems, creating new theories and new technologies in ways that would not be created by hypothesis-directed research. Thus, synthesis drives discovery and paradigm changes in ways that analysis cannot. Described here are the products that have arisen so far through the pursuit of one grand challenge in synthetic biology: Recreate the genetics, catalysis, evolution, and adaptation that we value in life, but using genetic and catalytic biopolymers different from those that have been delivered to us by natural history on Earth. The outcomes in technology include new diagnostic tools that have helped personalize the care of hundreds of thousands of patients worldwide. In science, the effort has generated a fundamentally different view of DNA, RNA, and how they work.
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Inspired by Nature 

Inspired by Nature  | SynBioFromLeukipposInstitute | Scoop.it
Retooling Biological Systems To Develop New Drugs And Therapies
Despite all that humankind has created, the natural engineering of biological systems never ceases to amaze. The human brain continues to outmatch manmade machines when it comes to pattern recognition: Finding the elusive “Where’s Waldo” is a breeze for us but not so much for artificial intelligence. And plants found in nature – simple organisms capable of making complex molecules – remain the basis for many drugs used today.
While drawing inspiration from “living” technology is far from new, building novel biological systems is here and now. An emerging discipline, synthetic biology brings together concepts from engineering, physics and computer science to create artificial biological processes to improve on nature’s original design. Much of the work in this fledgling field has centered on reprogramming cells by modifying their genetic code (or DNA) to serve specific purposes. Scientists are exploring innovative “synbio” processes that offer less expensive and faster methods for developing novel products, from environmentally-friendly fuel to reengineered immune cells that fight cancer.
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The Next Generation of Synthetic Biology Chassis: Moving Synthetic Biology from the Laboratory to the Field 

The Next Generation of Synthetic Biology Chassis: Moving Synthetic Biology from the Laboratory to the Field  | SynBioFromLeukipposInstitute | Scoop.it
Escherichia coli (E. coli) has played a pivotal role in the development of genetics and molecular biology as scientific fields. It is therefore not surprising that synthetic biology (SB) was built upon E. coli and continues to dominate the field. However, scientific capabilities have advanced from simple gene mutations to the insertion of rationally designed, complex synthetic circuits and creation of entirely synthetic genomes. The point is rapidly approaching where E. coli is no longer an adequate host for the increasingly sophisticated genetic designs of SB. It is time to develop the next generation of SB chassis; robust organisms that can provide the advanced physiology novel synthetic circuits will require to move SB from the laboratory into fieldable technologies. This can be accomplished by developing chassis-specific genetic toolkits that are as extensive as those for E. coli. However, the holy grail of SB would be the development of a universal toolkit that can be ported into any chassis. This viewpoint article underscores the need for new bacterial chassis, as well as discusses some of the important considerations in their selection. It also highlights a few examples of robust, tractable bacterial species that can meet the demands of tomorrow’s state-of-the-art in SB. Significant advances have been made in the first 15 years since this field has emerged. However, the advances over the next 15 years will occur not in laboratory organisms, but in fieldable species where the potential of SB can be fully realized in game changing technology.
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From the Community: GeneMods September Newsreel | PLOS Synthetic Biology Community

From the Community: GeneMods September Newsreel | PLOS Synthetic Biology Community | SynBioFromLeukipposInstitute | Scoop.it
From the Community: GeneMods September Newsreel
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10 mind-blowing things I learned at the Bulletproof Biohacking Conference

From stem cell therapy to light hacking to intel on the new Bulletproof Labs, here are 10 cool takeaways from Dave Asprey's annual wellness summit.
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Rewriting yeast central carbon metabolism for industrial isoprenoid production

Rewriting yeast central carbon metabolism for industrial isoprenoid production | SynBioFromLeukipposInstitute | Scoop.it
A bio-based economy has the potential to provide sustainable substitutes for petroleum-based products and new chemical building blocks for advanced materials. We previously engineered Saccharomyces cerevisiae for industrial production of the isoprenoid artemisinic acid for use in antimalarial treatments1. Adapting these strains for biosynthesis of other isoprenoids such as β-farnesene (C15H24), a plant sesquiterpene with versatile industrial applications2, 3, 4, 5, is straightforward. However, S. cerevisiae uses a chemically inefficient pathway for isoprenoid biosynthesis, resulting in yield and productivity limitations incompatible with commodity-scale production. Here we use four non-native metabolic reactions to rewire central carbon metabolism in S. cerevisiae, enabling biosynthesis of cytosolic acetyl coenzyme A (acetyl-CoA, the two-carbon isoprenoid precursor) with a reduced ATP requirement, reduced loss of carbon to CO2-emitting reactions, and improved pathway redox balance. We show that strains with rewired central metabolism can devote an identical quantity of sugar to farnesene production as control strains, yet produce 25% more farnesene with that sugar while requiring 75% less oxygen. These changes lower feedstock costs and dramatically increase productivity in industrial fermentations which are by necessity oxygen-constrained6. Despite altering key regulatory nodes, engineered strains grow robustly under taxing industrial conditions, maintaining stable yield for two weeks in broth that reaches >15% farnesene by volume. This illustrates that rewiring yeast central metabolism is a viable strategy for cost-effective, large-scale production of acetyl-CoA-derived molecules.
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Synthetic Biology Market is expected to Grow more than US$ 38 Billion by 2020 

Synthetic Biology Market is expected to Grow more than US$ 38 Billion by 2020  | SynBioFromLeukipposInstitute | Scoop.it
New York, September 29: Market Research Engine has published a new report titled as “Synthetic Biology Market (Synthetic DNA, Synthetic Genes, Synthetic Cells, XNA, Chassis Organisms, DNA Synthesis, Oligonucleotide Synthesis) - Global Industry...
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Twister ribozymes as highly versatile expression platforms for artificial riboswitches

Twister ribozymes as highly versatile expression platforms for artificial riboswitches | SynBioFromLeukipposInstitute | Scoop.it
The utilization of ribozyme-based synthetic switches in biotechnology has many advantages such as an increased robustness due to in cis regulation, small coding space and a high degree of modularity. The report of small endonucleolytic twister ribozymes provides new opportunities for the development of advanced tools for engineering synthetic genetic switches. Here we show that the twister ribozyme is distinguished as an outstandingly flexible expression platform, which in conjugation with three different aptamer domains, enables the construction of many different one- and two-input regulators of gene expression in both bacteria and yeast. Besides important implications in biotechnology and synthetic biology, the observed versatility in artificial genetic control set-ups hints at possible natural roles of this widespread ribozyme class.
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Synthetic Biology Publications: Top Ten

Synthetic Biology Publications: Top Ten | SynBioFromLeukipposInstitute | Scoop.it
The latest insights and analyses from SynbiCITE – synthetic biology industrial accelerator based at Imperial College, London, UK
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Synthetic Biology-Based Point-of-Care Diagnostics for Infectious Disease

Synthetic Biology-Based Point-of-Care Diagnostics for Infectious Disease | SynBioFromLeukipposInstitute | Scoop.it
Infectious diseases outpace all other causes of death in low-income countries, posing global health risks, laying stress on healthcare systems and societies, and taking an avoidable human toll. One solution to this crisis is early diagnosis of infectious disease, which represents a powerful way to optimize treatment, increase patient survival rate, and decrease healthcare costs. However, conventional early diagnosis methods take a long time to generate results, lack accuracy, and are known to seriously underperform with regard to fungal and viral infections. Synthetic biology offers a fast and highly accurate alternative to conventional infectious disease diagnosis. In this review, we outline obstacles to infectious disease diagnostics and discuss two emerging alternatives: synthetic viral diagnostic systems and biosensors. We argue that these synthetic biology-based approaches may overcome diagnostic obstacles in infectious disease and improve health outcomes.
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Human-Computer Interaction 

Human-Computer Interaction  | SynBioFromLeukipposInstitute | Scoop.it
Human-Computer Interaction - HCI Research. 21,477 likes · 273,246 talking about this. A free page for sharing and collecting latest informatio
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Targeted Gene Activation Using RNA-Guided Nucleases

The discovery of the prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) system and its adaptation for targeted manipulation of DNA in diverse species has revolutionized the field of genome engineering. In particular, the fusion of catalytically inactive Cas9 to any number of transcriptional activator domains has resulted in an array of easily customizable synthetic transcription factors that are capable of achieving robust, specific, and tunable activation of target gene expression within a wide variety of tissues and cells. This chapter describes key experimental design considerations, methods for plasmid construction, gene delivery protocols, and procedures for analysis of targeted gene activation in mammalian cell lines using CRISPR-Cas transcription factors.
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