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Wired Health Conference: Synthetic Biology

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47 min video
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Wired

"Craig Venter, CEO & President, Synthetic Genomics, shares the latest advancements in synthetic genomic research and technology. According to Venter, synthetic biological organisms" 

http://bit.ly/U6SeAF

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Orthogonality and burdens of heterologous AND gate gene circuits in E. coli

Synthetic biology approaches commonly introduce heterologous gene networks into a host to predictably program cells, with the expectation of the synthetic network being orthogonal to the host background. However, introduced circuits may interfere with the host’s physiology, either indirectly by posing a metabolic burden and/or through unintended direct interactions between parts of the circuit with those of the host, affecting functionality. Here we used RNA-Seq transcriptome analysis to quantify the interactions between a representative heterologous AND gate circuit and the host Escherichia coli under various conditions including circuit designs and plasmid copy numbers. We show that the circuit plasmid copy number outweighs circuit composition for their effect on host gene expression with medium-copy number plasmid showing more prominent interference than its low-copy number counterpart. In contrast, the circuits have a stronger influence on the host growth with a metabolic load increasing with the copy number of the circuits. Notably, we show that variation of copy number, an increase from low to medium copy, caused different types of change observed in the behaviour of components in the AND gate circuit leading to the unbalance of the two gate-inputs and thus counterintuitive output attenuation. The study demonstrates the circuit plasmid copy number is a key factor that can dramatically affect the orthogonality, burden and functionality of the heterologous circuits in the host chassis. The results provide important guide for future efforts to design orthogonal and robust gene circuits with minimal unwanted interaction and burden to their host.
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A CRISPR device to record time

A CRISPR device to record time | SynBioFromLeukipposInstitute | Scoop.it
Synthetic Biology
The CRISPR adaptation system has been used to record the sequence and ordering of exogenous oligonucleotides that are electroporated into cell populations. Sheth et al. engineered a system bypassing the use of exogenous DNA to directly record temporal signals. An input biological
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First-of-its-kind chemical oscillator offers new level of molecular control (w/video)

First-of-its-kind chemical oscillator offers new level of molecular control (w/video) | SynBioFromLeukipposInstitute | Scoop.it
Researchers successfully constructed a first-of-its-kind chemical oscillator that uses DNA components. DNA molecules that follow specific instructions could offer more precise molecular control of synthetic chemical systems, a discovery that opens the door for engineers to create molecular machines with new and complex behaviors.
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Spaghetti-like, DNA 'noodle origami' the new shape of things to come for nanotechnology

Spaghetti-like, DNA 'noodle origami' the new shape of things to come for nanotechnology | SynBioFromLeukipposInstitute | Scoop.it
For the past few decades, scientists have been inspired by the blueprint of life, DNA, as the shape of things to come for nanotechnology.
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Comprehensive computational design of ordered peptide macrocycles

Macrocycles by design
Macrocyclic peptides have diverse properties, including antibiotic and anticancer activities. This makes them good therapeutic leads, but screening libraries only cover a fraction of the sequence space available to peptides comprising D and L amino acids. Hosseinzadeh et al. achieved near-complete coverage in sampling the sequence space for 7- to 10-residue cyclic peptides and identified more than 200 designs predicted to fold into stable structures. Of 12 structures determined, nine were close to the computational models. They also sampled and designed 11- to 14-residue macrocycles, but without complete coverage. The designed macrocycles provide a path forward for engineering new therapeutics.

Science, this issue p. 1461
Abstract
Mixed-chirality peptide macrocycles such as cyclosporine are among the most potent therapeutics identified to date, but there is currently no way to systematically search the structural space spanned by such compounds. Natural proteins do not provide a useful guide: Peptide macrocycles lack regular secondary structures and hydrophobic cores, and can contain local structures not accessible with L-amino acids. Here, we enumerate the stable structures that can be adopted by macrocyclic peptides composed of L- and D-amino acids by near-exhaustive backbone sampling followed by sequence design and energy landscape calculations. We identify more than 200 designs predicted to fold into single stable structures, many times more than the number of currently available unbound peptide macrocycle structures. Nuclear magnetic resonance structures of 9 of 12 designed 7- to 10-residue macrocycles, and three 11- to 14-residue bicyclic designs, are close to the computational models. Our results provide a nearly complete coverage of the rich space of structures possible for short peptide macrocycles and vastly increase the available starting scaffolds for both rational drug design and library selection methods.
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Programming Morphogenesis through Systems and Synthetic Biology

Stem cell-derived multicellular systems and organoids have opened new opportunities to emulate and understand human development in vitro and provide novel patient specific tissue surrogates for disease modeling.
Single cell sequencing technologies and spatial tissue analysis provide a wealth of information on cellular fate and function and offer invaluable opportunities to decipher developmental processes.
Mammalian synthetic biology utilizes designer synthetic gene circuits to program cell fate and functions towards a desired outcome.
Engineering morphogenesis is an emerging area of science that integrates engineering principles with developmental biology to control and guide collective cell behaviors.
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Microfluidics for the masses | MIT News

Microfluidics for the masses | MIT News | SynBioFromLeukipposInstitute | Scoop.it
A new MIT-designed open-source website might well be the Pinterest of microfluidics. The site, Metafluidics.org, is a free repository of designs for lab-on-a-chip devices, submitted by all sorts of…
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UbiGate: a synthetic biology toolbox to analyse ubiquitination

Ubiquitination is mediated by an enzymatic cascade that results in the modification of substrate proteins, redefining their fate. This post-translational modification is involved in most cellular processes, yet its analysis faces manifold obstacles due to its complex and ubiquitous nature. Reconstitution of the ubiquitination cascade in bacterial systems circumvents several of these problems and was shown to faithfully recapitulate the process.
Here, we present UbiGate − a synthetic biology toolbox, together with an inducible bacterial expression system – to enable the straightforward reconstitution of the ubiquitination cascades of different organisms in Escherichia coli by ‘Golden Gate’ cloning.
This inclusive toolbox uses a hierarchical modular cloning system to assemble complex DNA molecules encoding the multiple genetic elements of the ubiquitination cascade in a predefined order, to generate polycistronic operons for expression.
We demonstrate the efficiency of UbiGate in generating a variety of expression elements to reconstitute autoubiquitination by different E3 ligases and the modification of their substrates, as well as its usefulness for dissecting the process in a time- and cost-effective manner.

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MIT iGEM team uses new CRISPR protein to target cancer-causing RNA splicing errors

MIT iGEM team uses new CRISPR protein to target cancer-causing RNA splicing errors | SynBioFromLeukipposInstitute | Scoop.it
Students built a construct that has the potential to guide a mutated form of Cas13a to a particular mRNA sequence to prevent incorrect RNA splicing. Further testing is necessary, but if successful, this construct could be used therapeutically in small cell lung cancer.
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Scientists shape DNA into doughnuts, teddy bears, and an image of the Mona Lisa

Scientists shape DNA into doughnuts, teddy bears, and an image of the Mona Lisa | SynBioFromLeukipposInstitute | Scoop.it
Scientists have made a big advance in building shapes out of the so-called building blocks of life. New techniques can shape DNA—the double-stranded helical molecule that encodes genes—into objects up to 20 times bigger than previously achieved, three separate groups report today. Together, the new approaches can make objects of virtually any shape: 3D doughnuts and dodecahedrons, cubes with teddy bear–shaped cutouts, and even a tiled image of the Mona Lisa. The techniques could someday lead to a bevy of novel devices for electronics, photonics, nanoscale machines, and possibly disease detection.

Scientists have been making shapes out of DNA since the 1980s, and those efforts took off in 2006 with the invention of a folding technique called DNA origami. It starts with a long DNA strand—called a scaffold—that has a precise sequence of the four molecular units, or nucleotides, dubbed A, C, G, and T, with which DNA spells out its genetic code. Researchers match patches of the scaffold to complementary strands of DNA called staples, which latch on to their targets in two separate places. Connecting those patches forces the scaffold to fold into a prescribed shape. A second version of the technology, introduced in 2012, uses only small strands of DNA—but no scaffolds—that assemble into Lego-like bricks that can then be linked together.

Both approaches have been wildly popular among nanotechnologists, allowing them to design shapes made from DNA from the bottom up. Researchers have also been able to coat their DNA objects with plastics, metals, and other materials to fashion tiny machine components, electronics, and photonic devices. But the size of conventional DNA objects has been limited to about 100 nanometers: Grow them any larger, and they become too floppy to take a particular shape or cannot make enough connections to their neighbors to get bigger. 
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UbiGate: a synthetic biology toolbox to analyse ubiquitination

Ubiquitination is mediated by an enzymatic cascade that results in the modification of substrate proteins, redefining their fate. This post-translational modification is involved in most cellular processes, yet its analysis faces manifold obstacles due to its complex and ubiquitous nature. Reconstitution of the ubiquitination cascade in bacterial systems circumvents several of these problems and was shown to faithfully recapitulate the process. Here, we present UbiGate - a synthetic biology toolbox, together with an inducible bacterial expression system - to enable the straightforward reconstitution of the ubiquitination cascades of different organisms in Escherichia coli by 'Golden Gate' cloning. This inclusive toolbox uses a hierarchical modular cloning system to assemble complex DNA molecules encoding the multiple genetic elements of the ubiquitination cascade in a predefined order, to generate polycistronic operons for expression. We demonstrate the efficiency of UbiGate in generating a variety of expression elements to reconstitute autoubiquitination by different E3 ligases and the modification of their substrates, as well as its usefulness for dissecting the process in a time- and cost-effective manner.
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Mass Spectrometry in Chemical Biology

Mass Spectrometry in Chemical Biology | SynBioFromLeukipposInstitute | Scoop.it
The field of synthetic biology aims to make use of the principles of engineering to understand and re-design biological systems, originating cells/organisms with predictable and novel functions to produce a wide range of chemicals such as fuels, drugs, agrochemicals and polymers. However, advances in the construction of biosynthetic pathways are hampered by bottlenecks such as deficient expression of proteins introduced by the new route, accumulation of toxic intermediates and activation of cell stress responses due to the disturbance caused by the new product. Mass spectrometry helps to elucidate the limitations in synthetic biology. This chapter will present some examples of studies in which mass spectrometry has played a major role, helping synthetic biologists to discover and identify limitations, leading to the optimization of synthetic pathways.
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Bio-computer powered by jellyfish DNA plays Tetris and other retro videogames

Bio-computer powered by jellyfish DNA plays Tetris and other retro videogames | SynBioFromLeukipposInstitute | Scoop.it
An Imperial alumus has developed a bio-pixel display that can play games such as Tetris, Snake or Pong using the protein that makes jellyfish glow

Bixel is a creative, educational tool that helps the public learn about synthetic biology, biotechnology and its applications. It was created by Cell-Free Technology, a start-up co-founded by Dyson School of Design Engineering graduate Helene Steiner.
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Multiplex recording of cellular events over time on CRISPR biological tape

A CRISPR device to record time
The CRISPR adaptation system has been used to record the sequence and ordering of exogenous oligonucleotides that are electroporated into cell populations. Sheth et al. engineered a system bypassing the use of exogenous DNA to directly record temporal signals. An input biological signal is transformed into the ratio of the frequency of incorporating trigger DNA to that of incorporating reference DNA into the genomes of a bacterial population. A multiplexing strategy enables simultaneous recording of three environmental signals with high temporal resolution.
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Engineers create stretchable camouflage skin inspired by octopuses

Engineers create stretchable camouflage skin inspired by octopuses | SynBioFromLeukipposInstitute | Scoop.it
Scientists have taken a page out of nature and created a type of stretchable skin that can quickly be turned into different shapes and textures.
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Artificial heart

ARTIFICIAL HEART A BIOENGINEERING APPLICATION PRESENTED BY B.AVINASH, V.RAVINDRA, B.ANIL KUMAR B.TECH III/IV IT G.V.P.C.O.E VISHAKAPATNAM .
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Demand for genetic engineering tools is heating up like crazy

Demand for genetic engineering tools is heating up like crazy | SynBioFromLeukipposInstitute | Scoop.it
The big-ticket acquisition of genetic design company Cell Design Labs signals a coming wave of precision cures.
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Single-stranded DNA and RNA origami

Single-stranded DNA and RNA origami | SynBioFromLeukipposInstitute | Scoop.it
Structured Abstract
INTRODUCTION
Self-folding of an information-carrying polymer into a compact particle with defined structure and function (for example, folding of a polypeptide into a protein) is foundational to biology and offers attractive potential as a synthetic strategy. Over the past three decades, nucleic acids have been used to create a variety of complex nanoscale shapes and devices. In particular, multiple DNA strands have been designed to self-assemble into user-specified structures, with or without the help of a long scaffold strand. In recent years, RNA has also emerged as a unique, programmable material, offering distinct advantages for molecular self-assembly. On the other hand, biological macromolecules, such as proteins (or protein domains), typically fold from a single polymer into a well-defined compact structure. The ability to fold de novo designed nucleic acid nanostructures in a similar manner would enable unimolecular folding instead of multistrand assembly and even replication of such structures. However, a general strategy to construct large [>1000 nucleotides (nt)] single-stranded origami (ssOrigami) remains to be demonstrated where a single-stranded nucleic acid folds into a user-specified shape.
RATIONALE
The key challenge for constructing a compact single-stranded structure is to achieve structural complexity, programmability, and generality while maintaining the topological simplicity of strand routing (to avoid putative kinetic traps imposed by knots) and hence ensuring smooth folding. The key innovation of our study is to use partially complemented double-stranded DNA or RNA and parallel crossover cohesion to construct such a structurally complex yet knot-free structure that can be folded smoothly from a single strand.
RESULTS
Here, we demonstrate a framework to design and synthesize a single DNA or RNA strand to efficiently self-fold into an unknotted compact ssOrigami structure that approximates an arbitrary user-prescribed target shape. The generality of the method was validated by the construction of 18 multikilobase DNA and 5 RNA ssOrigami, including a ~10,000-nt DNA structure (37 times larger than the previous largest discrete single-stranded DNA nanostructure) and a ~6000-nt RNA structure (10 times larger than the previous largest RNA structure). The raster-filling nature of ssOrigami permitted the experimental construction of programmable patterns of markers (for example, a “smiley” face) and cargoes on its surface, its single-strandedness enabled the demonstration of facile replication of the strand in vitro and in living cells, and its programmability allowed us to codify the design process and develop a web-based automated design tool.
CONCLUSION
The work here establishes that unimolecular DNA or RNA folding, similar to multicomponent self-assembly, is a fundamental, general, and practically tractable strategy for constructing user-specified and replicable nucleic acid nanostructures, and expands the design space and material scalability for bottom-up nanotechnology.

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Crispr Therapeutics Plans Its First Clinical Trial for Genetic Disease

Crispr Therapeutics Plans Its First Clinical Trial for Genetic Disease | SynBioFromLeukipposInstitute | Scoop.it
IN LATE 2012, French microbiologist Emmanuelle Charpentier approached a handful of American scientists about starting a company, a Crispr company. They included UC Berkeley’s Jennifer Doudna, George Church at Harvard University, and his former postdoc Feng Zhang of the Broad Institute—the brightest stars in the then-tiny field of Crispr research. Back then barely 100 papers had been published on the little-known guided DNA-cutting system. It certainly hadn’t attracted any money. But Charpentier thought that was about to change, and to simplify the process of intellectual property, she suggested the scientists team up.

It was a noble idea. But it wasn’t to be. Over the next year, as the science got stronger and VCs came sniffing, any hope of unity withered up and washed away, carried on a billion-dollar tide of investment. In the end, Crispr’s leading luminaries formed three companies—Caribou Biosciences, Editas Medicine, and Crispr Therapeutics—to take what they had done in their labs and use it to cure human disease. For nearly five years the “big three’ Crispr biotechs have been promising precise gene therapy solutions to inherited genetic conditions. And now, one of them says it’s ready to test the idea on people.

Last week, Charpentier’s company, Crispr Therapeutics, announced it has asked regulators in Europe for permission to trial a cure for the disease beta thalassemia. The study, testing a genetic tweak to the stem cells that make red blood cells, could begin as soon as next year. The company also plans to file an investigational new drug application with the Food and Drug Administration to treat sickle cell disease in the US within the first few months of 2018. The company, which is co-located in Zug, Switzerland and Cambridge, Massachusetts, said the timing is just a matter of bandwidth, as they file the same data with regulators on two different continents.

Both diseases stem from mutations in a single gene (HBB) that provides instructions for making a protein called beta-globin, a subunit of hemoglobin that binds oxygen and delivers it to tissues throughout the body via red blood cells. One kind of mutation leads to poor production of hemoglobin; another creates abnormal beta-globin structures, causing red blood cells to distort into a crescent or “sickle” shape. Both can cause anemia, repeated infections, and waves of pain. Crispr Therapeutics has developed a way to hit them both with a single treatment.

It works not by targeting HBB, but by boosting expression of a different gene—one that makes fetal hemoglobin. Everyone is born with fetal hemoglobin; it’s how cells transport oxygen between mother and child in the womb. But by six months your body puts the brakes on making fetal hemoglobin and switches over to the adult form. All Crispr Therapeutics’ treatment does is take the brakes off.

From a blood draw, scientists separate out a patient’s hematopoietic stem cells—the ones that make red blood cells. Then, in a petri dish, they zap ‘em with a bit of electricity, allowing the Crispr components to go into the cells and turn on the fetal hemoglobin gene. To make room for the new, edited stem cells, doctors destroy the patient's existing bone marrow cells with radiation or high doses of chemo drugs. Within a week after infusion, the new cells find their way to their home in the bone marrow and start making red blood cells carrying fetal hemoglobin.

According to company data from human cell and animal studies presented at the American Society of Hematology Annual Meeting in Atlanta on Sunday, the treatment results in high editing efficiency, with more than 80 percent of the stem cells carrying at least one edited copy of the gene that turns on fetal hemoglobin production; enough to boost expression levels to 40 percent. Newly minted Crispr Therapeutics CEO Sam Kulkarni says that’s more than enough to ameliorate symptoms and reduce or even eliminate the need for transfusions for beta-thalassemia and sickle cell patients. Previous research has shown that even a small change in the percentage of stem cells that produce healthy red blood cells can have a positive effect on a person with sickle cell diseases.

“I think it’s a momentous occasion for us, but also for the field in general,” says Kulkarni. “Just three years ago we were talking about Crispr-based treatments as sci-fi fantasy, but here we are.”

It was around this time last year that Chinese scientists first used Crispr in humans—to treat an aggressive lung cancer as part of a clinical trial in Chengdu, in Sichuan province. Since then, immunologists at the University of Pennsylvania have begun enrolling terminal cancer patients in the first US Crispr trial—an attempt to turbo-charge T cells so they can better target tumors. But no one has yet used Crispr to fix a genetic disease.
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DNA as a supramolecular building block

DNA as a supramolecular building block | SynBioFromLeukipposInstitute | Scoop.it
PhD student Willem Noteborn has investigated supramolecular structures. These can be useful for the loading of medicines and signalling molecules regarding, for example, cellular differentiation. In his thesis, he describe
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In Vivo Target Gene Activation via CRISPR/Cas9-Mediated Trans-epigenetic Modulation

Current genome-editing systems generally rely on inducing DNA double-strand breaks (DSBs). This may limit their utility in clinical therapies, as unwanted mutations caused by DSBs can have deleterious effects. CRISPR/Cas9 system has recently been repurposed to enable target gene activation, allowing regulation of endogenous gene expression without creating DSBs. However, in vivo implementation of this gain-of-function system has proven difficult. Here, we report a robust system for in vivo activation of endogenous target genes through trans-epigenetic remodeling. The system relies on recruitment of Cas9 and transcriptional activation complexes to target loci by modified single guide RNAs. As proof-of-concept, we used this technology to treat mouse models of diabetes, muscular dystrophy, and acute kidney disease. Results demonstrate that CRISPR/Cas9-mediated target gene activation can be achieved in vivo, leading to measurable phenotypes and amelioration of disease symptoms. This establishes new avenues for developing targeted epigenetic therapies against human diseases.
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CRISPR resources

CRISPR resources | SynBioFromLeukipposInstitute | Scoop.it
Biologists continue to hone their tools for deleting, replacing or otherwise editing DNA and a strategy called CRISPR has become one of the most popular ways to do genome engineering. Utilizing a modified bacterial protein and a RNA that guides it to a specific DNA sequence, the CRISPR system provides unprecedented control over genes in many species, including perhaps humans. This control has allowed many new types of experiments, but also raised questions about what CRISPR can enable.

At least one group has already used CRISPR on human embryos, sparking calls for a moratorium on similar work and an international summit at the end of 2015 to discuss the science and ethics of human gene editing. Meanwhile, CRISPR is making it much easier to generate genetically modified animals and plants, creating new regulatory issues that scientists, agencies politicians and, ultimately, society must address.
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Expansion of the Genetic Alphabet: A Chemist’s Approach to Synthetic Biology

The information available to any organism is encoded in a four nucleotide, two base pair genetic code. Since its earliest days, the field of synthetic biology has endeavored to impart organisms with novel attributes and functions, and perhaps the most fundamental approach to this goal is the creation of a fifth and sixth nucleotide that pair to form a third, unnatural base pair (UBP) and thus allow for the storage and retrieval of increased information. Achieving this goal, by definition, requires synthetic chemistry to create unnatural nucleotides and a medicinal chemistry-like approach to guide their optimization. With this perspective, almost 20 years ago we began designing unnatural nucleotides with the ultimate goal of developing UBPs that function in vivo, and thus serve as the foundation of semi-synthetic organisms (SSOs) capable of storing and retrieving increased information. From the beginning, our efforts focused on the development of nucleotides that bear predominantly hydrophobic nucleobases and thus that pair not based on the complementary hydrogen bonds that are so prominent among the natural base pairs but rather via hydrophobic and packing interactions. It was envisioned that such a pairing mechanism would provide a basal level of selectivity against pairing with natural nucleotides, which we expected would be the greatest challenge; however, this choice mandated starting with analogs that have little or no homology to their natural counterparts and that, perhaps not surprisingly, performed poorly. Progress toward their optimization was driven by the construction of structure–activity relationships, initially from in vitro steady-state kinetic analysis, then later from pre-steady-state and PCR-based assays, and ultimately from performance in vivo, with the results augmented three times with screens that explored combinations of the unnatural nucleotides that were too numerous to fully characterize individually. The structure–activity relationship data identified multiple features required by the UBP, and perhaps most prominent among them was a substituent ortho to the glycosidic linkage that is capable of both hydrophobic packing and hydrogen bonding, and nucleobases that stably stack with flanking natural nucleobases in lieu of the potentially more stabilizing stacking interactions afforded by cross strand intercalation. Most importantly, after the examination of hundreds of unnatural nucleotides and thousands of candidate UBPs, the efforts ultimately resulted in the identification of a family of UBPs that are well recognized by DNA polymerases when incorporated into DNA and that have been used to create SSOs that store and retrieve increased information. In addition to achieving a longstanding goal of synthetic biology, the results have important implications for our understanding of both the molecules and forces that can underlie biological processes, so long considered the purview of molecules benefiting from eons of evolution, and highlight the promise of applying the approaches and methodologies of synthetic and medical chemistry in the pursuit of synthetic biology.
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Synthetic biology-inspired therapies for metabolic diseases


Our ability to engineer mammalian cells with effective therapeutic behaviors has brought new hope for treating metabolic diseases. Synthetic gene networks have been customized to interface with the host metabolism, discriminate between healthy and diseased states, and respond by producing an adjusted dose of the therapeutic molecule. Such devices have the potential to treat a range of dysfunctions that are simply not addressable using conventional therapies. Recently, the repurposing of native signaling pathways has formed the basis of autonomous therapeutic programs genetically installed in mammalian cells and has greatly expanded the possibilities to effectively tackle metabolic disorders. Here, we outline network topologies that have been successfully validated in animal models of metabolic diseases and discuss future developments that will be important for bringing this technology closer to clinical application.
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Expanding DNA's alphabet lets cells produce novel proteins

Expanding DNA's alphabet lets cells produce novel proteins | SynBioFromLeukipposInstitute | Scoop.it
Scientists are expanding the genetic code of life, using man-made DNA to create a semi-synthetic strain of bacteria—and new research shows those altered microbes actually worked to produce proteins unlike those found i
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