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Team:Slovenia - 2012.igem.org

Team:Slovenia - 2012.igem.org | SynBioFromLeukipposInstitute | Scoop.it

*Bistable toggle switch in mammalian cells*

- great iGem project from Slovenia

"Biological drugs are being used ever more often as advanced drugs for the treatment of numerous diseases, due to their more specific mode of action. In current therapies the biological drugs are usually distributed more or less throughout the whole body, although each function should often be restricted to a specific organ or tissue. This can cause serious side effects, requires larger dosages and consequently raises the price of therapy. Our solution to this problem was to implant cells producing biological drugs inside the very tissue where the drug is required. The drug producing cells are safely sealed inside microcapsules that prevent cells from spreading throughout the body and protect them from destruction by cells of the host immune system. We constructed a device that allows implanted cells to produce different types of drugs while switching between those production states can be controlled from the outside by a physician, depending on the stage of the disease. We designed our device specifically for the therapy of hepatitis C or heart attack. Against hepatitis C the engineered cells produce a protein with antiviral activity, whose biological activity we have tested and confirmed. After the state of the cells is switched, a protein that improves liver regeneration would be produced. For the therapy after a heart attack we designed cells to suppress local inflammation and promote formation of new blood vessels only around the affected tissue. A physician may initiate self-destruction of the therapeutic cells and capsules by an outside stimulus when the therapy is complete or at any other given time. We believe our system to be safe, effective and applicable in the real world for the therapy of different types of diseases."
http://bit.ly/QbjlN5

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The US intelligence chief added gene editing to a list of threats that includes North Korea's nukes and Syria's chemical weapons

The United States' top intelligence official just added gene editing technology to a list of threats that includes North Korea's nukes and Syria's chemical weapons, MIT's Technology Review reported.

Director of National Intelligence James Clapper testified before the Senate Armed Services Committee on Tuesday about 2016's US Intelligence Community's Worldwide Threat Assessment.

Genome editing is a technology used to cut and paste DNA inside living cells.

In recent years, a technique known as CRISPR has been widely adopted because it is far easier and more precise than previous methods.

It has been touted for its potential to cure or eradicate diseases and modify crops, but critics worry it could lead to the creation of designer babies or rogue organisms.
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Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion

Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion | SynBioFromLeukipposInstitute | Scoop.it
Cells routinely compartmentalize enzymes for enhanced efficiency of their metabolic pathways. Here we report a general approach to construct DNA nanocaged enzymes for enhancing catalytic activity and stability. Nanocaged enzymes are realized by self-assembly into DNA nanocages with well-controlled stoichiometry and architecture that enabled a systematic study of the impact of both encapsulation and proximal polyanionic surfaces on a set of common metabolic enzymes. Activity assays at both bulk and single-molecule levels demonstrate increased substrate turnover numbers for DNA nanocage-encapsulated enzymes. Unexpectedly, we observe a significant inverse correlation between the size of a protein and its activity enhancement. This effect is consistent with a model wherein distal polyanionic surfaces of the nanocage enhance the stability of active enzyme conformations through the action of a strongly bound hydration layer. We further show that DNA nanocages protect encapsulated enzymes against proteases, demonstrating their practical utility in functional biomaterials and biotechnology.
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Genetically encoded sensors enable real-time observation of metabolite production.

Engineering cells to produce valuable metabolic products is hindered by the slow and laborious methods available for evaluating product concentration. Consequently, many designs go unevaluated, and the dynamics of product formation over time go unobserved. In this work, we develop a framework for observing product formation in real time without the need for sample preparation or laborious analytical methods. We use genetically encoded biosensors derived from small-molecule responsive transcription factors to provide a fluorescent readout that is proportional to the intracellular concentration of a target metabolite. Combining an appropriate biosensor with cells designed to produce a metabolic product allows us to track product formation by observing fluorescence. With individual cells exhibiting fluorescent intensities proportional to the amount of metabolite they produce, high-throughput methods can be used to rank the quality of genetic variants or production conditions. We observe production of several renewable plastic precursors with fluorescent readouts and demonstrate that higher fluorescence is indeed an indicator of higher product titer. Using fluorescence as a guide, we identify process parameters that produce 3-hydroxypropionate at 4.2 g/L, 23-fold higher than previously reported. We also report, to our knowledge, the first engineered route from glucose to acrylate, a plastic precursor with global sales of $14 billion. Finally, we monitor the production of glucarate, a replacement for environmentally damaging detergents, and muconate, a renewable precursor to polyethylene terephthalate and nylon with combined markets of $51 billion, in real time, demonstrating that our method is applicable to a wide range of molecules.
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Biosensors on demand

Biosensors are powerful tools in synthetic biology for engineering metabolic pathways or controlling synthetic and native genetic circuits in bacteria. Scientists have had difficulty developing a method to engineer “designer” biosensor proteins that can precisely sense and report the presence of specific molecules, which has so far limited the number and variety of biosensor designs able to precisely regulate cell metabolism, cell biology, and synthetic gene circuits.
But new research published in Nature Methods ("Engineering an allosteric transcription factor to respond to new ligands") by a team at Harvard’s Wyss Institute for Biologically Inspired Engineering and Harvard Medical School (HMS) has leveraged combination of computational protein design, in vitro synthesis and in vivo testing to establish a first-of-its-kind strategy for identifying custom-tailored biosensors.
"Our original motivation for developing customizable biosensors was to get a life or death feedback loop for metabolic engineering," said George Church, Ph.D., Wyss Institute Core Faculty member, Professor of Genetics at HMS, Professor of Health Sciences and Technology at Harvard and the Massachusetts Institute of Technology (MIT), and the senior author on the study. "This would essentially give us 'Darwinian evolution on steroids', where colonies of bacteria genetically programmed to output a desirable commodity molecule would rapidly become more efficient from generation to generation as only the best metabolic producers will be 'self-identified' for survival."
"This advance represents a powerful new way for us to access the chemical diversity of the biosphere by mining for new pathways to make useful molecules," said Srivatsan Raman, Ph.D., formerly a Postdoctoral Fellow at the Wyss Institute and HMS and currently Assistant Professor of Biochemistry at University of Wisconsin-Madison, who is the corresponding author on the study.
To develop the method, researchers chose as their test case a natural regulatory protein from E. coli called LacI. LacI is an allosteric transcription factor (aTF), which becomes active in response to sensing "inducer" metabolites or molecules in the bacterium’s environment, thereby triggering expression of a downstream gene. Using LacI, the team set out to develop a framework for re-engineering new biosensor variants that would respond to four inducer molecules (lactitol, sucralose, gentiobiose, and fucose) that cannot be metabolized by natural E. coli. Sucralose, for example, is a completely synthetic sugar molecule sold commercially as Splenda®.
To synthesize and identify the custom-made LacI variants for sensing these four new inducers, the team designed a novel workflow incorporating a combinatorial synthesis strategy that relies on computational protein design and the Wyss Institute’s custom DNA synthesis resources to build a variant library of potential new biosensor designs comprising hundreds of thousands of mutated LacI proteins.
Then, to identify the variants with the most specific responses to the four target molecules of interest, the team engineered groups of E. coli bacteria to express green fluorescent protein (GFP) when the desired molecule was detected, thereby making the bacteria fluoresce. Performing high-throughput in vivo screening of the sensor library in the engineered E. coli, the team identified the most effective variants by their high fluorescence, then filtered them out and genetically sequenced them to reveal the DNA profiles and design maps for transforming aTFs into custom-tailored sensors with high specificity.
The results are striking in that an optimized engineered aTF sensor can be identified for sensing any arbitrary molecule using this approach, opening new doors in synthetic biology by putting allosteric proteins in the control of genetic engineers.
"The LacI protein we chose to re-design into a custom biosensor is only one of thousands of different allosteric transcription factors that exist in nature," said Noah Taylor, a graduate researcher at the Wyss Institute who recently finished his Ph.D. in Biological and Biomedical Sciences at HMS, and the first author on the study. "The ability to engineer LacI using nothing more than sequence and structure information suggests we could find tens, hundreds, or even thousands of other biosensors that respond to different molecules."
Biosensors built using this approach provide feedback on how much of a certain metabolite is present inside a cell. Metabolically engineered bacteria can be outfitted with these custom aTFs, enabling them to monitor their own bioproduction of a desired chemical, pharmaceutical or biofuel. This allows sophisticated designs in which the lack of sufficient product could result in the death of an individual cell, eliminating it from the culture. In this way, powerful evolutionary methods can be harnessed for metabolic engineering.
Sensitive detection of metabolites within cells also presents a new paradigm for the way scientists can interrogate single cells. Until now, it has been very challenging to study the metabolic state of a single individual cell. But designer biosensors could be utilized as custom responders to metabolites of interest, giving insight into the metabolic states of live cells in close to real time.
"We are now utilizing the method to find biosensors for a variety of high-value targets, particularly those that can aid in protecting the environment," said Alexander Garruss, co-author on the study, who is a graduate researcher at the Wyss Institute and a Ph.D. candidate in Bioinformatics and Integrative Genomics at HMS.
Beyond measuring metabolites within cells, the combinatorial synthesis approach paves a path forward toward designing countless new and highly specific biological sensors for novel applications such as environmental monitoring, medical diagnostics, bioremediation, and precision gene therapies.
"The team’s ability to engineer custom biosensors for virtually any molecule is another triumph showing the power of synthetic biology, and its ability to generate valuable new tools to advance medicine and protect our environment," said Wyss Institute Founding Director Donald Ingber, M.D., who is also the Judah Folkman Professor of Vascular Biology at Harvard Medical School and the Vascular Biology Program at Boston Children’s Hospital and Professor of Bioengineering at the Harvard John A. Paulson School of Engineering and Applied Sciences.
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The art is alive at UC Irvine exhibit

The art is alive at UC Irvine exhibit | SynBioFromLeukipposInstitute | Scoop.it
The new exhibit at UC Irvine's Beall Center for Art + Technology is a place where art has come to life — literally.

The show, "Wetware: Art Agency Animation," opened Saturday, featuring nine artists who created works of synthetic biology art — pieces made out of biological or electronics materials.

Amino acids, contained bacteria, enzymes, microbial fuel cells, cell phone motors and other components were integrated into more than a dozen artworks.

"Nearly all the artwork here is alive," said Jens Hauser, one of the exhibit's curators. "It is artwork being an organism. It is not art for the market, but for the market of ideas. The food for thought."

Three of the nine artists developed their pieces for "Wetware" during a three-week UCI residency in October and November.

British artist Anna Dumitriu and a duo from Amsterdam, Evelina Domnitch and Dmitry Gelfand, were chosen from a pool of about 40 applicants when the university put out a call in July.

Originally, the residency program was meant for one artist. But when "Wetware's" curators, Hauser and David Familian, received the applications and project proposals during the summer, they were so impressed by the talent that they decided to feature more works.

During the three artists' stay in Irvine, they were paired with UCI researchers to develop their scientific art.

"This was the first time that our lab has worked with artists," said Elliot Hui, a UCI associate professor who worked with Domnitch and Gelfand in the Hui Lab for biological microtechnology. "Art can definitely help communicate scientific concepts, but in a more beautiful and relatable way."

The Hui Lab helped generate a liquid solution that would glow for Domnitch and Gelfand's piece, titled "Luminiferous Drift." The artwork was inspired by images of a swirling, hexagon-shaped jet stream above Saturn's north pole.

On the exhibit's opening day Saturday, Gelfand presented the duo's work to guests, using a syringe to pour drops of the solution into bath water in a spinning, cylindrical container. The solution — which includes materials such as horseradish enzyme, luminol, sodium hydroxide and hydrogen peroxide — glowed on contact with the water.

As it spun in the bath, the solution formed a hexagonal shape meant to visualize the same movements above Saturn.

"The rotating bath and a disc at the bottom are both spinning at different speeds, allowing it to create that hexagonal shape," Gelfand said. "We very much wanted our piece to be a guided, sensory experience."

Before the duo leaves Irvine next week, the artists plan to build a system that will automatically drip the solution into the bath, Gelfand said.

Dumitriu collaborated with UCI's Liu Laboratory for Synthetic Evolution for her artwork, which includes framed sheets of black velvet with yeast imprinted in the fibers and a necklace with beads arranged in the shape of an engineered antibody from the Liu Lab.

Lab members often described the antibody's components as being similar to beads. The metaphor inspired the idea for Dumitriu's 452-bead necklace, which also contains the 21 types of amino acids needed to make the actual engineered antibody, Dumitriu said. Within each bead is one amino acid, and the beads are arranged in the precise order as in the antibody.

"I wanted to use these objects to take people through a journey," Dumitriu said. "A lot of people are nervous to look at art because they think they don't know how to understand it. But I think the real key is to take time and ask what it is making you feel. That's how you understand art."

The exhibit also showcases the works of artists from Austria, Mexico and the United States.
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Towards the first data acquisition standard in Synthetic Biology.

This paper describes the development of a new data acquisition standard for synthetic biology. This comprises the creation of a methodology that is designed to capture all the data, metadata and protocol information associated with biopart characterization experiments. The new standard, called DICOM-SB, is a based on the highly successful Digital Imaging and Communications in Medicine (DICOM) standard in medicine. A data model is described which has been specifically developed for synthetic biology. The model is a modular, extensible data model for the experimental process, which can optimize data storage for large amounts of data. DICOM-SB also includes services orientated towards the automatic exchange of data and information between modalities and repositories. DICOM-SB has been developed in the context of systematic design in synthetic biology - which is based on the engineering principles of modularity, standardization and characterization. The systematic design approach utilizes the design, build, test and learn design cycle paradigm. DICOM-SB has been designed to be compatible with and complementary to other standards in synthetic biology, including SBOL. In this regard, the software provides effective interoperability. The new standard has been tested by experiments and data exchange between Nanyang Technological University in Singapore and Imperial College London.
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Optogenetic control of nuclear protein export

Optogenetic control of nuclear protein export | SynBioFromLeukipposInstitute | Scoop.it
Active nucleocytoplasmic transport is a key mechanism underlying protein regulation in eukaryotes. While nuclear protein import can be controlled in space and time with a portfolio of optogenetic tools, protein export has not been tackled so far. Here we present a light-inducible nuclear export system (LEXY) based on a single, genetically encoded tag, which enables precise spatiotemporal control over the export of tagged proteins. A constitutively nuclear, chromatin-anchored LEXY variant expands the method towards light inhibition of endogenous protein export by sequestering cellular CRM1 receptors. We showcase the utility of LEXY for cell biology applications by regulating a synthetic repressor as well as human p53 transcriptional activity with light. LEXY is a powerful addition to the optogenetic toolbox, allowing various novel applications in synthetic and cell biology.
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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...

Gerd Moe-Behrens's insight:
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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Yeast converts sugar to make renewable nylon - Futurity

Yeast converts sugar to make renewable nylon - Futurity | SynBioFromLeukipposInstitute | Scoop.it
A new hybrid way to convert sugar into nylon works at room temperature and uses inexpensive materials.

The technique combines a genetically engineered strain of yeast with a lead catalyst.

Previous attempts to combine biocatalysis and chemical catalysis to produce biorenewable chemicals have resulted in low conversion rates. That’s usually because the biological processes leave residual impurities that harm the effectiveness of chemical catalysts.

Zengyi Shao and Jean-Philippe Tessonnier, assistant professors of chemical and biological engineering at Iowa State University, are lead authors of a paper in the journal Angewandte Chemie International Edition that describes the successful hybrid conversion process.

Shao says it “opens the door to the production of a broad range of compounds not accessible from the petrochemical industry.”

Moving forward, the engineers will work to scale up their technology by developing a continuous conversion process, says Tessonnier.
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Chemical cages: New technique advances synthetic biology

Chemical cages: New technique advances synthetic biology | SynBioFromLeukipposInstitute | Scoop.it
Living systems rely on a dizzying variety of chemical reactions essential to development and survival. Most of these involve a specialized class of protein molecules--the enzymes.
In a new study, Hao Yan, director of the Center for Molecular Design and Biomimetics at Arizona State University's Biodesign Institute presents a clever means of localizing and confining enzymes and the substrate molecules they bind with, speeding up reactions essential for life processes.
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Engineering an allosteric transcription factor to respond to new ligands

Engineering an allosteric transcription factor to respond to new ligands | SynBioFromLeukipposInstitute | Scoop.it
Genetic regulatory proteins inducible by small molecules are useful synthetic biology tools as sensors and switches. Bacterial allosteric transcription factors (aTFs) are a major class of regulatory proteins, but few aTFs have been redesigned to respond to new effectors beyond natural aTF-inducer pairs. Altering inducer specificity in these proteins is difficult because substitutions that affect inducer binding may also disrupt allostery. We engineered an aTF, the Escherichia coli lac repressor, LacI, to respond to one of four new inducer molecules: fucose, gentiobiose, lactitol and sucralose. Using computational protein design, single-residue saturation mutagenesis or random mutagenesis, along with multiplex assembly, we identified new variants comparable in specificity and induction to wild-type LacI with its inducer, isopropyl β-D-1-thiogalactopyranoside (IPTG). The ability to create designer aTFs will enable applications including dynamic control of cell metabolism, cell biology and synthetic gene circuits.
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Meet the Radical Scientists Who Want to Grow Our Seafood In a Lab

Meet the Radical Scientists Who Want to Grow Our Seafood In a Lab | SynBioFromLeukipposInstitute | Scoop.it
In thirty years, meat that was grown in a laboratory might be a perfectly ordinary sight at your local grocery store. The technology to produce it exists, and it’s getting cheaper every day.

But while epicureans are already lining up to order the first stem cell burgers and meatless meatballs, another major protein source has yet to be added to this future food menu: seafood.

Fish, shellfish, and crustaceans are dietary staples for billions of people, and unsustainable fishing practices are decimating stocks worldwide. Some say fish farms are the answer, but these come with a host of environmental problems. That’s why a handful of scientists, entrepreneurs, artists and designers recently have begun to ponder a more radical solution: replacing nets with petri dishes.

“I think once people are more aware of the seafood supply chain, they’ll be looking for alternatives,” Dominique Barnes, co-founder of New Wave Foods told Gizmodo. “If you’re going to eat seafood, why not eat something that tastes just as good, with the same nutritional benefit, texture and flavor?”

Barnes’ company, a startup at the San Fransisco tech incubator IndieBio, made headlines last fall for its ambitious plan to create synthetic shrimp that’s indistinguishable from the real thing. The two-woman team is close to bringing its first “seafood” to market—and their success could pave the way for other entrepreneurs searching for a foothold in this emerging industry. But there are still a host of technical challenges to be overcome, and some experts question whether lab-grown seafood will ever be anything more than a technological novelty.

If you’ve heard anything about lab-grown meat, it’s probably thanks to a certain stem cell burger which created an international sensation in 2013. But efforts to grow meat in vitro can be traced back over a decade earlier—and one of the very first attempts involved fish.

In 2002, Morris Benjaminson, a professor emeritus at Touru University, received a small grant from NASA to explore the possibility of lab-grown meat, with the idea that future astronauts might use the technology to enjoy steak nights in space. In a rather grisly experiment, Benjaminson and his colleagues excised chunks of goldfish muscle from live fish and dunked them in vats of fetal bovine serum, a nutrient-rich cocktail brewed from the blood of unborn calves. After about a week, the severed fish chunks had grown in size by 14 percent and resembled small filets.
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TECNOx: A Latin American Syn Bio (and more) student competition | PLOS Synthetic Biology Community

TECNOx: A Latin American Syn Bio (and more) student competition | PLOS Synthetic Biology Community | SynBioFromLeukipposInstitute | Scoop.it
TECNOx: A Latin American Syn Bio (and more) student competition
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Integrative Systems and Synthetic Biology of Cell-Matrix Adhesion Sites.

The complexity of cell-matrix adhesion convolves its roles in the development and functioning of multicellular organisms and their evolutionary tinkering. Cell-matrix adhesion is mediated by sites along the plasma membrane that anchor the actin cytoskeleton to the matrix via a large number of proteins, collectively called the integrin adhesome. Fundamental challenges for understanding how cell-matrix adhesion sites assemble and function arise from their multi-functionality, rapid dynamics, large number of components and molecular diversity. Systems biology faces these challenges in its strive to understand how the integrin adhesome gives rise to functional adhesion sites. Synthetic biology enables engineering intracellular modules and circuits with properties of interest. In this review I discuss some of the fundamental questions in systems biology of cell-matrix adhesion and how synthetic biology can help addressing them.
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Researchers create synthetic biopathway to turn agriculture waste into 'green' products

Researchers create synthetic biopathway to turn agriculture waste into 'green' products | SynBioFromLeukipposInstitute | Scoop.it

http://www.sciencedaily.com/releases/2016/02/160208135451.htm Researchers at the University of Minnesota have engineered a new synthetic biopathway that can...

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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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