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DNA Fragments Assembly Based on Nicking Enzyme System

DNA Fragments Assembly Based on Nicking Enzyme System | SynBioFromLeukipposInstitute | Scoop.it
PLOS ONE: an inclusive, peer-reviewed, open-access resource from the PUBLIC LIBRARY OF SCIENCE. Reports of well-performed scientific studies from all disciplines freely available to the whole world.
Gerd Moe-Behrens's insight:

by
Rui-Yan Wang, Zhen-Yu Shi, Ying-Ying Guo, Jin-Chun Chen, Guo-Qiang Chen 

"A couple of DNA ligation-independent cloning (LIC) methods have been reported to meet various requirements in metabolic engineering and synthetic biology. The principle of LIC is the assembly of multiple overlapping DNA fragments by single-stranded (ss) DNA overlaps annealing. Here we present a method to generate single-stranded DNA overlaps based on Nicking Endonucleases (NEases) for LIC, the method was termed NE-LIC. Factors related to cloning efficiency were optimized in this study. This NE-LIC allows generating 3′-end or 5′-end ss DNA overlaps of various lengths for fragments assembly. We demonstrated that the 10 bp/15 bp overlaps had the highest DNA fragments assembling efficiency, while 5 bp/10 bp overlaps showed the highest efficiency when T4 DNA ligase was added. Its advantage over Sequence and Ligation Independent Cloning (SLIC) and Uracil-Specific Excision Reagent (USER) was obvious. The mechanism can be applied to many other LIC strategies. Finally, the NEases based LIC (NE-LIC) was successfully applied to assemble a pathway of six gene fragments responsible for synthesizing microbial poly-3-hydroxybutyrate (PHB)."

http://bit.ly/WqCL1h

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Predictability: The Brass Ring For Synthetic Biology

Predictability: The Brass Ring For Synthetic Biology | SynBioFromLeukipposInstitute | Scoop.it
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by

Lynn Yarris


"Predictability is often used synonymously with “boring,” as in that story or that outcome was soooo predictable. For practitioners of synthetic biology seeking to engineer valuable new microbes, however, predictability is the brass ring that must be captured. Researchers with the multi-institutional partnership known as BIOFAB have become the first to grab at least a portion of this ring by unveiling a package of public domain DNA sequences and statistical models that greatly increase the reliability and precision by which biological systems can be engineered."

 

http://1.usa.gov/13TKBF0

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Spanning high-dimensional expression space using ribosome-binding site combinatorics

Spanning high-dimensional expression space using ribosome-binding site combinatorics | SynBioFromLeukipposInstitute | Scoop.it
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Zelcbuch L, Antonovsky N, Bar-Even A, Levin-Karp A, Barenholz U, Dayagi M, Liebermeister W, Flamholz A, Noor E, Amram S, Brandis A, Bareia T, Yofe I, Jubran H, Milo R.

"Protein levels are a dominant factor shaping natural and synthetic biological systems. Although proper functioning of metabolic pathways relies on precise control of enzyme levels, the experimental ability to balance the levels of many genes in parallel is a major outstanding challenge. Here, we introduce a rapid and modular method to span the expression space of several proteins in parallel. By combinatorially pairing genes with a compact set of ribosome-binding sites, we modulate protein abundance by several orders of magnitude. We demonstrate our strategy by using a synthetic operon containing fluorescent proteins to span a 3D color space. Using the same approach, we modulate a recombinant carotenoid biosynthesis pathway in Escherichia coli to reveal a diversity of phenotypes, each characterized by a distinct carotenoid accumulation profile. In a single combinatorial assembly, we achieve a yield of the industrially valuable compound astaxanthin 4-fold higher than previously reported. The methodology presented here provides an efficient tool for exploring a high-dimensional expression space to locate desirable phenotypes."

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DNA tool kit goes live online

DNA tool kit goes live online | SynBioFromLeukipposInstitute | Scoop.it
Gerd Moe-Behrens's insight:

by
Ewen Callaway

"The latest shopping website is open for business, offering unusual wares: DNA tools to help biologists to engineer life.

 The DNA sequences — which allow precise control of gene activity in the bacterium Escherichia coli — are the first output of BIOFAB, based in Emery­ville, California, which calls itself “the world’s first biological design-build facility”. Launched in 2009 with a US$1.4-million grant from the US National Science Foundation, BIOFAB aims to advance synthetic biology by creating standard biological ‘parts’ in the form of DNA sequences that control gene expression. These standard sequences should allow biologists to engineer cells that can make medicines and perform other useful tasks simply by plugging in various sets of genes. The sequences are meant to overcome a key barrier to synthetic biology: genes inserted into an organism do not behave predictably, even in such a well-understood workhorse as E. coli. “You would think after a generation of genetic engineering, expressing genes with precision in an organism as well utilized as E. coliwould be pretty straightforward. It turns out it’s not,” says BIOFAB co-director Drew Endy, a synthetic biologist at Stanford University in California. Over the past three decades, scientists have amassed collections of these sequences and used them to express genes in which they are interested. Some sequences tend to be ‘strong’ and others ‘weak’, resulting in varying levels of RNA and protein being produced. But a team led by Endy and BIOFAB co-director Adam Arkin, of Lawrence Berkeley National Laboratory in Berkeley, California, has found that the activities of those sequences are far from predictable. In two papers published online this week in Nature Methods1, 2, the team reports inserting many different combinations of promoters and RBS sequences in front of genes encoding fluorescent proteins, and then measuring the level of protein that was made. “It was a bloody mess,” says Arkin, with each promoter–RBS combination having varying effects depending on the gene. He and Endy also cite an earlier finding that a scientist hoping to express a protein at a particular level has just a 50% chance of producing the required amount within a factor of two. Such hit-or-miss expression poses a major challenge to synthetic biologists who would like to create genetic circuits involving dozens of genes. As a solution, the BIOFAB team designed promoter and RBS sequences for E. colithat do not interfere with downstream DNA, so that their effects are independent of the specific gene they are paired with. The sequences should provide scientists with a much tighter grip on gene expression, offering around a 93% chance of hitting a desired level of expression within a factor of two2. Researchers can obtain the sequences for free online (see http://www.biofab.org/data), and Arkin says that some of his colleagues are already finding them useful. Endy and Arkin’s team also devised a statistical method1 to measure the variability in the performance of their promoter and RBS sequences, and indeed any genetic part to be used in synthetic-biology applications. The method should allow researchers to create a kind of specification sheet for each biological part, making it easier for scientists to develop and share their work. Randy Rettberg, a synthetic biologist at the Massachusetts Institute of Technology in Cambridge, who has worked with Endy on similar projects, says that more labs should follow BIOFAB’s lead and industrialize the production of biological parts. And synthetic biologist Alistair Elfick of the University of Edinburgh, UK, says that the BIOFAB products should help synthetic biologists to design bigger and more complicated circuits....."

http://bit.ly/ZHgtt6

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Library of synthetic transcriptional AND gates built with split T7 RNA polymerase mutants

Library of synthetic transcriptional AND gates built with split T7 RNA polymerase mutants | SynBioFromLeukipposInstitute | Scoop.it
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*Library of synthetic transcriptional AND gates built with split T7 RNA polymerase mutants*

by
David L. Shisa and Matthew R. Bennett

"The construction of synthetic gene circuits relies on our ability to engineer regulatory architectures that are orthogonal to the host’s native regulatory pathways. However, as synthetic gene circuits become larger and more complicated, we are limited by the small number of parts, especially transcription factors, that work well in the context of the circuit. The current repertoire of transcription factors consists of a limited selection of activators and repressors, making the implementation of transcriptional logic a complicated and component-intensive process. To address this, we modified bacteriophage T7 RNA polymerase (T7 RNAP) to create a library of transcriptional AND gates for use in Escherichia coli by first splitting the protein and then mutating the DNA recognition domain of the C-terminal fragment to alter its promoter specificity. We first demonstrate that split T7 RNAP is active in vivo and compare it with full-length enzyme. We then create a library of mutant split T7 RNAPs that have a range of activities when used in combination with a complimentary set of altered T7-specific promoters. Finally, we assay the two-input function of both wild-type and mutant split T7 RNAPs and find that regulated expression of the N- and C-terminal fragments of the split T7 RNAPs creates AND logic in each case. This work demonstrates that mutant split T7 RNAP can be used as a transcriptional AND gate and introduces a unique library of components for use in synthetic gene circuits."

http://bit.ly/10JMweE

comment: 
*Wetware advances*: *Biological logic gate built by splitting viral gene*

by
John Timmer 

"In recent years, researchers in the messy world of biology have been able to build systems that function like the clean, binary switches on computer chips—and we've covered a number of reports in this area. Unfortunately, most of these share a significant limitation: they rely on proteins from bacteria that act as switches to turn genes on and off under specific conditions. We know about only a limited number of these genetic switches, which may set a severe limit on the number of logical operations we can string together inside a cell.

 A paper in this week's PNAS describes a system that may allow us to get around this limitation. The new method takes a protein from a virus that infects bacteria and cuts it in two, making a pair of genes (A and B) that each produce part of the mature protein. The two parts then act as a biological version of an AND logic gate, with output (in the form of protein activity) present only when both A and B interact. When either or both A and B are missing, the output is off. In biological terms, the inputs usually involve a simple molecule that can be sensed by proteins inside a bacteria. This paper, for example, used two kinds of sugars (arabinose and lactose). When the sugars are present, they attach to proteins inside the cell, activating genes that are controlled by those proteins. To make an AND gate, you need to design a bit of biology that can respond to both of these signals—it should be active only when both a gene regulated by arabinose and a gene regulated by lactose are each active. This has been done in a variety of ways in the past, but the authors of the new paper (both faculty at Rice University) come up with a clever scheme for doing so. It's clever in part because it's so remarkably simple. The uses of a T7 bacteriophage The new work relies on a gene from a virus called the T7 bacteriophage that infects bacteria. Instead of relying on host proteins to ensure that its genes are turned into RNA (and thus into proteins), T7 carries its own gene for a protein that transcribes DNA into RNA. This gene, called (wait for it) T7 RNA polymerase, recognizes DNA sequences on the virus, sticks to them, and then starts copying the DNA nearby into RNA. If you move the sequences it recognizes somewhere else—into the bacterial genome, onto a construct you supply—it will start copying there instead. T7 RNA polymerase therefore makes a good output. When it's active, you can use it to turn on a variety of genes, thus coordinating a significant response to your logic. But how do you get T7 RNA polymerase to respond to the two different inputs required for an AND gate? You break it in two. Scientists who were studying T7 RNA polymerase had found that, during purification, it would sometimes get cut into two different pieces, one about four times the size of the other. Either of the parts on its own is inactive, but if you put them together they stick, and the resulting aggregate is active (that is, it would bind to the appropriate DNA sequences and start making RNA, albeit at a slightly slower rate than the intact protein). It turns out this also works if the two parts are encoded by completely separate genes. You can encode the larger T7 RNA polymerase fragment in a gene that responds to arabinose and the smaller fragment in a gene that responds to lactose. A functional T7 RNA polymerase will only be present when both sugars are present, so you've made your biological AND gate. By putting a fluorescent protein under the control of T7 RNA polymerase, the authors were able to show that this worked as expected. The cells glowed green only when both sugars were added. That on its own is pretty good, although not so much better than some previous work in the field. The great part of this system is its flexibility. Because T7 RNA polymerase has been studied extensively, researchers have identified a variety of mutations that alter the protein's ability to bond to specific DNA sequences. A single change in the right location can thus switch T7 RNA polymerase from sticking to (for example) a sequence that includes GACG to one that includes the sequence GCAT. Other changes in the T7 RNA polymerase can alter the sequence it recognizes even further. Instead of relying on different proteins for every logical operation you need to do (which will quickly exhaust your supply of tractable proteins), you can now build up logic using different forms of T7 RNA polymerase, each recognizing a somewhat different sequence. This doesn't necessarily help with alternate logic operations, like NOT, but having a larger array of potential tools can only make designing biological circuitry easier."

http://ars.to/YqqEhc

Fig http://bit.ly/16q2tqB

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Practicing medicine at the nanoscale

Practicing medicine at the nanoscale | SynBioFromLeukipposInstitute | Scoop.it
Gerd Moe-Behrens's insight:

New approaches to drug delivery offer hope for new, more targeted treatments.


ByAnne Trafton


"With the recent launch of MIT’s Institute for Medical Engineering and Science, MIT News examines research with the potential to reshape medicine and health care through new scientific knowledge, novel treatments and products, better management of medical data, and improvements in health-care delivery. 

 Modern medicine is largely based on treating patients with “small-molecule” drugs, which include pain relievers like aspirin and antibiotics such as penicillin. Those drugs have prolonged the human lifespan and made many life-threatening ailments easily treatable, but scientists believe the new approach of nanoscale drug delivery can offer even more progress. Delivering RNA or DNA to specific cells offers the promise of selectively turning genes on or off, while nanoscale devices that can be injected or implanted in the body could allow doctors to target drugs to specific tissues over a defined period of time."

http://bit.ly/Y87vEB

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Wearable Electronic Sensors Can Now Be Printed Directly on the Skin | MIT Technology Review

Wearable Electronic Sensors Can Now Be Printed Directly on the Skin | MIT Technology Review | SynBioFromLeukipposInstitute | Scoop.it
Gerd Moe-Behrens's insight:

*Electronic Sensors Printed Directly on the Skin*

New electronic tattoos could help monitor health during normal daily activities. By Mike Orcutt 

"Taking advantage of recent advances in flexible electronics, researchers have devised a way to “print” devices directly onto the skin so people can wear them for an extended period while performing normal daily activities. Such systems could be used to track health and monitor healing near the skin’s surface, as in the case of surgical wounds.  Skin signals: This device, applied directly to the skin, can record useful medical information. So-called “epidermal electronics” were demonstrated previously in research from the lab of John Rogers, a materials scientist at the University of Illinois at Urbana-Champaign; the devices consist of ultrathin electrodes, electronics, sensors, and wireless power and communication systems. In theory, they could attach to the skin and record and transmit electrophysiological measurements for medical purposes. These early versions of the technology, which were designed to be applied to a thin, soft elastomer backing, were “fine for an office environment,” says Rogers, “but if you wanted to go swimming or take a shower they weren’t able to hold up.” Now, Rogers and his coworkers have figured out how to print the electronics right on the skin, making the device more durable and rugged...."



http://bit.ly/W2KPX6

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Constructing Synthetic Biology, One Breadboard at a Time

Constructing Synthetic Biology, One Breadboard at a Time | SynBioFromLeukipposInstitute | Scoop.it
Gerd Moe-Behrens's insight:

By Paul Voosen
"If there’s a point that may be lost in my recent take on synthetic biology, published this week in The Chronicle Review, it’s this: Once you get past the inflated rhetoric, synthetic biology still oozes a revolutionary vibe.

 Last year, when I visited the lab of Jim Collins, one of the field’s founders, his team was coming off the creation of a plug-and-play “breadboarding” system for microbes. It’s an idea inspired by electrical engineering, where plastic “breadboards” serve as experimental bases for tweaking circuits without the permanence of soldering. Collins’s method allows much the same, but in bacteria. There are plenty of tools around for inserting bits of DNA into bugs with some precision. But given the messiness of life, things rarely work out right the first time around. The team’s method makes pulling biological parts out of the DNA much easier, said Raffi B. Afeyan, an author of the study describing the system, which appeared this past November in Nature Methods. “There’s a very long post-construction process of making things work the way you want them to,” Afeyan said. “We sort of tried to attack that by making constructed circuits very accessible.” It gets complex quickly, but essentially the team inserted 31 unique DNA signatures into the genome of an E. coli bacterium, each matched up to a protein known to cut DNA only at one targeted site. They then developed a suite of gene parts that could be swapped in and out of those 31 slots, allowing quick modification of genome designs gone awry. And as a demonstration, they modified Collins’s classic “toggle switch” design—its creation is detailed in the article—into a four-part loop in only five days. Now they’re developing a similar tool set for human-style cells. “We found that it really speeds things up for us,” Afeyan said. In my reporting, one eminent scientist compared synthetic biology to the early days of organic chemistry. It’s an apt analogy, and one you can see in Collins’s plug-and-play system, or the “switchboard” that his lab also created recently. Work regularly appears defining how synthetic biology should be conducted, as much as it is creating applications that benefit society. Another example: Just last month, Timothy K. Lu, an assistant professor at MIT and one of Collins’s former students, published work describing bacteria that can run simple Boolean logic—AND, OR, NOT, etc.—and then stash the results in their own DNA, treating their genetic code like a personal hard drive, its memory lasting for at least 90 generations. (Here’s more detail.) Such logic-and-memory systems will be crucial for persistent computing to become a reality in synthetic biology, Lu told Nature. “To make this a really rigorous engineering discipline, we need to move towards frameworks that allow you to program cells in a more scalable fashion,” he said. “We wanted to show you can assemble a bunch of simple parts in a very easy fashion.” Of course, Collins and Lu have applications in mind, too. Right now, Lu is collaborating with the Walter Reed National Military Medical Center in hopes of treating combat veterans plagued with antibiotic-resistant skin infections. Several years ago, Collins and Lu developed modified viruses that raise the efficacy of antibiotics. Perhaps they can help end festering pain that can accompany blast wounds; right now, though, the work is limited to animal models. Meanwhile, Collins is eyeing engineered probiotics, the beneficial bugs that sit in the human gut. He has a grant from the Bill & Melinda Gates Foundation to arm those bugs with a trigger that will detect the bacteria that cause cholera, spitting out amino acids to attack the microbe. The culture would be introduced every few days, through yogurt. It would be “a synthetic self-sentinel,” Collins said, “sitting there waiting to detect and respond to a cholera infection, hoping to fight back the cholera before it took over your gut.” The eventual goal, many years away, is the possibility of modifying the gut’s bacteria permanently, eliminating the need for regular doses. Then you can start to modify microbiomes in the gut or lung to deal with allergies, asthma—maybe even dietary issues. After all, even the toolmakers must dream...."



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Quality control for synthetic biology

Gerd Moe-Behrens's insight:

"Predictable activity is what synthetic biologists seek as they combine DNA parts they have designed in order to re-create a biological process. Two methods published online this week in Nature Methods propose how to first assess the quality of current biological parts and then how to design an element that performs reliably.

 One of the ongoing challenges in synthetic biology is that parts that work well in one local DNA neighborhood or genomic context fail to perform in another. Drew Endy, Adam Arkin, and colleagues tackled this problem of reliability with two studies. First they introduce a statistical framework to assess the quality of bacterial transcription and translation control elements. They assign to each part a quality score that predicts its performance across different contexts. Then they design a control element to drive the expression of a gene of interest that performed reliably over a 1,000-fold dynamic range. These methods will take most of the guess work out of synthetic circuit design. Once a large number of elements are characterized, researchers can more confidently select the part they need to achieve the desired level of expression, without having to worry that neighboring DNA will interfere with or even silence the part."


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What is your favorite scientific paper of all time?

What is your favorite scientific paper of all time? | SynBioFromLeukipposInstitute | Scoop.it
Gerd Moe-Behrens's insight:

My personal answer:

Leonard M. Adelman, Molecular Computation of Solutions To Combinatorial Problem, Science, 266: 1021-1024, (Nov. 11) 1994.

Adelman’s paper basically kick started the field of biological computer .
A small instance of the' Hamiltonian path problem' is encoded in molecules of DNA and solved in a test tube using the tools of molecular biology. This Hamiltonian path problem is in principle similar o the following: Imagine you wish to visit 7 cities connected by a set of roads. How can you do this by stopping in each city only once? This is apparently the first example of computation carried out at the molecular level and suggests the possibility of fundamental connections between biology and computer science.

http://bit.ly/10o83pg

Picture form homepage
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Quantum Biology at the Cellular Level

Quantum Biology at the Cellular Level | SynBioFromLeukipposInstitute | Scoop.it
Gerd Moe-Behrens's insight:

 

Right in line with the discussion initialized by the extra edition of Science about quantum computer: *Quantum Biology at the Cellular Level-elements of the research program* byBordonaro M, Ogryzko V. "Quantum biology is emerging as a new field at the intersection between fundamental physics and biology, promising novel insights into the nature and origin of biological order. We discuss several elements of QBCL (Quantum Biology at Cellular Level)-a research program designed to extend the reach of quantum concepts to higher than molecular levels of biological organization. We propose a new general way to address the issue of environmentally-induced decoherence and macroscopic superpositions in biological systems, emphasizing the 'basis-dependent' nature of these concepts. We introduce the notion of 'formal superposition' and distinguish it from that of Schroedinger's cat (i.e., a superposition of macroscopically distinct states). Whereas the latter notion presents a genuine foundational problem, the former one contradicts neither common sense nor observation, and may be used to describe cellular 'decision-making' and adaptation. We stress that the interpretation of the notion of 'formal superposition' should involve non-classical correlations between molecular events in a cell. Further, we describe how better understanding of the physics of Life can shed new light on the mechanism driving evolutionary adaptation (viz., 'Basis-Dependent Selection', BDS). Experimental tests of BDS and the potential role of synthetic biology in closing the 'evolvability mechanism' loophole are also discussed." http://bit.ly/WT6fBY ref and fig http://bit.ly/Y1JdLM 

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Parallel In Vivo DNA Assembly by Recombination: Experimental Demonstration and Theoretical Approaches

Parallel In Vivo DNA Assembly by Recombination: Experimental Demonstration and Theoretical Approaches | SynBioFromLeukipposInstitute | Scoop.it
Gerd Moe-Behrens's insight:

 by
Shi Z, Wedd AG, Gras SL.

"The development of synthetic biology requires rapid batch construction of large gene networks from combinations of smaller units. Despite the availability of computational predictions for well-characterized enzymes, the optimization of most synthetic biology projects requires combinational constructions and tests. A new building-brick-style parallel DNA assembly framework for simple and flexible batch construction is presented here. It is based on robust recombination steps and allows a variety of DNA assembly techniques to be organized for complex constructions (with or without scars). The assembly of five DNA fragments into a host genome was performed as an experimental demonstration."

http://bit.ly/WzeTL9

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Biotechnology: Rewriting a genome

Biotechnology: Rewriting a genome | SynBioFromLeukipposInstitute | Scoop.it
Gerd Moe-Behrens's insight:

by
Emmanuelle Charpentier& Jennifer A. Doudna

"A bacterial enzyme that uses guide RNA molecules to target DNA for cleavage has been adopted as a programmable tool to site-specifically modify genomes of cells and organisms, from bacteria and human cells to whole zebrafish."

http://bit.ly/WPTWXd


Fig ref http://bit.ly/MyJGkY

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keith martin's curator insight, September 16, 2013 1:30 PM

there taking the dna out of zebrafish and changeing it. there changing the genome. they used the bacterial enzyme that guids rna to dna as a tool to change genomes of cells in a orginism. Its like they take the dna that gives the fish black stripes and alter it to blue and then inject it in another fish and the fish becomes white and blue.

Mau Rea's curator insight, September 22, 2014 5:32 AM

The future of Biotechnology

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Online learning: Campus 2.0

Online learning: Campus 2.0 | SynBioFromLeukipposInstitute | Scoop.it
Gerd Moe-Behrens's insight:

by
M. Mitchell Waldrop

"When campus president Wallace Loh walked into Juan Uriagereka's office last August, he got right to the point. “We need courses for this thing — yesterday!”

 Uriagereka, associate provost for faculty affairs at the University of Maryland in College Park, knew exactly what his boss meant. Campus administrators around the world had been buzzing for months about massive open online courses, or MOOCs: Internet-based teaching programmes designed to handle thousands of students simultaneously, in part using the tactics of social-networking websites. To supplement video lectures, much of the learning comes from online comments, questions and discussions. Participants even mark one another's tests.MOOCs had exploded into the academic consciousness in summer 2011, when a free artificial-intelligence course offered by Stanford University in California attracted 160,000 students from around the world — 23,000 of whom finished it. Now, Coursera in Mountain View, California — one of the three researcher-led start-up companies actively developing MOOCs — was inviting the University of Maryland to submit up to five courses for broadcast on its software platform. Loh wanted in. “He was very clear,” says Uriagereka. “We needed to be a part of this...."


http://bit.ly/12TXxva

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Supramolecular Construction of Optoelectronic Biomaterials

Supramolecular Construction of Optoelectronic Biomaterials | SynBioFromLeukipposInstitute | Scoop.it
Gerd Moe-Behrens's insight:

by
Tovar JD.

"Peptide self-assembly is a powerful method to create functional nanoscale materials such as optoelectronically relevant organic nanostructures. The enormous potential that may come from bringing π-conjugated electronic function into biological environments is poised to impact cell and tissue engineering, biosensors, and related biomedical applications. However, very little synthetic guidance is available with respect to uniting these two different materials sets in a generally applicable manner. In this Account, I describe my group's work to synthesize and assemble peptidic nanostructures built around organic electronic elements. The Account begins with a very brief background to the area of supramolecular electronics, followed by a description of areas where these nanomaterials could be useful in biology. I then discuss the synthetic approaches that we utilized to embed a variety of π-electron units directly within peptide backbones. A key supramolecular challenge with respect to subsequent self-assembly of these new molecules is balancing electrostatic contributions within the resulting nanomaterials, because the suitable geometries for stabilizing peptide assemblies may not necessarily correspond to those suitable for maximizing intermolecular π-electron interactions. Regardless of the respective magnitudes of these two major influences, the assembly paradigm is fairly robust. Variation of the π-electron units and the peptide sequences that make up the "peptide-π-peptide" triblock molecules consistently leads to fairly uniform tape-like nanostructures that maintain strong electronic coupling among the component π-electron units. We explored a diverse range of π-electron units spanning fluorescent oligo(phenylene vinylene)s, electron-accepting rylene diimides, and hole-transporting oligothiophenes. I then describe the characterization of the nanomaterials that form after molecular self-assembly in order to understand their internal structures, electronic interactions, and morphologies as existing within self-supporting hydrogel matrices. I also describe how a facile shearing process provided globally aligned macroscopic collections of one-dimensional electronic fibrils in hydrogel matrices. These general assembly processes influence intermolecular π-stacking among the embedded chromophores, and the assemblies themselves can facilitate the covalent cross-linking and polymerization (for example, of reactive diyne units). The latter offers an exciting possibility to create peptidic nanostructures comprised of single polymer chains. Finally, I discuss electronic properties as manifested in the interactions of transition dipoles within the nanomaterials and electrical properties resulting from field-effect gating. The ability to tune the observable electrical properties of the nanostructures externally will allow for their transition to in vitro or in vivo platforms as a powerful new approach to regulating biological interactions at the nanoscale."

http://bit.ly/ZM1NZJ

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Precise and reliable gene expression via standard transcription and translation initiation elements

Precise and reliable gene expression via standard transcription and translation initiation elements | SynBioFromLeukipposInstitute | Scoop.it
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by

Vivek K Mutalik,Joao C Guimaraes,Guillaume Cambray,Colin Lam,Marc Juul Christoffersen,Quynh-Anh Mai,Andrew B Tran,Morgan Paull,Jay D Keasling,Adam P Arkin& Drew Endy

 

"An inability to reliably predict quantitative behaviors for novel combinations of genetic elements limits the rational engineering of biological systems. We developed an expression cassette architecture for genetic elements controlling transcription and translation initiation in Escherichia coli: transcription elements encode a common mRNA start, and translation elements use an overlapping genetic motif found in many natural systems. We engineered libraries of constitutive and repressor-regulated promoters along with translation initiation elements following these definitions. We measured activity distributions for each library and selected elements that collectively resulted in expression across a 1,000-fold observed dynamic range. We studied all combinations of curated elements, demonstrating that arbitrary genes are reliably expressed to within twofold relative target expression windows with ~93% reliability. We expect the genetic element definitions validated here can be collectively expanded to create collections of public-domain standard biological parts that support reliable forward engineering of gene expression at genome scales."

http://bit.ly/Xq362r

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Craig Venter close to creating synthetic life

Craig Venter close to creating synthetic life | SynBioFromLeukipposInstitute | Scoop.it
Gerd Moe-Behrens's insight:

 by Andy Coghlan

"For the first time we are close to creating artificial life from scratch for the first time. So says Craig Venter, founder of the J. Craig Venter Institute in Rockville, Maryland, and famed for creating the first cell with a synthetic genome.

 "We think we're close, but we've not submitted a paper yet," he said at the Global Grand Challenges summit in London this week. Venter announced in 2010 that he had brought to life an almost completely synthetic version of the bacterium Mycoplasma mycoides, by transplanting it into the vacant shell of another bacterium. Venter's latest creation, which he has dubbed the Hail Mary Genome, will be made from scratch with genes he and his institute colleagues, Clyde Hutchison and Hamilton Smith, consider indispensable for life. The team is using computer simulations to better understand what is needed to create a simple, self-replicating cell. "Once we have a minimal chassis, we can add anything else to it," he says. Venter's quest to engineer algae to produce more oil than usual is also going well. "We've been able to increase photosynthesis threefold, meaning that we get three times as much energy per photon [of sunlight] as from natural algae," he says. He also announced that his programme to scour the oceans for novel microscopic life has so far turned up 80 million genes new to biology."


http://bit.ly/Y8iaiE

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Metabolic engineering of Escherichia coli: A sustainable industrial platform for bio-based chemical production

Metabolic engineering of Escherichia coli: A sustainable industrial platform for bio-based chemical production | SynBioFromLeukipposInstitute | Scoop.it
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Chen X, Zhou L, Kangming T, Kumar A, Singh S, Prior BA, Wang Z.

"In order to decrease carbon emissions and negative environmental impacts of various pollutants, more bulk and/or fine chemicals are produced by bioprocesses, replacing the traditional energy and fossil based intensive route. The Gram-negative rod-shaped bacterium, Escherichia coli has been studied extensively on a fundamental and applied level and has become a predominant host microorganism for industrial applications. Furthermore, metabolic engineering of E. coli for the enhanced biochemical production has been significantly promoted by the integrated use of recent developments in systems biology, synthetic biology and evolutionary engineering. In this review, we focus on recent efforts devoted to the use of genetically engineered E. coli as a sustainable platform for the production of industrially important biochemicals such as biofuels, organic acids, amino acids, sugar alcohols and biopolymers. In addition, representative secondary metabolites produced by E. coli will be systematically discussed and the successful strategies for strain improvements will be highlighted. Moreover, this review presents guidelines for future developments in the bio-based chemical production using E. coli as an industrial platform."

http://bit.ly/YYJTS5

Fig http://bit.ly/YjL7Hu

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Can't Burn This: DNA Shows Surprising Flame-Retardant Properties

Can't Burn This: DNA Shows Surprising Flame-Retardant Properties | SynBioFromLeukipposInstitute | Scoop.it
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by
NADIA DRAKE

"In addition to building organisms and storing Shakespeare’s sonnets, DNA could also keep your favorite nerd-shirt from going up in flames.

 Normally, cotton fabrics are highly flammable. But when scientists tried to set fire to cotton coated with herring sperm DNA, the fabric refused to burn, the team reported in Journal of Materials Chemistry A. “DNA can be considered as a natural flame retardant and suppressant,” said materials scientist Giulio Malucelli, whose lab at Italy’s Politecnico di Torino, Alessandria branch, tested the fire-retardant properties of DNA. “It could work also on other synthetic fabrics, or thin or thick plastic films.”  Malucelli’s lab tested whether the macromolecule could stop fires by using DNA extracted from herring sperm. The team dissolved the DNA in water, coated cotton fabrics with it, let them dry, and tried to light them up. The coating behaved similarly to ammonium polyphosphate, a flame retardant commonly used on polymeric materials such as polyurethanes (found in foams and Spandex) and polyolefins (found in flexible foams and electrical insulation). DNA’s chemical structure makes it ideal for the flame-stopping job. When heated, its phosphate-containing backbone produces phosphoric acid, which chemically removes water from cotton fibers while leaving behind a flame-resistant, carbon-rich residue. The nitrogen-containing bases release ammonia — which dilutes flammable gases and inhibits combustion reactions — and can act as “blowing agents,” which help turn the carbon-rich deposits into a slow-burning protective layer. Ultimately, these ingredients stop combustion by forming either a carbon-rich foam, or a protective, glassy carbon coating called char. “I was surprised, and then as I looked at the chemical structure of DNA, it started to become obvious why DNA works as a fire retardant,” said Alexander Morgan, a flame retardant materials scientist at the University of Dayton Research Institute. ”You probably get a mix of the glassy carbon and carbon foam forming during burning of DNA on the fabric.”...."



 http://bit.ly/10JonEQ

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When a Scientific Metaphor Becomes a Burden

When a Scientific Metaphor Becomes a Burden | SynBioFromLeukipposInstitute | Scoop.it
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By Paul Voosen
"In any science, it’s hard to talk to the outside world without resorting to metaphor and analogy. That is especially true for the nascent field of synthetic biology, which promises to apply the ideas of engineering to life, as I detail this week in The Chronicle Review. At some level, really, synthetic biology is nothing but an extended metaphor. Yet such metaphors, designed to convey complex science to the public, could be why the expectations of synthetic biology have gone so far beyond its capabilities. By “debiologizing” the work, the metaphors of computing and Lego bricks suggest an advanced understanding of the function, reliability, and purpose of living organisms that is often at odds with what’s known in biology. At least, that’s the case made by Eleonore Pauwels, a research scholar who has studied synthetic biology for the past few years at the Woodrow Wilson International Center for Scholars. “If researchers … are aware of the relative weakness of the analogy around the ‘software of life,’” she writes in a new study, out soon in BioScience, “the narratives produced in its wake might affect not only public perceptions and trust but might also have broader ramifications that would influence debates on safety assessment and ownership.” No one scientist, communicator, or journalist is responsible for how synthetic biology has been conveyed to the public. But when the Wilson Center conducted several focus groups on synthetic biology in recent years, they discussed in particular one paragraph, from a well-known article by The New Yorker’s Michael Specter: [Scientists] in the field, who have formed bicoastal clusters in the Bay area and in Cambridge, Massachusetts, see cells as hardware and genetic code as the software required to make them run. Synthetic biologists are convinced that with enough knowledge, they will be able to write programs to control those genetic components, programs that would let them not only alter nature but guide evolution as well. The hype building off such easy metaphors pains some scientists. Take Timothy S. Gardner, who helped invent the field with Jim Collins back in the late 1990s. Gardner is vice president for research and development at Amyris, one of the most prominent synthetic-biology start-ups. I reached him on the phone last year, while he was checking out the firm’s new fermenting facilities in Brazil. He’s frustrated with how the public sees the field. People seem to think scientists can literally design an organism from scratch, he said. And he can’t blame them if they think designer dinosaurs are just a few years off. “That’s fueled by the hype a lot of scientists generate,” he said. “They are intentionally blurring the line between science fiction and science fact. … Ultimately we do a disservice to ourselves to blur that line.” At times, such hype seems almost part of the scientific process. It’s happened before. See nanotechnology. Or biotechnology. It will happen again. See Big Data. It’s all part of a modern regime of innovation built on “technoscientific promises,” as Pauwels put it. Indeed, based on the center’s research, which amounts to several large national polls and a cluster of focus groups, the public may be more savvy than expected. When briefed on synthetic biology—by 2010 one-quarter of people had heard of the discipline—the public expressed neither candid optimism nor unilateral rejection. People were intrigued by the potential for doing good. And yes, they also expressed wariness of the field, but they were less worried about scientists’ ”playing God” than about their failure to admit uncertainty. Amyris’s Gardner rarely has the time to publish, given how he’s been pushing the 100 researchers that work under him. (“We’ve literally been in a sprint for five years,” he said.) But this year, he did write a short essay for Trends in Biotechnology, laying out his thoughts on the field. It still has revolutionary implications, he writes. But its most profound result could be a way of communicating, as biologists finally learn to standardize how they talk about biological function. The processes used to “characterize, archive, and communicate the function of biological parts are wholly inadequate,” he writes. They must do better. There’s another metaphor that can be applied to synthetic biology, he adds. And this one might be a bit more apt, given where things are: “Relative to its ambitions,” he writes, “synthetic biology is where aerospace engineering was in the 1800s—pre-flight.”..." 



http://bit.ly/ZBQNxU

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Quantitative estimation of activity and quality for collections of functional genetic elements

Quantitative estimation of activity and quality for collections of functional genetic elements | SynBioFromLeukipposInstitute | Scoop.it
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Vivek K Mutalik, Joao C Guimaraes, Guillaume Cambray, Quynh-Anh Mai, Marc Juul Christoffersen, Lance Martin, Ayumi Yu, Colin Lam, Cesar Rodriguez, Gaymon Bennett, Jay D Keasling, Drew Endy & Adam P Arkin

"The practice of engineering biology now depends on the ad hoc reuse of genetic elements whose precise activities vary across changing contexts. Methods are lacking for researchers to affordably coordinate the quantification and analysis of part performance across varied environments, as needed to identify, evaluate and improve problematic part types. We developed an easy-to-use analysis of variance (ANOVA) framework for quantifying the performance of genetic elements. For proof of concept, we assembled and analyzed combinations of prokaryotic transcription and translation initiation elements in Escherichia coli. We determined how estimation of part activity relates to the number of unique element combinations tested, and we show how to estimate expected ensemble-wide part activity from just one or two measurements. We propose a new statistic, biomolecular part 'quality', for tracking quantitative variation in part performance across changing contexts."

http://bit.ly/14P3PYw

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Designing DNA with Semantics: Using Semantic Technologies for Synthetic Biology

Designing DNA with Semantics: Using Semantic Technologies for Synthetic Biology | SynBioFromLeukipposInstitute | Scoop.it
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Michal Galdzicki 

Evren Sirin  Tuesday, June 4, 2013 01:30 PM - 02:15 PM Level: Technical - Introductory "Synthetic Biology is an emerging interdisciplinary field that takes an engineering approach to the design and development of novel biological devices and systems for useful purposes ranging from the production of anti-malarial drugs, to improved antibiotics, to tumor-tracking bacteria, and to engineering new biofuels. The main idea behind Synthetic Biology is to treat DNA sequences as Lego bricks and put these bricks together in different ways to build a new DNA strand that will perform a specific function.In this talk, we will describe how Semantic Technologies are being used to address many challenges synthetic biologists face today in the management of information throughout the synthetic biology research lifecycle. We will describe the RDF-based Synthetic Biology Open Language (SBOL), a standard for sharing and reusing synthetic biology designs that we have been helping to develop over the last several years, and SBPkb, a public SPARQL endpoint populated with more than 20 thousand synthetic biology parts. We will describe our early successes applying the principles of version control management from software engineering to annotate versions of synthetic biology designs using RDF. We will explain how Semantic Technologies are crucial for our ultimate goal of building intelligent CAD tools that allow researchers not only leverage reusable libraries of components, but also to carry out intelligent component retrieval, error detection, workflow management, and decision support for the design and assembly of new biological constructs. Dr. Evren Sirin is the Chief Technology Officer of Clark & Parsia, LLC. He is responsible for the ongoing design, maintenance, and implementation of the OWL 2 reasoner Pellet along with other C&P products based on semantic technologies. His areas of expertise include automated reasoning for Web ontologies, Description Logic (DL) reasoning, and AI planning. Before joining C&P, Dr. Sirin was a graduate research assistant at the MINDSWAP research group directed by Prof. Jim Hendler and received his PhD degree in Computer Science from University of Maryland, College Park in 2006. He authored many publications in top-tier journals and conferences about the Semantic Web and contributed to the standardization efforts for OWL and OWL-S. Michal is a Senior Fellow in the Department of Biomedical Informatics and Medical Education at the University of Washington. His research is focused on informatics for synthetic biologists. He works on the Synthetic Biology Information Lifecycle Management for the Enterprise project with Professors John Gennari and Herbert Sauro in collaboration with Dr. Evren Sirin from Clark and Parsia, LLC. Michal completed his PhD in Biomedical and Health Informatics at the University of Washington in 2012. As part of his dissertation research he co-founded the Synthetic Biology Open Language (SBOL) an information exchange standard for biological engineering. Michal continues to serve as an SBOL Editor. His previous research at the Children’s Hospital Boston was on the genetic basis of Autism. He received a BS in Biology from the University of Maryland in 2002 and a MS in Bioinformatics from Boston University in 2005."http://bit.ly/12G9EMp ;
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Fluorescent proteins and in vitro genetic organization for cell-free synthetic biology

Fluorescent proteins and in vitro genetic organization for cell-free synthetic biology | SynBioFromLeukipposInstitute | Scoop.it
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Roberta Lentini , Michele Forlin , Laura Martini , Cristina Del Bianco , Amy C Spencer , Domenica Torino , and Sheref S Mansy

"To facilitate the construction of cell-free genetic devices, we evaluated the ability of 17 different fluorescent proteins to give easily detectable fluorescence signals in real-time from in vitro transcription-translation reactions with a minimal system consisting of T7 RNA polymerase and E. coli translation machinery, i.e. the PUREsystem. The data were used to construct a ratiometric fluorescence assay to quantify the effect of genetic organization on in vitro expression levels. Synthetic operons with varied spacing and sequence composition between two genes that coded for fluorescent proteins were then assembled. The resulting data indicated which restriction sites and where the restriction sites should be placed in order to build genetic devices in a manner that does not interfere with protein expression. Other simple design rules were identified, such as the spacing and sequence composition influences of regions upstream and downstream of ribosome binding sites and the ability of non-AUG start codons to function in vitro."

http://bit.ly/12DfUo5

see also
Cell-free synthetic biology: Thinking outside the cell http://bit.ly/YjBmWH
Fig taken from this ref.

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The Future of Quantum Information Processing

The Future of Quantum Information Processing | SynBioFromLeukipposInstitute | Scoop.it
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Have a look at this awesome special edition of science. I am wondering about the possibilities of a quantumbiocomputer.

*The Future of Quantum Information Processing*

Introduction by:
Jelena Stajic

"In a world overwhelmed by increasing amounts of data, finding new ways to store and process information has become a necessity. Conventional silicon-based electronics has experienced rapid and steady growth, thanks to the progressive miniaturization of its basic component, the transistor, but that trend cannot continue indefinitely.

In conventional devices, information is stored and manipulated in binary form: The elementary components of these devices—the so-called bits—have two states, each of which encodes the binary 0 or 1. To move beyond the binary system, one can exploit the laws of quantum mechanics. A quantum-mechanical object with two energy levels at its disposal can occupy either of those two levels, but also an arbitrary combination ("superposition") of the two, much like an electron in a two-slit experiment can go through both slits at once. This results in infinitely many quantum states that a single quantum bit, or "qubit," can take; together with another strange property of quantum mechanics—entanglement—it allows for a much more powerful information platform than is possible with conventional components....."


 http://bit.ly/Y1JdLM ;

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A Formalized Design Process for Bacterial Consortia That Perform Logic Computing

A Formalized Design Process for Bacterial Consortia That Perform Logic Computing | SynBioFromLeukipposInstitute | Scoop.it
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*A formalized design process for bacterial consortia that perform logic computing*

by
Ji W, Shi H, Zhang H, Sun R, Xi J, Wen D, Feng J, Chen Y, Qin X, Ma Y, Luo W, Deng L, Lin H, Yu R, Ouyang Q.

"The concept of microbial consortia is of great attractiveness in synthetic biology. Despite of all its benefits, however, there are still problems remaining for large-scaled multicellular gene circuits, for example, how to reliably design and distribute the circuits in microbial consortia with limited number of well-behaved genetic modules and wiring quorum-sensing molecules. To manage such problem, here we propose a formalized design process: (i) determine the basic logic units (AND, OR and NOT gates) based on mathematical and biological considerations; (ii) establish rules to search and distribute simplest logic design; (iii) assemble assigned basic logic units in each logic operating cell; and (iv) fine-tune the circuiting interface between logic operators. We analyzed gene circuits with inputs ranging from two to four, comparing our method with the pre-existing ones. Results showed that this formalized design process is more feasible concerning numbers of cells required. Furthermore, as a proof of principle, an consortium that performs XOR function, a typical complex computing operation, was designed. The construction and characterization of logic operators is independent of "wiring" and provides predictive information for fine-tuning. This formalized design process provides guidance for the design of microbial consortia that perform distributed biological computation."

 http://bit.ly/Y1jXVG

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