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[genet-news] SCIENCE & TECHNOLOGY: Transgenic worms make tough fibers



                                  PART 1


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TITLE:   TRANSGENIC WORMS MAKE TOUGH FIBERS

SOURCE:  Technology Review, USA

AUTHOR:  Katherine Bourzac

URL:     http://www.technologyreview.com/biomedicine/26623/?p1=A2

DATE:    27.10.2010

SUMMARY: "Researchers have been trying to make artificial spider silk for decades. Now a startup claims to have overcome one of the main challenges in synthesizing the lightweight, stronger-than-steel fibers. Kraig Biocraft Laboratories has made genetically modified silkworms that produce fibers incorporating spider-silk proteins. The resulting fibers are much stronger, more flexible, and finer than silk made by normal silkworms."

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TRANSGENIC WORMS MAKE TOUGH FIBERS

A startup says it's jumped one of the big hurdles on the way to making artificial spider silk.

Researchers have been trying to make artificial spider silk for decades. Now a startup claims to have overcome one of the main challenges in synthesizing the lightweight, stronger-than-steel fibers.

Kraig Biocraft Laboratories has made genetically modified silkworms that produce fibers incorporating spider-silk proteins. The resulting fibers are much stronger, more flexible, and finer than silk made by normal silkworms. The company says it believes it will be able to match the properties of spider silk within five years. The company hopes to sell the first generation of fibers to companies that will make stronger everyday silk products. Its ultimate goal is to mass-produce artificial spider silk, which could be used to make very strong and lightweight products including bulletproof vests, composite materials for vehicles and sports equipment, and even new construction materials.

Spiders make many varieties of silk, and many of these fibers are stronger than steel. Mimicking such silk and developing ways of producing it industrially has long been a goal of materials scientists. But spiders are too aggressive to be farmed, so researchers have made transgenic animals that make the spider proteins. But that isn't enough, because simply producing the protein components of these materials is not enough--you have to mimic the way spiders put them together by spinning a thread.

"Genetic engineers have been focused on making organisms that produce as much spider-silk protein as possible, but this is like dumping a load of bricks in the yard and asking why you don't have a house," says Kim Thompson, founder and CEO of Kraig Biocraft, based in Lansing, Michigan. Bacteria, for example, can be made to produce spider-silk proteins, and the Canadian biotech company Nexia even succeeded in creating goats that excreted high levels of spider-silk proteins in their milk. But they lacked the means to assemble these proteins into usable silk.

Other groups have created transgenic silkworms that make spider silk, but the worms didn't integrate the foreign proteins into the fiber structure, and fiber's mechanical properties didn't significantly improve over what natural silkworms make. The worms' natural systems for spinning fibers are tailored to their own natural proteins. "There's no reason the silkworms would necessarily include the spider protein in their fiber," says Randy Lewis, professor of molecular biology at the University of Wyoming. Lewis has sequenced several spider-silk genes.

Kraig Biocraft had to alter the spider-silk proteins so that they would not just be made by the silkworms but also integrated into the structural matrix of the silk fibers. These proteins have chemical structures that fit together tightly to give silk fibers their integrity and strength. The company licensed Lewis's gene sequences and modified them. Lewis says he's verified that these spider proteins are chemically integrated into the core of the fiber.

The company's system for making transgenic silkworms was developed by Malcolm Fraser, professor of biological sciences at the University of Notre Dame. Fraser's methods for genetic engineering in insects and other animals were used to make the first artificial spider-protein-producing silkworms in the year 2000.

Kraig Biocraft has made 20 new varieties of transgenic silkworms by injecting silkworm embryos with Fraser's DNA constructs incorporating the modified spider genes, then screening to find worms that have integrated the new genes into their genomes. 

The company has not disclosed the results of mechanical tests performed on the fibers made by the engineered silkworms, nor has it released information about how much of the spider protein the animals make. But representatives claim that all of the animals make silks stronger and more flexible than natural silkworm silk and that one variety makes particularly strong fibers that the company has deemed "monster silk."

"We don't have much control over how much [spider] protein the transgenics express--some express a little, some a lot," says Fraser. "We're currently working on methods to ensure optimal transgenics."

David Kaplan, chair of biomedical engineering at Tufts University, who has developed biomedical applications for silkworm silk, notes that Kraig Biocraft has not yet published its results in a scientific journal. Kaplan adds that a transgenic silkworm is a promising system for making spider fibers, but notes that over the long-term, large-scale industrial production may not be as viable as growing silk-producing bacteria in vats. The fiber-making process remains a challenge for approaches using bacteria, but researchers at the University of California, Berkeley, are working on microfluidics and other systems designed to mimic the silkworm's fiber-spinning capabilities.

Kraig Biocraft's Thompson says the company's first product is likely to be based on the first versions of silk, which aren't strong enough for specialized industrial applications. These products will target the $4-billion-a-year raw-silk market in 2011. The company will then pursue industrial applications using much stronger silk.



                                  PART 2

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TITLE:   MAKING SPIDER-STRENGTH MATERIALS

SOURCE:  Technology Review, USA

AUTHOR:  Katherine Bourzac

URL:     http://www.technologyreview.com/biomedicine/25922/

DATE:    03.08.2010

SUMMARY: "Researchers have been trying to make artificial spider silk--a lightweight, tougher-than-steel material that could have countless industrial applications--for decades. In an important step toward that goal, researchers at Tufts University have created genetically engineered microbes that produce more of the proteins needed to make spider silk than ever before."

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MAKING SPIDER-STRENGTH MATERIALS

Genetically modified bacteria can produce enough proteins for super-strong spider silk.

Researchers have been trying to make artificial spider silk--a lightweight, tougher-than-steel material that could have countless industrial applications--for decades. In an important step toward that goal, researchers at Tufts University have created genetically engineered microbes that produce more of the proteins needed to make spider silk than ever before.

Dragline silk--the type spiders use for the rims and spokes of their webs--is tougher and far lighter than steel. Engineered bacteria can produce the proteins needed to synthesize this silk, which is spun together to make fibers. However, previous efforts to make spider silk using bacteria have been hamstrung for several reasons. First, researchers have had an incomplete picture of the dragline silk gene sequence. And second, they've had limited success in modifying the bacteria to produce enough of the proteins.

David Kaplan, chair of the biomedical engineering department at Tufts University, has pioneered the application of silkworm silk in medical devices, biodegradable electronics, optical devices, and adhesives. He believes that spider silk, which is stronger than the silkworm variety, could open up new applications, but says, "It hasn't been explored as much because we haven't had enough material." Spiders are aggressive and territorial and thus can't be farmed like silkworms.

Bioengineers have had only modest success in getting microbes to make spider-silk proteins. Chemical giant DuPont tried unsuccessfully to develop a bacteria-produced silk product in the 1990s. Part of the problem is that spider silk is made from a very large protein with a highly repetitive genetic sequence, making it hard to decode, says Christopher Voigt, professor of pharmaceutical chemistry at the University of California, San Francisco.

Last year, researchers using new sequencing technologies produced the first complete genetic sequence for spider silk. Before that, researchers were forced to use truncated silk genes, and fibers made using these genes were not as strong and tough as natural silk.

Even with the full dragline silk gene sequence, producing artificial silk is a challenge. Making enough of the protein requires a larger amount of starting material than the bacteria naturally contain. Working with researchers at the Korea Advanced Institute of Science and Technology in Daejeon and Seoul National University, Kaplan added the full silk gene to E. coli and then altered the bacteria's protein-making pathway so that it makes sufficient quantities of the amino acids needed to enable silk production. Previously, engineered bacteria have only been able to produce tens of milligrams of the protein per liter. Kaplan's E. coli yield one to two grams per liter.

"They've clearly shown that E. coli can make these large proteins, and engineered them to have the resources to do it," says Randy Lewis, professor of molecular biology at the University of Wyoming. Lewis predicts that it will be possible to use a bacterial system to produce kilogram quantities of artificial spider silk within a few years.

Kaplan says that's his plan. "We'd like to turn it into a continuous production process," he says.

Kaplan says what's needed now are more energy-efficient methods for making the proteins into fibers. Using spinning methods similar to those used to make polymer fibers such as polyester, his group has created fibers from the team's proteins with properties comparable to natural dragline silk in terms of strength, elasticity, and toughness. However, because spider-silk proteins are finicky and insoluble in water, spinning them into fibers requires high-temperature processing and harsh solvents.

The fibers "take a huge amount of energy to put together," says Kaplan. Materials scientists would like to make silk fibers the way spiders do: at ambient temperatures, with no harsh solvents.

A novel approach to the problem is being pursued by Luke Lee, director of the molecular nanotechnology center at the University of California, Berkeley. He is designing spinning systems that incorporate microfluidic channels designed to provide the salt- and solvent- gradients found in spider glands. A company called Refactored Materials, founded by students of Lee's and Voigt's, is also working on the spinning problem.



                                  PART 3

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TITLE:   SPIDER MAN

SOURCE:  Arizona State University, USA

AUTHOR:  Diane Boudreau

URL:     http://researchstories.asu.edu/stories/spider-men-1712

DATE:    29.10.2010

SUMMARY: "Scientists have come up with ingenious ways to get around this problem. They have genetically engineered silkworms, E. coli, and even goats to produce spider silk. Unfortunately, while these organisms produce the same proteins that spiders make, they don?t have the same mechanical properties as the natural product. They aren?t as strong, for instance, or as flexible. This is where the ASU researchers? expertise comes in."

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SPIDER MAN

When Peter Parker was bitten by a radioactive spider in a laboratory, he became Spider-Man, a superhero with the ability to spin strong, flexible webs. Jeff Yarger and Gregory Holland are hoping to harness spider power in their research lab, as well. But these real-world spider men aren?t waiting around for bizarre lab accidents. The ASU chemists are studying the molecular structure of spider silk in an effort to produce materials ranging from bulletproof vests to artificial tendons. They also want to mimic spiders? eco-friendly methods in the process.

?Everybody?s familiar with silk, because they?re familiar with silkworm silk. The silk trade has been around for a long time. But spider silk has a much larger variety in its properties,? says Yarger, a professor of chemistry and biochemistry in the College of Liberal Arts and Sciences.

While silkworms produce only one kind of fiber, a single spider can produce up to six different varieties. For instance, there is ultra-strong dragline silk, used to make the framework of a web. Some spiders produce draglines that have more tensile strength than Kevlar. Then there is flagelliform, a highly elastic silk that makes up the spirals of a web. This fiber can sometimes stretch up to 200 times its original length. Spiders use other silks for wrapping their prey, making egg sacs for their young, and other purposes.

None of this is new information. ?So why if it?s been known for hundreds of years has it never been used?? asks Yarger. The reason is that spiders don?t produce silk in large quantities.

?You can put lots of silkworms in a small area and genetically modify them to go from the larval state to a moth in 20-30 days. Spiders take longer. But let?s get to the crux of it?spiders don?t like each other. They eat each other,? he explains. This of course eliminates the possibility of farming them en masse.

Scientists have come up with ingenious ways to get around this problem. They have genetically engineered silkworms, E. coli, and even goats to produce spider silk. Unfortunately, while these organisms produce the same proteins that spiders make, they don?t have the same mechanical properties as the natural product. They aren?t as strong, for instance, or as flexible.

This is where the ASU researchers? expertise comes in. Yarger is the director of the Magnetic Resonance Research Center at ASU. Holland is an assistant research professor in the Department of Chemistry and Biochemistry. Both scientists examine the molecular structure of spider silks.

?We use a suite of magnetic resonance tools?NMR and MRI?along with other physical characterization methods such as x-ray diffraction, Raman, and infrared spectroscopy,? explains Holland.

Many groups are interested in the results of this work. Holland has received a grant from the Department of Defense to study ultra-strong materials that could be used for products like body armor. Yarger collaborates with Randy Lewis, a molecular biologist at the University of Wyoming, on a grant from the National Institutes of Health. Lewis is leading a study of flagelliform silk for use in making artificial tendons. In addition to being very flexible, spider silk is highly biocompatible, meaning that it doesn?t produce allergic reactions in humans.

Additionally, Yarger is funded by the National Science Foundation to develop new NMR techniques for analyzing biopolymers. Yarger has spent his career seeking ways to understand amorphous materials like polymers on a molecular level. While crystalline materials are highly structured, amorphous materials are largely disordered. Polymers are a type of amorphous material. Biopolymers are polymers produced by living things.

?Spider silk is the one we focus on in the grant, but the goal is to make these techniques applicable to a range of biopolymers,? says Yarger. ?The two that we?re most familiar with in the human body are collagen and elastin?both protein-based biopolymers.?

Green lynx spiders are commonly found in Arizona. Photo by Pete Zrioka.Green lynx spiders are commonly found in Arizona. Photo by Pete Zrioka.

The main focus of Yarger?s work is using NMR to determine how artificial silk differs from natural silk, and why. The goal is to help scientists reproduce it more accurately, with the desired characteristics.

To understand how the same molecule can have different properties, think of diamonds and graphite. Both have the same composition?carbons connected together?but they have incredibly different characteristics. You don?t see jewelers selling graphite engagement rings, after all.

?They are made up of the same constituent. Carbon is carbon, you would say?but it?s not. How carbon is arranged in three-dimensional space can greatly affect its structure, whether you have graphite or diamonds or buckyballs,? explains Yarger.

Spiders work some rather interesting chemistry within their tiny bodies. They hold the proteins for making webs inside glands in their abdomens, where they are dissolved in solution. When the spider wants to spin a web, it pulls the proteins through a series of ducts and out a set of spinnerets at the base of its abdomen.

?They?ve taken this aqueous protein solution and they?ve pulled an ultra-strong fiber that is no longer soluble in the medium it was in,? says Yarger. ?When it rains outside, webs don?t dissolve.?

?One of the main goals of the research is to understand the biochemistry involved in transforming the soluble proteins in the spider?s gland to an insoluble super-fiber at the spider?s spinneret,? adds Holland. ?It is our hope that a better understanding of this process will help our group and research groups around the world spin fibers that more closely resemble native spider silk.?

Spider silk offers benefits beyond simply providing useful materials. Imitating the natural process will allow scientists to create products in an environmentally friendly way.

?Most of the strong polymer materials we make right now?things like Kevlar and Teflon?very few of these are made using sustainable, environmentally friendly chemistry methods,? notes Yarger. ?They usually use toxic organic solvents and lots of them. Spiders do it out of a very natural, green aqueous solution. If we could reproduce some of that, it would go a long way to an environmentally friendly, sustainable way of producing ultra-strong polymers.?

Behind the Magnetic Resonance Research Center, in the basement of ASU?s Interdisciplinary Science and Technology 1 building, there is a room full of spiders. Each spider has a name taped to its cage, like ?Beyonce,? the golden orb weaver. These big, yellow-bellied arachnids create intricate webs that can span several meters. Because of their size, the webs need to be incredibly strong to resist breaking when faced with struggling prey.

A spider enjoys a snack while waiting to be ?milked? of its silk. Photo by Pete Zrioka.A spider enjoys a snack while waiting to be ?milked? of its silk. Photo by Pete Zrioka.

There are also silver-backed Argiopes, common garden spiders found in California. These build smaller webs than the orb weavers, but their silk is highly elastic.

Across the aisle are collections of two local spiders, captured by students. One is the bright green lynx spider, a jumping spider you can find on cactus leaves. These spiders only produce dragline silk. Yarger is looking at whether the dragline silk from more primitive spiders, which only produce one type of silk, is evolutionarily conserved among later spiders that produce many.

The other local catch is the infamous black widow, highly venomous with dragline silk similar to that of the orb weavers. While imported spiders can only be obtained during certain seasons, the researchers can collect local spiders like black widows all year long.

Yarger says he often receives requests to analyze silk from other species of spiders, as well.

?We?re one of the few groups in the world that?s studying the molecular structure of silk. We have entomologists and arachnologists contact us quite often with some unique spider species, asking if we would be interested in looking at their silk,? he says. ?What they don?t realize is that these are big proteins with very complicated structures, and there are multiple proteins in one silk. We don?t spend a day determining the structure?we spend a year or two.?

As a result, the group has to be extremely choosy about what kinds of silk it will analyze. Yarger also adds that spiders and silkworms aren?t the only silk-producing creatures. He raises insects called webspinners, for example. These creatures enshroud themselves in an extremely fine silk so that predators can?t get at them.

?It?s a very strong silk and much thinner than spider silk?nanoscopic in size. We?d like to understand the nanostructure of these,? he says.

Graduate and undergraduate students help with the work in Yarger?s lab. They learn to do everything from collecting spiders and ?milking? them for silk, as well as working with the instruments and analyzing data.

?I look at it as, you need a complete education,? says Yarger, who received his own Ph.D. at ASU. ?You can?t just say, ?I worked with spider silk? and not have done some of the basics involved in that.?



                                  PART 4

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TITLE:   SPIDER SILK FROM BACTERIA - GENETICALLY ENGINEERED E. COLI MEANS NO SPIDER FARMS

SOURCE:  The Chemical Engineer Today, USA

AUTHOR:  Helen Tunnicliffe

URL:     http://www.tcetoday.com/tcetoday/NewsDetail.aspx?nid=12995

DATE:    29.07.2010

SUMMARY: "ESCHERICHIA COLI bacteria have been genetically engineered to produce artificial spider dragline silk, which is five times stronger than steel and has multiple potential applications. Sang Yup Lee at the Korea Advanced Institute of Science and Technology led the research to find a viable alternative to spider-farming, which is virtually impossible due to spiders? territorial behaviour."

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SPIDER SILK FROM BACTERIA - GENETICALLY ENGINEERED E. COLI MEANS NO SPIDER FARMS

Strength, extensibility, and stiffness were found to be very similar to natural spider dragline silk

ESCHERICHIA COLI bacteria have been genetically engineered to produce artificial spider dragline silk, which is five times stronger than steel and has multiple potential applications.

Sang Yup Lee at the Korea Advanced Institute of Science and Technology (KAIST) led the research to find a viable alternative to spider-farming, which is virtually impossible due to spiders? territorial behaviour. The E. coli bacterium was an ideal target, as it is widely used in industry and its optimum growing conditions are well-known.

Firstly, the researchers identified the portion of spider DNA which coded for silk protein expression in the spider Nephila clavipes and inserted this into the DNA of E. coli. Initial attempts to get the bacteria to produce spider dragline silk proteins were unsuccessful, as the silk is rich in the amino acid glycine and the proteins have a high molecular weight and a highly repetitive nature. High levels of stress response proteins from the bacteria were observed.

Lee tells tce that analysis of enzymes within E. coli and experiments with increasing the levels of different elements within the process led to the researchers increasing the levels of tRNAGly, an important coding structure for the protein. The genes coding for the two types of tRNAGly were overexpressed in plasmids, small rings of coding DNA found within bacteria. Raising the tRNAGly pool led to increased cell growth of 30?50% and much higher production of the highest-weight silk proteins, which was further increased by raising the levels of glycine available.

?We could obtain appreciable expression of the 285 kilodalton spider silk protein, which is the largest recombinant silk protein ever produced in E. coli. That was really incredible,? says Lee.

Yields of protein were 0.5?2.7 g/l, but Lee says that in unpublished work they have achieved yields of 4 g/l. This is comparable to a previously reported method in tce for bee silk.

Lyophilised silk proteins were dissolved and extruded into a 90% (v/v) methanol solution and after spinning, left in the coagulation solution for 20 minutes, before being cut into 50 mm lengths. These were hand-drawn to lengths of 250 mm and dried at room temperature. The fibres were then tested for strength, extensibility, and stiffness, and were found to be very similar to natural spider dragline silk.

?We have offered an overall platform for mass production of native-like spider dragline silk. This platform would enable us to have broader industrial and biomedical applications for spider silk. Moreover, many other silk-like biomaterials such as elastin, collagen, byssus, resilin, and other repetitive proteins have similar features to spider silk protein. Thus, our platform should also be useful for their efficient bio-based production and applications,? says Lee.

Proceedings of the National Academy of Sciences doi: 10.1073/pnas.1003366107



                                  PART 5

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TITLE:   IF SPIDERS AND WORMS CAN DO IT, WHY CAN'T WE?

SOURCE:  National Science Foundation, USA (NSF)

AUTHOR:  Press Release 10-129

URL:     http://www.nsf.gov/news/news_summ.jsp?org=NSF&cntn_id=117415&preview=false

DATE:    29.06.2010

SUMMARY: "no one knows how exactly the spiders and silk worms actually make silk. Scientists have determined they don?t secrete the stuff, but instead pull it out of special glands in very specific ways. Spiders, for example, pull it with their legs, while silkworms perform a ?figure eight? dance with their heads to create the silk threads. Despite this knowledge, Omenetto and Kaplan write, ?there are still significant knowledge gaps in understanding how to reverse-engineer silk protein fibers.?"

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IF SPIDERS AND WORMS CAN DO IT, WHY CAN'T WE?

Future research could spin up new medical and materials breakthroughs based on silk, but obstacles remain in quest to replicate natural silk production, scientists say in this week?s edition of Science

Imagine a material that is tougher than Kelvar or steel, yet remarkably flexible. It?s something you can easily find in your attic or a lingerie store. It?s as instantly recognizable today as it was to our early ancestors, yet we still aren?t sure exactly how it?s made.

The miracle thread in question is natural silk, the ubiquitous fibers made by spiders and silkworms, which has been used throughout history for items ranging from stockings and parachutes to surgical sutures. Today scientists and engineers are creating a number of useful materials based on silk research. But many researchers believe these applications may just be the start of a whole web of useful new products and devices, if only we had a better understanding of just how these small creatures spin their precious thread. In recent years, researchers have worked to gain a better understanding of what silk is and how it?s made, with the goal of being able to consistently replicate and enhance its production synthetically. In the July 30 edition of the journal Science, two Tufts University researchers, Fiorenzo G. Omenetto and David L. Kaplan, review the state of silk research, the challenges that remain, and why synthetic silk production is so appealing.

According to Omenetto and Kaplan, scientists understand that silk is ?a relatively simple protein processed from water.? Research has established what those proteins are, and they have determined that the properties of silk can vary a great deal depending on factors such as the outside temperature, how fast the silk is spun, and the exact type of silk created.

But no one knows how exactly the spiders and silk worms actually make silk. Scientists have determined they don?t secrete the stuff, but instead pull it out of special glands in very specific ways. Spiders, for example, pull it with their legs, while silkworms perform a ?figure eight? dance with their heads to create the silk threads. Despite this knowledge, Omenetto and Kaplan write, ?there are still significant knowledge gaps in understanding how to reverse-engineer silk protein fibers.?

The spiders and silkworms have also figured out another neat trick that, according to Omenetto and Kaplan, still evades the capabilities of their would-be mechanical copycats. When the scientists try to store silk proteins in the lab, they find they must do so under exacting conditions, or material will quickly begin to crystallize. Nature?s silk makers, on the other hand, don?t seem to have this problem. They can store the raw silk materials internally at a variety of temperatures for days and even weeks without encountering the crystallization problem, and at this point in time, the authors write, no one is sure how they do it.

One goal of silk research, Omenetto and Kaplan write, is to find a way to genetically engineer other organisms to produce custom-designed silk proteins that could then be used to produce synthetic silk for specific purposes on a large scale. This has led to genetically modified mushrooms, bacteria and even goats that are able to produce silk protein, yet none of the actual silk produced from these modified organisms matches the qualities of the stuff produced by spiders and silk worms. Once these issues are overcome, however, Omenetto and Kaplan believe that someday, plants could be modified to produce silk as a crop, like cotton is harvested today.

So why all of this focus on silk? Omenetto and Kaplan say that figuring out how to replicate and modify silk could lead to new breakthroughs in medicine, among other fields. Although silk is used in sutures today, the authors explain, it has to be coated in wax, which prevents the sutures from being gradually absorbed into the body. Modified silks could be wax free, Omenetto and Kaplan write, and could be used to safely administer drugs within the body or even create ?degradable and flexible electronic displays for improved physiological recording? of a person?s body. These and other intriguing possibilities await, Omenetto and Kaplan say, if we can just figure out how exactly the spider spins that web.




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