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  • richardmitnick 1:13 pm on June 18, 2018 Permalink | Reply
    Tags: , , , , Synthetic Biology   

    From Lawrence Berkeley National Lab: “Faster, Cheaper, Better: A New Way to Synthesize DNA” 

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    From Lawrence Berkeley National Lab

    June 18, 2018
    Julie Chao
    JHChao@lbl.gov
    (510) 486-6491

    1
    Sebastian Palluk (left) and Daniel Arlow of the Joint BioEnergy Institute (JBEI) have pioneered a new way to synthesize DNA sequences. (Credit: Marilyn Chung/Berkeley Lab)

    In the rapidly growing field of synthetic biology, in which organisms can be engineered to do things like decompose plastic and manufacture biofuels and medicines, production of custom DNA sequences is a fundamental tool for scientific discovery. Yet the process of DNA synthesis, which has remained virtually unchanged for more than 40 years, can be slow and unreliable.

    Now in what could address a critical bottleneck in biology research, researchers at the Department of Energy’s Joint BioEnergy Institute (JBEI), based at Lawrence Berkeley National Laboratory (Berkeley Lab), announced they have pioneered a new way to synthesize DNA sequences through a creative use of enzymes that promises to be faster, cheaper, and more accurate. The discovery, led by JBEI graduate students Sebastian Palluk and Daniel Arlow, was published in Nature Biotechnology in a paper titled De novo DNA Synthesis Using Polymerase-Nucleotide Conjugates.

    “DNA synthesis is at the core of everything we try to do when we build biology,” said JBEI CEO Jay Keasling, the corresponding author on the paper and also a Berkeley Lab senior faculty scientist. “Sebastian and Dan have created what I think will be the best way to synthesize DNA since [Marvin] Caruthers invented solid-phase DNA synthesis almost 40 years ago. What this means for science is that we can engineer biology much less expensively – and in new ways – than we would have been able to do in the past.”

    The Caruthers process uses the tools of organic chemistry to attach DNA building blocks one at a time and has become the standard method used by DNA synthesis companies and labs around the world. However, it has drawbacks, the main ones being that it reaches its limit at about 200 bases, partly due to side reactions than can occur during the synthesis procedure, and that it produces hazardous waste. For researchers, even 1,000 bases is considered a small gene, so to make longer sequences, the shorter ones are stitched together using a process that is failure-prone and can’t make certain sequences.

    Buying your genes online

    A DNA sequence is made up of a combination of four chemical bases, represented by the letters A, C, T, and G. Researchers regularly work with genes of several thousand bases in length. To obtain them, they either need to isolate the genes from an existing organism, or they can order the genes from a company.

    “You literally paste the sequence into a website, then wait two weeks,” Arlow said. “Let’s say you buy 10 genes. Maybe nine of them will be delivered to you on time. In addition, if you want to test a thousand genes, at $300 per gene, the costs add up very quickly.”

    Palluk and Arlow were motivated to work on this problem because, as students, they were spending many long, tedious hours making DNA sequences for their experiments when they would much rather have been doing the actual experiment.

    “DNA is a huge biomolecule,” Palluk said. “Nature makes biomolecules using enzymes, and those enzymes are amazingly good at handling DNA and copying DNA. Typically our organic chemistry processes are not anywhere close to the precision that natural enzymes offer.”


    Faster, Cheaper, Better Way to Make DNA

    Thinking outside the box

    The idea of using an enzyme to make DNA is not new – scientists have been trying for decades to find a way to do it, without success. The enzyme of choice is called TdT (terminal deoxynucleotidyl transferase), which is found in the immune system of vertebrates and is one of the few enzymes in nature that writes new DNA from scratch rather than copying DNA. What’s more, it’s fast, able to add 200 bases per minute.

    In order to harness TdT to synthesize a desired sequence, the key requirement is to make it add just one nucleotide, or DNA building block, and then stop before it keeps adding the same nucleotide repeatedly. All of the previous proposals envisioned using nucleotides modified with special blocking groups to prevent multiple additions. However, the problem is that the catalytic site of the enzyme is not large enough to accept the nucleotide with a blocking group attached. “People have basically tried to ‘dig a hole’ in the enzyme by mutating it to make room for this blocking group,” Arlow said. “It’s tricky because you need to make space for it but also not screw up the activity of the enzyme.”

    Palluk and Arlow came up with a different approach. “Instead of trying to dig a hole in the enzyme, what we do is tether one nucleotide to each TdT enzyme via a cleavable linker,” Arlow said. “That way, after extending a DNA molecule using its tethered nucleotide, the enzyme has no other nucleotides available to add, so it stops. A key advantage of this approach is that the backbone of the DNA – the part that actually does the chemical reaction – is just like natural DNA, so we can try to get the full speed out of the enzyme.”

    Once the nucleotide is added to the DNA molecule, the enzyme is cleaved off. Then the cycle can begin again with the next nucleotide tethered to another TdT enzyme.

    Keasling finds the approach clever and counterintuitive. “Rather than reusing an enzyme as a catalyst, they said, ‘Hey, we can make enzymes really inexpensively. Let’s just throw it away.’ So the enzyme becomes a reagent rather than a catalyst,” he said. “That kind of thinking then allowed them to do something very different from what’s been proposed in the literature and – I think – accomplish something really important.”

    They demonstrated their method by manually making a DNA sequence of 10 bases. Not surprisingly, the two students were initially met with skepticism. “Even when we had first results, people would say, ‘It doesn’t make sense; it doesn’t seem right. That’s not how you use an enzyme,’” Palluk recalled.

    The two still have much work to do to optimize their method, but they are reasonably confident that they will be able to eventually make a gene with 1,000 bases in one go at many times the speed of the chemical method.

    Berkeley Lab has world-renowned capabilities in synthetic biology, technology development for biology, and engineering for biological process development. A number of technologies developed at JBEI and by the Lab’s Biosciences Area researchers have been spun into startups, including Lygos, Afingen, TeselaGen, and CinderBio.

    “After decades of optimization and fine-tuning, the conventional method now typically achieves a yield of about 99.5 percent per step. Our proof-of-concept synthesis had a yield of 98 percent per step, so it’s not quite on par yet, but it’s a promising starting point,” Palluk said. “We think that we’ll catch up soon and believe that we can push the system far beyond the current limitations of chemical synthesis.”

    “Our dream is to make a gene overnight,” Arlow said. “For companies trying to sustainably biomanufacture useful products, new pharmaceuticals, or tools for more environmentally friendly agriculture, and for JBEI and DOE, where we’re trying to produce fuels and chemicals from biomass, DNA synthesis is a key step. If you speed that up, it could drastically accelerate the whole process of discovery.”

    JBEI is a DOE Bioenergy Research Center funded by DOE’s Office of Science, and is dedicated to developing advanced biofuels. Other co-authors on the paper are: Tristan de Rond, Sebastian Barthel, Justine Kang, Rathin Bector, Hratch Baghdassarian, Alisa Truong, Peter Kim, Anup Singh, and Nathan Hillson.

    See the full article here .


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  • richardmitnick 3:16 pm on July 17, 2017 Permalink | Reply
    Tags: A key building block for controlling microbiomes, Controlling gene expression across bacterial colonies, , , Master clock, Programming the clock, Synthetic Biology,   

    From UCSD Jacobs School of Engineering: “Scientists at the UC San Diego Center for Microbiome Innovation invent new tool for the Synthetic Biologist’s toolbox” 

    UC San Diego bloc

    UC San Diego


    Jacobs School of Engineering

    July 10, 2017
    Mario Aguilera
    Scripps Institute of Oceanography
    Phone: 858-534-3624
    scrippsnews@ucsd.edu

    Researchers at the University of California San Diego have invented a new method for controlling gene expression across bacterial colonies. The method involves engineering dynamic DNA copy number changes in a synchronized fashion. The results were published in the July 10, 2017 online edition of Nature Genetics.

    Until now, methods for controlling or programming bacterial cells involved transcriptional and post-transcriptional regulation. UC San Diego researchers led by Jeff Hasty, a professor of bioengineering and biology and member of the UC San Diego Center for Microbiome Innovation, describe a new method, which involves cutting circular pieces of bacterial DNA called plasmids, effectively destroying the DNA and turning off regulation.

    The study also demonstrates how DNA concentration can be increased to turn on a synthetic gene circuit. By controlling DNA copy number, researchers can effectively regulate gene expression.

    Synthetic Biology – which can involve altering biological systems for some purpose – is emerging as an engineering discipline. The field was firmly established in 2000, with the description of synthetic biological circuits in which parts of a cell are designed to perform functions, similar to the way an electronic circuit works. Also similar to an electronic circuit, the task performed by a biological circuit can be turned on and off. At the same time, researchers described the making of a “genetic clock”, which involves placing genes in a particular order so that they’ll be turned on at a specific time. This approach has also helped researchers understand natural “oscillators”, such as our sleep-wake cycle.

    Since these early inventions, Hasty and his team have shown how engineered cellular oscillations can be synchronized within a bacterial colony using plasmids, synthetically designed by the researchers themselves. Now, the team is adding a new tool to the Synthetic Biologist’s toolbox – a “master clock” of sorts that will allow researchers to coordinate subprocesses in bacterial cells.

    “This remarkable achievement is a key building block for controlling microbiomes”, said Rob Knight, professor of pediatrics at UC San Diego with a joint appointment in computer science and engineering. Knight leads the Center for Microbiome Innovation. “By controlling different strains with the same master clock, or by giving different strains their own clocks, we can start to engineer population-level dynamics to control specific microbiome functions.”

    Examples of these functions might include interaction with host cells at particular times of day, such as timed release of neurotransmitters produced by the bacteria, or interactions with other bacteria such as antifungal production triggered by a meal rich in sugar.

    Programming the clock

    The researchers used an endonuclease from Saccharomyces cerevisiae, a species of yeast, expressed alongside a plasmid containing the nuclease recognition sequence to temporarily reduce the plasmid’s copy number below natural levels.

    “We found that plasmid replication is so strong that we couldn’t cut them all,” said Hasty. “This was good news, because it meant we could down-regulate gene expression, but not eliminate it.”

    The researchers reasoned that the method could be used to regulate an entire suite of genes and promoters, and tested their idea using a previously constructed circuit to produce sustained cycling of DNA plasmid concentration across a colony of E. coli cells.

    The circuit works by using a small molecule, known as AHL, to coordinate gene expression across a colony of bacterial cells. Once on, the genes driven by the promoter are also activated, including the AHL-producing gene itself. Thanks to this positive feedback loop, the more AHL accumulates, the more it is produced. Because AHL is small enough to diffuse between cells and turn on the promoter in neighboring cells, the genes activated by it would also be produced in high amounts, leading to a phenomenon known as quorum sensing.

    Hasty and his team employed the endonuclease to reduce the number of these plasmids present in the colony and used this mechanism as negative feedback to driving the oscillations in gene expression. Using quorum sensing, the feedback system was coupled across the colony of cells.

    “We observed regular oscillations of gene expression in microfluidic chambers at different colony length scales and over extended time periods,” said Hasty. “By incorporating elements for both positive and negative copy number regulation, we were able to improve the robustness of the circuit.”

    See the full article here .

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    About the Jacobs School
    Innovation Happens Here

    The UC San Diego Jacobs School of Engineering is a premier research school set apart by our entrepreneurial culture and integrative engineering approach.

    The Jacobs School’s Mission:

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    Engineering for the global good
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    Our education models focus on deep and broad engineering fundamentals, enhanced by real-world design and research, often in partnership with industry. Through our Team Internship Program and GlobalTeams in Engineering Service program, for example, we encourage students to develop their communications and leadership skills while working in the kind of multi-disciplinary team environment experienced by real-world engineers.

    We are home to exciting research centers, such as the San Diego Supercomputer Center, a national resource for data-intensive computing; our Powell Structural Research Laboratories, the largest and most active in the world for full-scale structural testing; and the Qualcomm Institute, which is the UC San Diego division of the California Institute for Telecommunications and Information Technology (Calit2), which is forging new ground in multi-disciplinary applications for information technology.

    Located at the hub of San Diego’s thriving information technology, biotechnology, clean technology, and nanotechnology sectors, the Jacobs School proactively seeks corporate partners to collaborate with us in research, education and innovation.

    The University of California, San Diego (also referred to as UC San Diego or UCSD), is a public research university located in the La Jolla area of San Diego, California, in the United States.[12] The university occupies 2,141 acres (866 ha) near the coast of the Pacific Ocean with the main campus resting on approximately 1,152 acres (466 ha).[13] Established in 1960 near the pre-existing Scripps Institution of Oceanography, UC San Diego is the seventh oldest of the 10 University of California campuses and offers over 200 undergraduate and graduate degree programs, enrolling about 22,700 undergraduate and 6,300 graduate students. UC San Diego is one of America’s Public Ivy universities, which recognizes top public research universities in the United States. UC San Diego was ranked 8th among public universities and 37th among all universities in the United States, and rated the 18th Top World University by U.S. News & World Report ‘s 2015 rankings.

     
  • richardmitnick 12:25 pm on December 4, 2015 Permalink | Reply
    Tags: , , Synthetic Biology   

    From MIT: “Bringing synthetic biology education to life” 


    MIT News

    December 4, 2015
    Rob Matheson

    1
    Founded by Natalie Kuldell, an instructor in the Department of Biological Engineering, BioBuilder equips middle and high schools with synthetic biology kits and curricula. Here, students participate in a BioBuilder lab. Photo: Apples and Honey Photography

    Synthetic biology — which involves engineering biological systems for new uses — has become an increasingly prominent, and promising, field of study in colleges and universities worldwide.

    Research has yielded, for example, viruses that attack harmful bacteria, yeasts that produce biofuels, and engineered microorganisms capable of detecting toxins in the environment, among many other innovations.

    Yet high school students rarely learn about synthetic biology at all, says Natalie Kuldell, an instructor of biological engineering at MIT. The issue, she says, is lack of accessible, hands-on curricula for such a rapidly developing field.

    “With subjects like physics, for instance, you can demonstrate engineering by building Rube Goldberg machines or model bridges,” Kuldell says. “But it’s hard to think how to bring engineering to biology for high school students.”

    In partnership with high school teachers, Kuldell launched the BioBuilder Educational Foundation in 2011 to provide schools with lab kits and lesson plans — adapted from her own MIT curriculum and MIT research labs — to boost interest and innovation in the field.

    Today, more than 400 teachers in 43 states are using the formal BioBuilder curriculum. Some have also adapted the coursework for middle school students. Many other teachers worldwide incorporate some of the material, which is freely available online, into their lesson plans. Recently, BioBuilder and Boston’s Museum of Science received a National Science Foundation (NSF) grant to develop synthetic biology kits to send to more than 200 other museums around the country next summer.

    “It’s certainly taken off,” Kuldell says, adding: “Teachers love learning, and love teaching, and BioBuilder lets them do both.”

    A new side of biology

    To use BioBuilder, students start off reading comic books and watching animations that present a specific research problem and experiment. Then it’s off to the lab, where groups of students are provided kits to conduct their own experiments.

    BioBuilder offers five labs, which include engineering bacteria to emit specific colors or odors, or building circuits. Kits include all necessary tools and materials, such as droppers, centrifuge tubes, circuit parts, and bacteria such as E. coli to study.

    Teachers can decide which BioBuilder lessons to teach, and how. Some teachers run full semester-long classes, and some pick and choose a few lessons each year; others use it as an afterschool program or final project.

    David Mangus, head of a new biotechnology program at Brockton High School in Brockton, Massachusetts, has been using several BioBuilder lessons as well as the “Biology by Design” unit — where students must write mock grant proposals — as capstone projects. BioBuilder, he says, has shown students a new side of biology. “It’s a different way of thinking about life,” he says. “It’s what we can do with it, instead of just experiencing it.”

    A favorite lab among students, Kuldell says, is called “Eau That Smell,” which involves examining bacteria engineered to smell like ripe bananas at a certain stage of their growth. Students must then modify the bacteria to emit that specific odor at different growth stages. “They do a lot of quantitative and qualitative measurements, start thinking about what data they believe, how to improve the system, and how to better engineer a system,” she says.

    Many students also enjoy “What a Colorful World,” a lab where students study E. coli programmed to change colors. In it, students must consider how different cellular chassis (structures) might be engineered to control the emitted color, “just as a car’s chassis must be tailored to the engine it will house,” Kuldell says. When the labs are complete, students may share any data through an online portal run by BioBuilder.

    Kuldell also hosts an afterschool BioBuilderClub for students and teachers around the world, where students can engineer their own biotechnology designs. Students also have the opportunity to present their work to each other and to synthetic biologists throughout the year. This encourages students to really get involved and “own their data,” Kuldell says.

    Impact beyond the classroom

    Over the years, BioBuilder has served as a launchpad for notable projects and influenced the career choices of some students.

    Last year, a team of high school students from the Taipei American School in Taiwan used their BioBuilder classwork to engineer an extracellular protein that binds to and inhibits Granzyme B, an enzyme that causes tissue damage. For their project, the students won the grand prize for the high school track at this year’s International Genetically Engineered Machine (iGEM) Competition in Boston, the world’s top synthetic biology competition for college and high school students.

    At Brockton High School, sophomore Aysha Cheretakis, one of Mangus’ students, has become a founding member of the school’s BioBuilderClub, which is currently engineering an organism to detect Rohypnol (a drug commonly referred to as “roofies”) in drinks, which they hope will be used in crime labs.

    Cheretakis became interested in synthetic biology after the “real-life laboratory experience” of BioBuilder’s “What a Colorful World” lab. “It’s not just stuff we read out of a book. In the lab we can apply our knowledge to the real world,” she says.

    For Michael Sheets, a graduate of Tyngsborough High School in Tyngsborough, Massachusetts, BioBuilder was the gateway to studying microbiology at Olin College. The program, he adds, also gave him a “fantastic boost” in his college research: “The knowledge and skills I gained have stayed with me and been immensely useful in my education and internships since, and I’m sure will continue to aid me throughout my career.”

    Building BioBuilder

    Kuldell launched BioBuilder as part of a NSF grant, called SynBERC, which provided 10 years of funding for synthetic biology research.

    The grant funded Kuldell’s partnership with several area high school teachers who came to MIT for summer research. Together they developed an online educational platform for synthetic biology, based on research at MIT and elsewhere. Teachers who were interested in bringing those lessons to their labs and classrooms came to a professional development workshop at MIT and returned to their schools across the nation to pilot the program.

    Soon the BioBuilder curriculum and professional development program grew in popularity and Kuldell turned to the MIT Venture Mentoring Service (VMS) for support in launching the program as a nonprofit. Among other things, they helped Kuldell form BioBuilder’s successful “training the trainers” model, where high school teachers not only learn the curriculum at workshops but also learn to teach the curriculum to other teachers at their own schools.

    “VMS were so important in helping me think about how to make something sustainable, how to speak about it, how to bring in partners, and develop partnerships,” she says. “They were invaluable.”

    Next up for Kuldell is establishing a teaching lab for BioBuilder at LabCentral, a shared incubator for biotech startups in Cambridge. BioBuilder recently moved headquarters there, after years of operating out of MIT’s Department of Biological Engineering. The proposed BioBuilder@LabCentral Innovation Lab would help connect students and teachers with entrepreneurs in the area. She also hopes to launch a program there where community college students can fulfill part of a synthetic biology certificate program to help them land jobs at biotech firms.

    “The same way we make connections with teachers in middle and high schools, we hope to make connections with students to get them from coursework into careers,” she says.

    See the full article here .

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  • richardmitnick 3:42 pm on November 8, 2014 Permalink | Reply
    Tags: , , Synthetic Biology   

    From LBL: “Synthetic Biology for Space Exploration” 

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    Berkeley Lab

    November 5, 2014
    Lynn Yarris (510) 486-5375

    Does synthetic biology hold the key to manned space exploration of the Moon and Mars? Berkeley Lab researchers have used synthetic biology to produce an inexpensive and reliable microbial-based alternative to the world’s most effective anti-malaria drug, and to develop clean, green and sustainable alternatives to gasoline, diesel and jet fuels. In the future, synthetic biology could also be used to make manned space missions more practical.

    “Not only does synthetic biology promise to make the travel to extraterrestrial locations more practical and bearable, it could also be transformative once explorers arrive at their destination,” says Adam Arkin, director of Berkeley Lab’s Physical Biosciences Division (PBD) and a leading authority on synthetic and systems biology.

    “During flight, the ability to augment fuel and other energy needs, to provide small amounts of needed materials, plus renewable, nutritional and taste-engineered food, and drugs-on-demand can save costs and increase astronaut health and welfare,” Arkin says. “At an extraterrestrial base, synthetic biology could make even more effective use of the catalytic activities of diverse organisms.”

    aa
    Adam Arkin is a leading authority on synthetic and systems biology.

    Arkin is the senior author of a paper in the Journal of the Royal Society Interface that reports on a techno-economic analysis demonstrating “the significant utility of deploying non-traditional biological techniques to harness available volatiles and waste resources on manned long-duration space missions.” The paper is titled Towards Synthetic Biological Approaches to Resource Utilization on Space Missions. The lead and corresponding author is Amor Menezes, a postdoctoral scholar in Arkin’s research group at the University of California (UC) Berkeley. Other co-authors are John Cumbers and John Hogan with the NASA Ames Research Center.

    One of the biggest challenges to manned space missions is the expense. The NASA rule-of-thumb is that every unit mass of payload launched requires the support of an additional 99 units of mass, with “support” encompassing everything from fuel to oxygen to food and medicine for the astronauts, etc. Most of the current technologies now deployed or under development for providing this support are abiotic, meaning non-biological. Arkin, Menezes and their collaborators have shown that providing this support with technologies based on existing biological processes is a more than viable alternative.

    am
    Amor Menezes

    “Because synthetic biology allows us to engineer biological processes to our advantage, we found in our analysis that technologies, when using common space metrics such as mass, power and volume, have the potential to provide substantial cost savings, especially in mass,” Menezes says.

    In their study, the authors looked at four target areas: fuel generation, food production, biopolymer synthesis, and pharmaceutical manufacture. They showed that for a 916 day manned mission to Mars, the use of microbial biomanufacturing capabilities could reduce the mass of fuel manufacturing by 56-percent, the mass of food-shipments by 38-percent, and the shipped mass to 3D-print a habitat for six by a whopping 85-percent. In addition, microbes could also completely replenish expired or irradiated stocks of pharmaceuticals, which would provide independence from unmanned re-supply spacecraft that take up to 210 days to arrive.

    “Space has always provided a wonderful test of whether technology can meet strict engineering standards for both effect and safety,” Arkin says. “NASA has worked decades to ensure that the specifications that new technologies must meet are rigorous and realistic, which allowed us to perform up-front techno-economic analysis.”

    mb
    Microbial-based biomanufacturing could be transformative once explorers arrive at an extraterrestrial site. (Image courtesy of Royal Academy Interface)

    The big advantage biological manufacturing holds over abiotic manufacturing is the remarkable ability of natural and engineered microbes to transform very simple starting substrates, such as carbon dioxide, water biomass or minerals, into materials that astronauts on long-term missions will need. This capability should prove especially useful for future extraterrestrial settlements.

    “The mineral and carbon composition of other celestial bodies is different from the bulk of Earth, but the earth is diverse with many extreme environments that have some relationship to those that might be found at possible bases on the Moon or Mars,” Arkin says. “Microbes could be used to greatly augment the materials available at a landing site, enable the biomanufacturing of food and pharmaceuticals, and possibly even modify and enrich local soils for agriculture in controlled environments.”

    The authors acknowledge that much of their analysis is speculative and that their calculations show a number of significant challenges to making biomanufacturing a feasible augmentation and replacement for abiotic technologies. However, they argue that the investment to overcome these barriers offers dramatic potential payoff for future space programs.

    “We’ve got a long way to go since experimental proof-of-concept work in synthetic biology for space applications is just beginning, but long-duration manned missions are also a ways off,” says Menezes. “Abiotic technologies were developed for many, many decades before they were successfully utilized in space, so of course biological technologies have some catching-up to do. However, this catching-up may not be that much, and in some cases, the biological technologies may already be superior to their abiotic counterparts.”

    This research was supported by the National Aeronautics and Space Administration (NASA) and the University of California, Santa Cruz.

    See the full article here.

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