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  • richardmitnick 1:13 pm on June 18, 2018 Permalink | Reply
    Tags: , , Joint BioEnergy Institute (JBEI) based at Lawrence Berkeley National Laboratory (Berkeley Lab), ,   

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

    Berkeley Logo

    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 1:01 pm on November 14, 2017 Permalink | Reply
    Tags: Advanced Biofuels and Bioproducts Process Demonstration Unit (ABPDU), , , , , Here we’re cultivating an entire community of microbes to access enzymes that we couldn’t get from isolates, Joint BioEnergy Institute (JBEI) based at Lawrence Berkeley National Laboratory (Berkeley Lab), , Metagenomic analysis, New types of cellulases enzymes that help break down plants into ingredients that can be used to make biofuels and bioproducts   

    From LBNL: “To Find New Biofuel Enzymes, It Can Take a Microbial Village” 

    Berkeley Logo

    Berkeley Lab

    November 14, 2017
    Sarah Yang
    scyang@lbl.gov
    (510) 486-4575

    A new study led by researchers at the Department of Energy’s Joint BioEnergy Institute (JBEI), based at Lawrence Berkeley National Laboratory (Berkeley Lab), demonstrates the importance of microbial communities as a source of stable enzymes that could be used to convert plants to biofuels.

    1
    This 50-milliliter flask contains a symbiotic mix of bacteria derived from compost that was maintained for three years. (Credit: Steve Singer/JBEI)

    The study, recently published in the journal Nature Microbiology, reports on the discovery of new types of cellulases, enzymes that help break down plants into ingredients that can be used to make biofuels and bioproducts. The cellulases were cultured from a microbiome. Using a microbial community veers from the approach typically taken of using isolated organisms to obtain enzymes.

    The scientists first studied the microbial menagerie present in a few cups of municipal compost. Metagenomic analysis at the DOE Joint Genome Institute (JGI) of the microbiome helped reveal that 70 percent of the enzymatic activity originated from cellulases produced by a cluster of uncultivated bacteria in the compost. They found that the enzymes easily broke down the cellulose in plant biomass into glucose at temperatures up to 80 degrees Celsius.

    2
    This chart shows the bacterial composition of the community in the bioreactor after two weeks of culturing. (Credit: Sebastian Kolinko/JBEI)

    “Here we’re cultivating an entire community of microbes to access enzymes that we couldn’t get from isolates,” said study principal investigator Steve Singer, senior scientist in Berkeley Lab’s Biological Systems and Engineering Division and director of Microbial and Enzyme Discovery at JBEI. “Some microbes are difficult to culture in a lab. We are cultivating microbes living in communities, as they occur in the wild, which allows us to see things we don’t see when they are isolated. This opens up the opportunity to discover new types of enzymes that are only produced by microbes in communities.”

    The bacterial population, Candidatus Reconcilibacillus cellulovorans, yielded cellulases that were arranged in remarkably robust carbohydrate-protein complexes, a structure never before observed in isolates. The stability of the new cellulase complexes makes them attractive for applications in biofuels production, the study authors said.

    “The enzymes persist, even after a decline in bacterial abundance,” said Singer, who compared the microbial community with sourdough starters fermented from wild yeast and friendly bacteria. “We kept the microbial community cultivation going for more than three years in the lab.”

    3
    A bioreactor at ABPDU was used to scale the growth of a mixture of bacteria from 50 milliliters to 300 liters. (Credit: Roy Kaltschmidt/Berkeley Lab).

    This stability is a key advantage over other cellulases that degrade more rapidly at high temperature, the researchers said.

    To determine whether the enzyme production can be scalable for industrial applications, JBEI scientists collaborated with researchers from the Advanced Biofuels and Bioproducts Process Demonstration Unit (ABPDU) at Berkeley Lab, a scale-up facility established by DOE to help accelerate the commercialization of biofuels research discoveries.

    Researchers at JBEI, a DOE Bioenergy Research Center, were able to produce 50-milliliter samples, but in about six weeks, the scientists at ABPDU scaled the cultures to a volume 6,000 times larger – 300 liters – in industrial bioreactors.

    The study’s lead author is Sebastian Kolinko, who worked on the study as a JBEI postdoctoral researcher.

    Other co-authors on this study include researchers from Taipei Medical University, the University of Georgia, the Manheim University of Applied Sciences, and Technical University of Braunschweig in Germany.

    JGI is a DOE Office of Science User Facility. This work was primarily supported by the DOE Office of Science and the DOE Office of Energy Efficiency and Renewable Energy.

    See the full article here .

    Please help promote STEM in your local schools.

    STEM Icon

    Stem Education Coalition

    A U.S. Department of Energy National Laboratory Operated by the University of California

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    DOE Seal

     
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