Genetic Engineering & Biotechnology News

AUG 2017

Genetic Engineering & Biotechnology News (GEN) is the world's most widely read biotech publication. It provides the R&D community with critical information on the tools, technologies, and trends that drive the biotech industry.

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14 | AUGUST 2017 | | Genetic Engineering & Biotechnology News what the cell was doing to stitch together our pieces of DNA." Synthesizing Whole Chromosomes This stitching approach seemed promising at first, but it had multiple shortcomings. "We needed something faster," explains Dr. Gibson. "Our goal was to synthesize a whole chromosome within a year, and we still did not have a meth- od that worked." Dr. Gibson's group started exploring the possibility of us- ing purified enzymes to link DNA fragments. "Ultimately, it became pretty simple to do this, provided the DNA frag- ments were designed to overlap each other, allowing the ends to come together specifically," explains Dr. Gibson. This process requires three enzymes: an exonuclease, which degrades the ends of the DNA fragments; a DNA polymerase, which fills gaps and anneals and repairs dam- aged DNA; and a DNA ligase, which covalently joins the molecules. This in vitro recombination method has become known as Gibson Assembly. "We used this method to synthesize whole chromosomes, but we also used natural homologous recombination ma- chinery in yeast," elaborates Dr. Gibson. This machinery, he adds, is remarkable because it enables yeast to take up and assemble dozens of overlap- ping large DNA fragments into large pieces. These key discoveries set the stage for the creation of synthetic cells. When scientists at the JCVI announced the creation of the first synthetic cell in 2010, they credited their success to the combined use of whole- genome synthesis and transplantation technologies. Gene-synthesis technologies provided crucial assistance when the JCVI, SGI, and Novartis collaborated to accelerate the generation of synthetic flu vaccines. Conventional influ- enza vaccine preparation currently requires at least a month after a viral sample has been obtained, and shortening this time would significantly improve epidemic and pandemic preparedness. "We teamed up and showed that we can synthesize in- fluenza viruses for vaccine production using DNA synthesis in less than a week," asserts Dr. Gibson. As part of this col- laboration, Dr. Gibson and colleagues carried out the viral nucleic acid synthesis. "What we needed was to completely change the way we synthesize genes," explains Dr. Gibson. The chemical synthesis approach used by Dr. Gibson and colleagues combined enzymatic cell-free gene synthe- sis with enzymatic error correction. The chemical synthesis of DNA is error-prone, with an error being introduced ap- proximately once every 500–1000 base pairs of DNA, and this precludes the synthesis of large genomic regions. To en- sure that the DNA of the influenza virus genome fragments is copied both accurately and efficiently, Dr. Gibson and colleagues used an approach that can increase the overlap between the nucleotides, incorporate an enzymatic correc- tion step, and concomitantly assemble a larger number of oligonucleotides. "We ended up developing a process using endonucleases and exonucleases, where the enzymes could identify and cleave mutations, so that only the error-free DNA molecules were left," states Dr. Gibson. This process not only generated high-quality influenza virus genes, it also reduced the time between receiving the DNA sequences and generating highly accurate influenza genes to about 16 hours. "These genes," maintains Dr. Gib- son, "were demonstrated to rescue flu virus by Novartis, which used them in vaccine production." The technologies that catalyzed these rapid, robust, and accurate DNA-synthesis methods became the driving force behind the digital-to-biological converter (DBC), a bio- manufacturing unit developed by Dr. Gibson and his col- leagues at SGI. "As a proof of concept, we demonstrated that we could build a DBC that could accept digitized DNA information," states Dr. Gibson. This information may originate anywhere in the world, be conveyed via email, and arrive at the DBC. The digital DNA sequence informa- tion can then be converted into biological material such as DNA, RNA, and proteins. The DBC is not commercially OMICS Feature See DNA Synthesis on page 16 DNA Fab Keeps Getting More Fab Continued from page 1 The Gibson Assembly method has allowed investigators to construct whole chromosomes in vitro, in a single reaction. The method covalently joins DNA fragments that have single-stranded overhangs. ChrisChrisW / Getty Images Gene-synthesis technologies provided crucial assistance when the JCVI, SGI, and Novartis collaborated to accelerate the generation of synthetic flu vaccines.

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