Science Concentrates BIOLOGICAL CHEMISTRY
Stepping up gene synthesis Emulsions allow multiple genes to be assembled simultaneously from pools of oligonucleotides Microarray synthesis of DNA oligonucleotides costs a fraction of a penny per base. But such methods can’t make pieces longer than about 200 bases. And stitching those oligonucleotide fragments into gene-length sequences typically must be done one strand at a time, losing the multiplex advantage of the array synthesis. A team led by Calin Plesa, Angus M. Sidore, and Sri Kosuri of UCLA now reports a method called DropSynth that synthesizes multiple genes in parallel from a pool of microarray-generated oligonucle-
Oligonucleotides synthesized on microarrays are captured by barcoded microbeads and enclosed in emulsion droplets, where they are assembled into gene-length sequences. otides (Science 2018, DOI: 10.1126/science. aao5167). The researchers design the oligonucleotides so that each piece needed for a particular gene has the same “barcode,” or identifying stretch of gene sequence. They then use microbeads tagged with a sequence complementary to that barcode to pull those oligonucleotides out of an oligonucleotide pool. “Each microbead probably has millions of copies of the barcode,” Kosuri says. That means that each microbead will have many copies of each fragment in the gene. The UCLA team processes the microbeads in a water-in-oil emulsion, with one bead in each emulsion droplet. “The way
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C&EN | CEN.ACS.ORG | JANUARY 8, 2018
we generate the emulsion is actually pretty simple,” Kosuri says. “You just take an oil and your oligo mixture and vortex it for 30 seconds. You don’t need any specialized equipment.” Once in the emulsion, enzymes clip the oligonucleotides from the microbeads. The pieces are assembled into the gene by a method called polymerase cycling assembly, and the resulting genes are recovered from the emulsion. The genes are placed on plasmids containing an assembly barcode. The researchers used nextgen sequencing methods to sequence the assembled genes, using the assembly barcodes to flag genes that were stitched together properly. The main benefit of the multiplexing approach, Kosuri says, is that a gene pool is now accessible for approximately the same price as an oligonucleotide pool. “We’re not adding much to the cost by doing an emulsion assembly,” he says. The method’s drawback is that the resulting gene library is “messy,” Kosuri says. The efficiency of making any particular gene product is only about 5%. “I don’t think this is going to be the new way that a gene synthesis company might do synthesis, because most users want an exact sequence,” Kosuri says. But he does expect DropSynth will allow individual labs to make large libraries of genes. “Say you want to design 1,000 proteins by gene design; you couldn’t really synthesize that without lots of money.” “As a field, cost-effective methods for synthesizing DNA—particularly genesized DNA fragments—have really lagged behind methods for sequencing DNA. This has been a bottleneck for many areas, but in particular for the field of protein design,” says Jay Shendure of the University of Washington. “If this method proves robust, it represents a major advance and will facilitate protein design and many other goals.”—CELIA ARNAUD
Food additive boosts pathogen In the early 2000s, epidemic strains of the bacterium Clostridium difficile, which can cause life-threatening diarrhea, began to emerge in the U.S., Canada, and several European countries, and have since spread around the world. But why two virulent types of the bacterium, known as RT027 and RT078, became so prevalent so quickly has been a mystery. Now, researchers led by Robert A. Britton at Baylor College of Medicine believe they have identified a chemical culprit: the disaccharide trehalose. Britton’s team found that both RT027 and RT078 thrive on small amounts of trehalose, a sweetener that retains water and is added to pasta, ice cream, and ground beef. In RT027, a single amino acid change in one protein makes it possible for the bacterium to grow on low levels of trehalose. The researchers also found that improved trehalose metabolism in RT027 leads to higher levels of a C. difficile toxin. Independently, RT078 has acquired four different genes that enhance its ability to consume and metabolize the disaccharide (Nature 2018, DOI: 10.1038/nature25178). Scientists had known about RT027 and RT078 long before the outbreaks in the early 2000s, but because of production costs, trehalose was not used as a food additive before that time. A novel enzymatic method for making the disaccharide from starch brought its cost down from around $700 per kg to about $3.00 per kg, and trehalose received approval as a food additive in the U.S. in 2000 and in Europe in 2001. The resulting addition of trehalose to people’s diets, the authors believe, has contributed to the deadly C. difficile epidemics.—BETHANY
HALFORD
C R E D I T: COU RTESY O F CA L I N P LESA & S R I RA M KO S U R I
SYNTHETIC BIOLOGY
