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ICL Scientists Describe New Method for Open-source, Modular DNA Assembly

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NEW YORK (GenomeWeb) – Assembling useful bits of DNA into plasmids like Lego bricks has been feasible for a while now; the hard part is breaking them up, adding new bits, and putting them back together. But soon that will be just as easy, according to a paper published earlier this month in ACS Synthetic Biology.

In that paper, scientists from Imperial College London described Biopart Assembly Standard for Idempotent Cloning (BASIC), a method for piecing together DNA into plasmids that can easily be unpacked, modified, and repacked with other strands. This could be a boon for synthetic biologists trying to build new gene circuits in organisms, or anybody else that is cloning large amounts of DNA, Geoff Baldwin, an ICL professor of synthetic biology and senior author on the study, told GenomeWeb.

"It's a format that parts are kept in. To standardize assembly, you have to standardize the DNA," Baldwin said. "The key features are that we simplified this process and made it very accurate. All of the parts are maintained in the same format so you only ever have a single protocol; if all of your parts are in the same format, they return to you in the same format."

BASIC relies on the use of standardized pairs of linker oligonucleotides that clip into the DNA parts to be assembled, and to each other. In addition to forming structural connections between DNA sequences, the linkers act both as barcodes and PCR priming sites.

It all starts with a DNA part, perhaps a gene "A" that a scientist wants to incorporate into a plasmid with others. This DNA part "A" is tagged with both a short oligonucleotide prefix and suffix that feature overhangs of four and six base pairs, respectively, as well as a restriction enzyme recognition site. The scientist then adds in two linkers, one to ligate to the prefix overhang and one for the suffix overhang. Each linker itself has a 21 base pair overhang that will later anneal to its complementary mate, which would be attached to a different DNA part.

"Linker one only ever pairs with linker one and not linker two," Baldwin said. "They're designed to optimize their uniqueness and also designed not to have unexpected biological function."

With three sets of matching linkers, the scientist could create a plasmid joining DNA parts "A," "B," and "C."

Moreover, if the scientist likes the "ABC" plasmid and wants to incorporate that into a larger plasmid, he or she could do so by clipping one of the junctions at the restriction enzyme recognition sites, and adding new linkers at those ends. It's an idea borrowed from computer science called "idempotence," where the output of a process is maintained now matter how many times the operation is performed. BASIC allows linkers to be added over and over.

"It makes the process very amenable to automation," Baldwin said.

The linkers also work as a watermark during sequencing. "If we want to test 'has this colony got all of the parts in the right order,' we can do PCR validations and have primers that are specific to the linkers," Baldwin said, because the linkers are standardized.

Baldwin and Ellis present BASIC as an efficient and accurate way to piece together DNA. Assemblies with four DNA parts were up to 99.7 percent accurate, after double antibiotic selection; assemblies with seven parts were up to 90 percent accurate.

Even though the price of DNA synthesis is falling as methods evolve and genome databases have more useful parts than ever to string together, Baldwin said size is still the limiting factor for the kinds of gene circuits synthetic biologists want to build.

"It's still kind of difficult if you want to build a system, to get the whole thing made through DNA synthesis," Baldwin said. Building a gene circuit often requires several cycles of designing, building, and testing. "To get it working optimally, the way you want it to, you may need to test thousands of constructs. To get this purely using DNA synthesis is cost prohibitive."

With over 100 linkers currently available and potentially many more to come, they will not be the limiting factor on the size of DNA constructs. "There's a lot of possible barcodes," Tom Ellis, also of ICL and an author on the paper, said. "I expect that it's almost an infinite number."

In theory, a set of several hundred linkers could enable scientists to make constructs on the scale of microbial genomes, Ellis said. Building a genome of half a million base pairs, with 300 to 400 genes would require an equivalent number of linker sets, which would be on the same order of magnitude as is already available.

BASIC has this potential because it combines some of the most attractive features of existing methods.

BioBricks, the first-generation synthetic DNA assembly method, works in much the same way as BASIC, but it can only clip together two DNA parts at a time. Newer methods can string together more sequences, but Baldwin said they require designing custom junctions. "We're back to bespoke design and assembly," he said.

BASIC offers several advantages, namely automation, and therefore speed; however, it's not because the method is quicker, but because it reduces the amount of prep work involved in arranging new genes to put into organisms.

"If you say, 'I want to introduce new genes,' then you have to go through a variety of steps of ordering new things or designing new things," Ellis said. "With standard methods and a PhD student doing it with their hands, it would be half a year's worth of work." A DNA assembly method such as BASIC could allow an automated platform to do that work in day or so, he said.

"It's very good for people who have projects where they want to get a toolkit in [to a cell] and try a lot of things. BASIC increases throughput by allowing it to be done in a parallel way. You can try a whole lot of things out on the same day," Ellis said.

Speed and throughput are not the only ways BASIC could accelerate progress in synthetic biology.

Baldwin said that he and his colleagues have developed BASIC as an open-source technology for anybody to use. "We don't want to have a patent thicket around DNA assembly that inhibits inventive work. The value should be in what you build, not how you build it," he said. BASIC is an industrial application that could increase innovation because it would allow people, both in academia and the private sector, to share their DNA parts.

"If we all make our pieces of DNA in the same way, then sharing them becomes easy," Baldwin said.

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