March 31, 2015

Assembling a Genome, Piece by Piece

Producing metabolic intermediates may help identify more antibiotics to counter pathogens like MRSA (green cells; image credit Flickr/NIAID

Metabolic pathways are multi-step endeavors that process one molecule to another, all in the service of cellular health. But not every intermediate waypoint is always seen; they may be consumed quickly as enzymatic reactions progress toward their energy-yielding or food-procuring objectives, rendering potentially useful products invisible to scientists looking for new antibiotics or industrial chemicals. Molecular surveys for these “natural products” soon became redundant; “we were identifying the same compound over and over,” explains Justin Bingham, SGI-DNA’s Director of Business Development. “A lot of that is because novel compounds are produced via secondary cryptic pathways, and those compounds are missed in ordinary screens.” To track these molecules down, scientists are now able to clone specific steps of a pathway into a new organism – one whose survival doesn’t depend on the enzymatic product – and collect the previously ephemeral intermediate for downstream tests.

This promising new approach to natural product characterization is becoming increasingly important, as the rate of new antibiotic discoveries has been decreasing. And it requires a lot of novel DNA, as multiple genes are frequently ported into a host organism. To produce these long stretches of sequence, SGI-DNA has automated its Gibson assembly protocol, the game-changing technology that enables pieces of double stranded DNA to be pieced together in a contiguous chain. Double stranded DNA fragments with overlapping sequence stretches are inserted into a reaction, and exonuclease enzymes chew back one end of each piece, exposing complementary sequences that link up, joining the two fragments together. As a result, “you don’t need restriction enzyme sites to leverage insertion of DNA,” says Bingham. “You just need to know what’s upstream and downstream; you can assemble DNA into anything.”

In 2010, researchers at the J. Craig Venter Institute (SGI’s non-profit sister organization) made headlines when they inserted a full, synthetically reconstructed Mycoplasma mycoides genome into an inactivated, zombie cell. The cell was revived, prompting hyperbolic cries of the first synthetic life form. It may not have been an entirely lab-created organism, but the implications were profound: scientists were able to recapitulate an entire genome with enough biochemical accuracy to maintain production of all essential – and presumably some non-essential – gene products. The M. mycoides episode served as a method development experiment for the Gibson Assembly, which has subsequently gained favor among researchers working on a range of experiments. Now, with the Gibson ultra method, SGI-DNA is able to sew together up to 15 different fragments – each up to 120 kb in length, or about 120 genes – in a single reaction.

SGI-DNA is also incorporating software-based analyses to mine vast databases of DNA sequence. “We have the ability to archive sequencing projects,” explains Bingham, “and use sophisticated design tools to go back and study how those designs work.” It’s an important acknowledgement that past sequencing efforts produced enormous amounts of incompletely examined data, whose value grows as new information comes to light. And by promoting the Archetype software as a community tool, SGI-DNA is hoping its clients will broaden the search space for enzymes based on patterns between different organisms. “You can basically run large scale comparative genomics across multiple genomes to identify trends,” says Bingham, “and use those trends to make novel designs.”

Then, with tools like the Gibson Assembly, “it becomes very easy to insert variation in a pathway or a genome because you have all the parts,” explains Bingham. “The range of diversity you’ll be able to sample is enormous, and we can’t wait to see what new products the scientific community comes up with.”

*This article is part of a special series on DNA synthesis that also appears on Wired.com

Justin Bingham of SGI-DNA will be speaking at SynBioBeta’s London Conference! For more info, click here!