Published online 10 October 2010 | Nature | doi:10.1038/news.2010.526

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A simplified recipe for synthesizing genes aids synthetic biology.

A fast method of making small synthetic genomes could be used to create vaccines. iStockphoto.com / Kirsty Pargeter

Take eight tiny DNA strands just 60 nucleotides long, combine with a master mix of enzymes and reagents, and incubate at 50ºC for an hour. By following this simple recipe, researchers could synthesize the genome of a mouse mitochondrion — an organelle that acts as the energy factory for plant and animal cells — in just five days.

The technique gives synthetic biologists the simplest tool yet for designing and constructing gene sequences to make synthetic vaccines or pharmaceuticals, or to turn microbial cells into alternative energy sources.

Earlier this year, researchers at the J. Craig Venter Institute in Rockville, Maryland, and San Diego, California, reported that they had built a complete bacterial genome from scratch and used it to 're-boot' a cell1. The latest study, published online in Nature Methods today by a subset of those scientists, is based on a similar idea — of stitching together a genome from smaller segments2.

To construct the synthetic bacterial genome, the researchers started with about 1,100 1,000-base-long DNA segments, ordered from a sequencing company, and introduced them into a yeast cell, which stitched them together. "But now we have a method where we could make those 1-kilobase pieces ourselves," says Daniel Gibson, who led both studies.

A major problem with purchasing long pieces of DNA from companies, Gibson explains, is that no one has figured out how to string nucleotides together with great accuracy. "Those errors are going to get incorporated into your final product," he says. The only way to ensure there are no errors in the synthesised genome is to sequence all the segments, which slows down the process.

Verifying the sequence in smaller pieces is much easier, so the researchers started with the shorter, 60-nucleotide chunks. They incubated these single-stranded snippets, eight at a time, in a concoction that they had perfected to join the fragments together — leaving them with 75 double-stranded pieces.

They then introduced those pieces in the bacterium Escherichia coli, and devised an automated process to sequence the resulting clones, pulling out the ones true to the sequence of the genome they were trying to build.

"We let the E. coli filter out the error," says Gibson.

The researchers finished by stitching these faithful copies into ever-larger segments in three additional steps to produce the 16.3-kilobase final product.

Viral catch-up

The technique offers "a more efficient way to construct relatively large pieces of DNA that are error-free," says Ron Weiss, a synthetic biologist at the Massachusetts Institute of Technology in Cambridge. "I think it's extending the possibility at the basic level for constructing large genetic circuits."

That efficiency could be put to good use, says Gibson. Last week, the J. Craig Venter Institute partnered with the company Synthetic Genomics in La Jolla, California, also founded by Venter, to create a start-up called Synthetic Genomics Vaccines that will use synthetic biology techniques to make vaccines.

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"The influenza virus mutates, and that's why you have a different vaccine every year," Gibson says. "With a method like this, we can keep up with that mutation, and we can synthesize a new virus very rapidly to make a vaccine against it."

The team started with mitochondrial genomes, he adds, because errors in their sequences are at the root of many diseases for which there is currently no treatment.

So far, he notes, "although we made a synthetic mitochondrial genome, we have not shown it was functional". But showing that a synthetic genome can correct malfunctions in cells with mitochondrial deficiencies could open the way for devising treatments for this group of diseases.