So far, there have been two primary routes to reengineering an organism's genome. The first is to start with an existing form of life and tweak it a bit, eliminating a few genes and adding in some others. The second is to start from scratch, building a new genome up from short stretches of DNA made with a machine. There are advantages to each approach, but a paper in Science provides a third option: wholesale editing of entire genomes.

Making a collection of individual tweaks gives you fine control over the properties of an organism, but can be time-consuming. Individual genes may have to be deleted or replaced, additional ones can be added to the genome, and others may be hosted on smaller pieces of DNA that are maintained by the organism in question. It can be technically challenging to perform this long series of modifications, and it's certainly time consuming. The alternative, building a genome from scratch, can allow you to make a series of extensive modifications as part of a single process. But at the moment, it only works on extremely simple organisms that don't have any of the complex biochemical pathways we're likely to want to reengineer.

The new approach provides something that's a bit in between the two, allowing researchers to edit DNA sequences scattered around the genome, allowing wide-scale modification of genomes as complex as the one carried by E. coli.

The paper suggests a reason for the authors to do this: they're interested in making a bacteria that uses a synthetic amino acid to make proteins (normally, living things are restricted to using only 20 amino acids, but there are many other members of this class of chemicals known). In DNA and RNA, individual amino acids are encoded by a set of three bases called a codon. So, for example, the bases AGG encode an amino acid called arginine, while AGC encodes serine. Three codons, UAA, UAG, and UGA, tell the cell where the protein ends (they're called stop codons).

To reengineer the genetic code, the team needed to free up one of the existing codons. It's not possible to simply start using AGG to encode something else, since thousands of proteins are using that to put arginines in specific locations. So the authors decided to take a simpler target: one of the stop codons, UAG. These are still important, but there happen to be only 314 instances of UAG stop codons in the entire E. coli genome, and the authors decided to replace them with UAA. Still, that's 314 individual locations within the genome that they need to replace, which is well beyond anything that anyone had attempted previously.

To do it, they divided the genome up into 32 segments, each with 10 stop codons to target. They first relied on a technique they had developed earlier, called MAGE, which involves growing E. coli in solutions that contain small segments of DNA containing the modified sequence. Over several generations, these modified sequences get incorporated into the cells' genomes. Not every cell would pick up all 10, but it's possible to screen for those that have and pull out individual cells that carry them all.

This left them with 32 new bacterial strains, each with a segment carrying 10 mutations. From there, the authors started combining the genomes that had modifications in adjacent segments. To do this, they took advantage of the bacterial version of sex, called conjugation. They inserted some DNA that initiates conjugation next to the start of a segment, and then mixed the strains and selected for those that carried both segments. This cut the number of strains down to 16, and another round got it down to eight, each of them carrying 80 individual mutations.

And there, for some reason, the authors stopped. It's a technical paper, and the technique is clearly working, but it's still a bit surprising that Science accepted the paper without making them take the process through to its end point.

Still, the cells seem largely unperturbed by carrying 80 of these changes, so chances are the final combinations can be performed (in fact, given the time it takes to publish anything, I expect that they already have if they can be). Even if the cells don't tolerate larger number of changes all that well, the authors can just grow them for a few thousand generations and wait for tolerance to evolve. Once they have all 314 in place, they should be set up for the really interesting part, adding in a 21st amino acid. That will require a few modified enzymes, though, which may have to be added back to the bacteria the old-fashioned way.

In the meantime, however, synthetic biologists might find this approach very interesting, since it can be used for much more than simply swapping stop codons—it should enable researchers to edit wholesale changes into the E. coli genome, and possibly those of some other bacteria. That could be enough to rewire entire biochemical pathways or optimize the production of some critical biomolecules.

Science, 2011. DOI: 10.1126/science.1205822 (About DOIs).