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If you make a living doing genetic engineering of some sort (which I guess is most of our readership), you’ll be no stranger to the regulations and procedures surrounding the growth and disposal of genetically modified organisms (GMOs). These regulations apply to everything you do, whether it’s creating an army of radioactive gorillas or simply throwing out some E. coli that happens to have a plasmid which encodes β-lactamase. If you’re not a gene tinkerer, then you should realize that all molecular biology, and indeed most modern biological research, depends on genetic modification of something. This is extremely well regulated, and even if these organisms managed to escape into the wild there wouldn’t be anything to worry about. Divorced from the comfort of the lab, the ubiquitous E. coli K12 strain simply cannot thrive, unlike its naturally occurring pathogenic cousin O157:H7, say.

The biggest risk is that bacteria could escape from the lab and somehow manage to survive long enough to transfer an antibiotic resistance gene to something altogether more nasty. This is why the European Food Safety Authority, for example, restricts the use of common lab antibiotic markers in commercially released GMOs. So we spend a lot of time and money autoclaving and bleaching to reduce the already tiny risk even further. But now, all those regulations might be superseded by a different type of GMO, one that uses an unnatural form of DNA.

Rupert Mutzel at the Freie Universität Berlin and colleagues set out to deliberately change how an E. coli strain builds its DNA. Instead of incorporating thymine (T), they wanted its DNA to contain 5-chlorouracil (χ), in addition to guanine, adenine and cytosine (G, A and C). They chose to replace thymine with 5-chlorouracil for a number of reasons, but a major one was because thymine is the only canonical nucleobase unique to DNA; RNA contains uracil (U) instead. There were also technical reasons for choosing the 5-chloro— form instead of the more common bromo—, which can also substitute for thymine: primarily because its 3D structure resembles thymine. Chloride is also freely available in nature but does not appear to contribute to any natural nucleotide building block.

Highly evolved

And this is where it gets clever. Instead of trying to work out how to make a bacterium build χ and incorporate it into DNA, Mutzel and co. knocked out its ability to make T (making it dependent on T in the culture medium) and applied strong selective pressure, forcing the bacteria to evolve. They did this using an apparatus they patented nearly 10 years ago, which is specifically designed to evolve new life forms. This essentially consists of two growth chambers, allowing a growing culture to be grown continually for extended periods of time, while at the same time preventing biofilm growth by regular sterilizing of the apparatus with sodium hydroxide.

Automated measuring of the culture density is coupled to a system that adds fresh media automatically (while simultaneously removing old culture to keep the volume constant)–so the bacteria can grow continuously. They used two media variants: a stressing medium containing 5-chlorouracil but no thymine, and a relaxing medium containing just thymine (or later in the experiment, both). Above a certain density threshold (that is, if the bacteria were growing fast) the system delivered stressing medium. Below this threshold (that is, if the bacteria were dying), the system delivered relaxing medium. This creates a strong selective pressure for the bacteria to adapt to the stressing medium, that is the one that contains 5-chlorouracil instead of thymine.

After nearly six months of this experiment, when the bacteria had been through either 1,000 or 2,000 generations (a number that depended on their doubling time), the researchers found they could isolate two strains that could survive without thymine. And unlike the “arsenic life” researchers, these guys used mass spectroscopy to demonstrate that the ersatz molecule really was incorporated into DNA.

The paper, Chemical Evolution of a Bacterium’s Genome, is published in Angewandte Chemie, and you can read a free evaluation by Chantal Abergel over at the usual place. As the accompanying editorial notes, in a sentiment echoed in Chantal’s evaluation, it has intriguing possibilities for making genetic modification even safer than it is already:

Complete genetic isolation, which is only possible with cells containing “xeno-DNA” as genetic material or alternative/different genetic codes, should prevent horizontal gene transfer between species. In this way, a genetic firewall against the natural DNA-based world could be established.

In other words, not only might the laboratory tool and model organism of the near-future not be able to survive outwith the confines of the laboratory, but even if it did, it wouldn’t be able to pass its alien DNA to its natural cousins.

Of course, being genetically modified itself (and subject to the EU legislation), no doubt we will still have to contend with regulations galore.

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Huge thanks to Maggie Middleton for help with this post.