I'm a sucker for synthetic biology in non-traditional (non E. coli) model organisms, so I was pretty excited by the news of a recent paper on expanding the genetic code of a worm. "Worms!? With unnatural genes!?" you ask? Yes, and yes, (and yes, it's a pretty awesome paper) but it's actually probably simpler not as terrifying as you might think.

Worms!?

Most synthetic biology happens in microbes, and I haven't heard a lot about work in the tiny nematode worm C. elegans besides a couple iGEM teams last year. C. elegans is an awesome model organisms for molecular biology, genetics, and developmental biology. They are also tiny (1 millimeter long), easy to take care of in the lab, grow fast, and are relatively easy to engineer, making them according to an old review, "as close to being a microbe as it would seem possible for an animal to be." In fact, this closeness to microbes is what makes it possible to expand their genetic code.

Expanded Genetic Code!?

There are 4 bases in DNA that make 64 three-letter codons, which map onto the 20 amino acids in the genetic code. Not wanting to be limited by this set, synthetic biologists have expanded the code by making different DNA bases, 4-letter codons, and incorporating unnatural amino acids into proteins. So far, all of these projects are much more about understanding the chemistry of life than about making new biological behaviors. By altering DNA bases we can better understand how DNA is copied and processed inside living cells and make in vitro diagnostic tools (here's a PDF of an interesting review that includes some of this research). Making new amino acids can likewise be used to create new chemical tools to study the function of proteins, either purified or inside of a living cell.

In all of these cases, after the chemistry is done to create the new bases or amino acids, the biological engineering happens at the level of the gene expression machinery. The proteins that copy DNA have to be mutated to accept different bases, ribosomes have to evolve to recognize 4-base codons, and new tRNAs have to be made to attach new amino acids. Usually the engineered tRNAs don't swap unnatural amino acids for natural ones, but add the unnatural amino acid at one of the three stop codons—TAG, TAA, or TGA—that tell the ribosome that it has reached the end of the protein.

As with many synthetic biology ideas, nature got here first. Mutations in tRNAs that cause the ribosome to "read through" and incorporate an amino acid at one of the three stop codons form a large class of mutations known as nonsense suppressors that play an important role in the history of molecular biology and the decoding of the genetic code. These mutations were discovered when some mutated bacterial viruses were isolated that could only infect certain mutant bacterial strains. These viruses had "nonsense mutations" that put a stop codon in the middle of an important protein, preventing that protein from being fully made and functional. When these viruses infected a bacterial strain that put an amino acid instead of stopping in the middle of the protein the viral mutation was "suppressed" because the protein could be made at its full length. The first stop codon to be discovered this way, TAG, is known as the "amber" codon because the mutated virus was isolated by a grad student named Harris Bernstein, whose last name means "amber" in German. The other two mutations were named ochre (TAA) and opal (TGA) to keep with the color theme.

Worms with an Expanded Genetic Code!?

Adding a tRNA that incorporates an unnatural amino acid instead of a stop codon is thus essentially introducing a nonsense suppressor mutation. In many microorganisms amber suppressor mutations occur naturally at different levels of efficiency (the percentage of stop codons that are replaced) and can survive just fine with some of their proteins a little elongated, making it relatively easy to expand the genetic code of bacteria and yeast this way. Synthetic biologists have more recently added these tRNAs to animal cells growing in culture with varying levels of success and replacement efficiency, but most animals don't survive such a drastic mutation.

C. elegans isn't like most animals, however, and it is the only multicellular organism where amber suppressors were identified and introduced into the germ line, with up to 30% suppression efficiency. Because of this and the many genetic tools available, Sebastian Greiss and Jason Chin at Cambridge University were able to introduce a new tRNA into C. elegans that could incorporate one of two unnatural amino acids at the TAG codon. To test whether or not this worked they designed a gene that had two fluorescent proteins fused together with a stop codon in between. Without their tRNA only the first fluorescent protein would be made by the ribosome and the worm would be green. When they add their tRNA to the worm and the unnatural amino acid in the worm's food, both proteins are made and the worm is green and red.

What can this be used for besides making a kind of splotchy red worm at so-so efficiency? Unnatural amino acids in purified proteins are useful to modify the protein chemistry for structural biology and enzymology studies, and inside live cells can be used to follow how proteins move and interact. In a whole animal, these tools can be used to see how proteins are involved in how the brain works and how the embryo develops. Worms are already used in these kinds of studies because they have tiny but complicated brains and every one of their nearly 1000 cells have been mapped through development. These aren't totally unnatural worms, they are worms that are slightly modified to help us better understand how natural worms work.