Scientific Method / Science & Exploration

Researchers replace one of yeast’s chromosomes with a synthetic one

Undergrads in "Build-A-Genome" class help get rid of all the junk DNA.

The synthetic chromosome, with the site of every single change marked. Areas denoted in tan were deleted entirely.
Illustration by Lucy Reading-Ikkanda

A few years ago, researchers managed a technical tour-de-force: starting with short DNA sequences that were chemically synthesized in a machine, they built up an artificial bacterial genome and used it to replace the normal copy in living bacteria. But their artificial genome had only minor differences from the original, mostly tags that allowed its presence to be detected.

Today, a large international team of researchers took a major step beyond that. Like the team that worked in bacteria, they started with nothing but short, chemically synthesized pieces of DNA. Using those, they built up an entire chromosome in yeast, eventually replacing the yeast's normal copy. Although this involved less DNA than the bacterial genome, the team made radical changes to the DNA normally found in yeast, deleting most of the sequences that might be considered non-essential. Despite the elimination of 15 percent of the chromosome, the synthetic version worked fine, and the resulting yeast were difficult to distinguish from their normal peers.

In many ways, baker's yeast is a bit like a eukaryotic version of bacteria. Although it's got a collection of linear chromosomes in its nucleus (bacteria lack a nucleus and have a single, circular chromosome), the genome is very compact, with little in the way of the superfluous sequences that seem to make up the majority of the vertebrate genomes. It also has some of the features that make genetics so convenient in bacteria: it can carry extra genes in short, circular pieces of DNA called plasmids, and it's easy to shuffle DNA from these plasmids into the yeast's chromosomes.

All of those conveniences were called upon to build up the synthetic chromosome. Short pieces of DNA made by chemical synthesis were combined into short pieces called "building blocks," which were 750 base pairs long. Each of these were stuffed into a bacterial plasmid. This work was considered so routine that the researchers relied on undergrads who enrolled in a class called "Build-a-Genome."

These building blocks were then dumped into a yeast cell with a partial yeast plasmid. Partial overlaps among the sequences ensured that the only way a viable yeast plasmid could emerge was if the yeast DNA repair system recombined them all, creating a 2,000- to 4,000-base-long fragment of chromosome called a "minichunk," inserted inside the yeast plasmid. These minichunks were then recombined into the normal chromosome, replacing the yeast's normal sequence. As more and more minichunks were added, the chromosome's normal sequence was gradually replaced by the artificial one.

The researchers targeted yeast chromosome III, which is normally 316,617 bases long. But they engineered their replacement to be significantly different. Some of the chromosome is composed of repetitive DNA and transposons that serve no known function; the researchers deleted that. They also got rid of introns, pieces of DNA that interrupt the normal coding sequence of genes but are spliced out of the mature RNA used to make proteins. Also on the chopping block: extra copies of genes that are found on other chromosomes (primarily tRNA genes). Backup copies of the genes that determine the yeast's mating type (yes, yeast have the equivalent of sexes) were also removed. In essence, the researchers created a junk-free chromosome.

When all was said and done, the chromosome had shrunk to 272,871 bases long, a drop of about 15 percent. (Amazingly, only 10 minor errors were introduced during this entire process. Good going undergrads!) Yeast carrying the synthetic chromosome were mostly indistinguishable from those with the normal one, with the primary exception being slower growth when a specific chemical was present.

Over the years, geneticists have determined that only 1,000 of the yeast's 6,000 genes are essential for its viability. Each of the remaining 5,000 can be deleted without obvious effect. But in many cases, two genes that can be deleted individually without any harm cause lethality when they're both missing. This can happen when they're part of the same network—lose one, and the network compensates; lose both, and it can't.

To get a better grip on this, the researchers flanked every single inessential gene on their synthetic chromosome with sites that allowed them to be deleted at will. Unfortunately, attempting to do so tended to cause large-scale changes in the synthetic chromosome, including the loss of DNA, which was invariably fatal. They may have to go back to the drawing board on that experiment.

But one of the other changes they did suggests that they have larger plans. Translation of RNA into proteins terminates at one of three "stop codons," sequences of three nucleotides that don't encode any of the amino acids that act as protein building blocks. The researchers switched every single instance of the stop codon TAG with the stop codon TAA. Essentially, on this chromosome at least, they freed up TAG to encode something entirely new. (They also did something similar with a redundant codon, meaning they have two that can encode different things.)

If they are able to repeat this process with the rest of the yeast chromosomes (there are 16 in all), they'll be able to get the cells to use TAG to encode an artificial amino acid, one not previously used by life. In essence, they'll ultimately be able to make a synthetic genome that helps them build a partly synthetic organism. In this sense, the project is far more ambitious than the early work in bacteria, even though it changed far less DNA in this preliminary effort.

Science, 2014. DOI: 10.1126/science.1249252  (About DOIs).

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