Build your own genome

Without it, we wouldn’t have bread, beer, chocolate, wine or whisky. And biofuel, insulin and antibiotics rely on its presence for their production. But as the Swiss Army Knife for science, one humble genome found in brewer’s yeast could hold the key to unlocking our understanding of all living organisms.

The ease with which Saccharomyces cerevisiae can be grown in culture and with which its relatively simple genome can be manipulated and altered has made it a mainstay of biotechnology and molecular biology. So when an international team of scientists were looking to build a synthetic genome, they knew S. cerevisiae would be the perfect candidate.

“It was clear that despite technology moving quickly in synthetic biology and DNA synthesis, it would be a massive undertaking for just one group alone to build the S. cerevisiae genome from scratch,” says Professor Tom Ellis, Professor of Synthetic Genome Engineering in Imperial’s Faculty of Engineering. And so it was that after a chance meeting at a synthetic biology conference in California between Ellis and Professor Jef Boeke (then at Johns Hopkins University and now at New York University), Ellis joined Boeke in an international team trying to make the synthetic genome.

Working together at Imperial, Ellis and Dr Benjamin Blount, now at Nottingham University, took on the specific task of building a large section of the S. cerevisiae genome – chromosome 11.

Thanks to genome sequencing efforts, the genetic ‘program’ for S. cerevisiae is already known. But while the genome of S. cerevisiae has been sequenced and studied more rigorously than almost any other organism, synthetic biology opens up a whole new avenue of exploration, enabling scientists to examine how the organism functions – and how to tweak and alter that function with incredible precision.

The ‘rungs’ of the DNA double helix are formed by four nucleotide bases pairing together: adenine (A), thymine (T), guanine (G) and cytosine (C). “The best way to think about it is to consider DNA as a programming language; it has its As and Ts and Gs and Cs, just like computers go with ones and zeros,” Ellis says. The challenge for Ellis and his colleagues across the consortium, then, is to construct the yeast DNA into the correct program.

Chromosome 11 consists of around 700,000 base pairs, or pairs of DNA nucleotides. The first step in synthetic chromosome assembly is to split up that entire long sequence into a set of smaller sequences that can be made from chemicals by companies specialising in synthesising DNA. These companies chemically synthesise DNA molecules about 250 bases long, then link them together into longer lengths going up to 10,000 base pairs. Ellis and colleagues take these longer DNA molecules and link them into even longer pieces of around 50,000 base pairs. 

“We then take that DNA and we put it into the yeast cell using a very specific reaction that will swap the equivalent bit of the yeast genome, putting our synthesised bit in and taking out the natural bit,” Ellis says. At this point, they need to check there are no errors in the sequence, and the best way to do that is by checking the yeast organism still functions in a range of conditions. This ‘debugging’ is vital, and is the reason why the team only swaps in a smaller section of the genome each time – in effect ‘walking along the genome’ and gradually making it more and more synthetic. Debugging in synthetic biology is just as time-consuming and arduous as it is in software programming: “We spent about three and a half years on building it, and about three and a half years on debugging,” Ellis says.

Once that entire process is complete, and the synthetic chromosome has been thoroughly debugged, the fun part starts, says Blount.

“We can use an in-built gene-shuffling system to make lots and lots of random changes on a scale that was previously impossible,” he says. This can involve removing existing genes, adding in extra copies, changing how and when they function, or adding in completely new genes and seeing what happens.

For example, Blount is particularly interested in using the synthetic genome to build strains of S. cerevisiae that can perform specific biotechnological functions, such as making biological molecules with pharmaceutical potential or that can act as a more efficient or sustainable biological replacement for a chemical industrial process. And because the synthetic gene-shuffling system is so rapid, it doesn’t take long for the results of those genetic tweaks to become clear. “The great thing about this system is it allows you to quickly prototype whether this is going to be a feasible thing and whether it’s working,” Blount says.

The intended genetic changes in the synthetic chromosome are very specific and targeted. And having synthetic chromosomes has also provided the opportunity to insert DNA sequences that accelerate evolution in S. cerevisiae, so that people can observe what random or useful changes come from genome evolution. The key to this is inserting a particular genetic sequence – called loxPsym – approximately every 2,000 to 3,000 base pairs along the chromosome. For the majority of the time, this sequence does nothing and the yeast ignores it. But when scientists flick the genetic switch for production of an enzyme called Cre, which is not normally made by yeast, this enzyme interacts with the loxPsym sequences and causes them to rearrange or delete the DNA between the loxPsym sites.

This random scrambling of DNA within and between chromosomes is a massively sped-up version of what normally happens with mating, or the exchange of genetic material between two individuals. Bits of DNA are swapped or deleted or duplicated, in exactly the same fashion as happens to the genome during sexual reproduction. “Now you have a million different versions of the genome of the regions that are synthetic,” Ellis says.

Some of those scrambled yeast genomes will be viable, some won’t. Some will be better at certain tasks than others. “That can teach you about what are the essential genes and what are the nonessential genes, and eventually maybe use this system to build a minimal genome for yeast,” Ellis says.

From being the first fully sequenced eukaryotic genome to the first fully synthetic genome, S. cerevisiae has opened some extraordinary and exciting new avenues of scientific study. It has been a profound learning experience, as each error, failure and challenge reveals more about how the yeast genome works.

And it’s just the first step. Work has already started on the next iteration of this synthetic genome, incorporating more changes to its fundamental structure that will help scientists to a deeper understanding of the structure and function, not just of this genome but those of all living organisms.

“From a fundamental science point of view,” says Blount, “this work asks, and hopefully answers, a few of those really important questions about how genomes work.”