April 03, 2014
Everyone who reads our blog knows how awesome the yeast Saccharomyces cerevisiae is. Without this little workhorse we would almost certainly not understand ourselves as well as we do now. It is an indispensable tool in figuring out how eukaryotes work.
And of course yeast is much more than that. It makes our bread fluffy and our drinks alcoholic. It can be manipulated into making medicines like artemisinin, a powerful anti-malarial drug, or biofuels or whatever else we can think of. It is the Swiss Army Knife of useful organisms.
Even with all of this fanfare, everyone knows yeast has its limitations. It is a powerful tool but it could be improved. For example, it would be nice if researchers could more easily manipulate its DNA to speed up the introduction of beneficial traits, add new biosynthetic pathways, or to do the kinds of experiments that will help one day cure cancer or Alzheimer’s disease. This is where Sc2.0 comes in.
Sc2.0 is an idea that has been kicking around for the last decade or so. First proposed by Ron Davis of Stanford University, the idea is to synthesize artificial yeast chromosomes to make yeast more useful. Eventually the idea would be to recreate every yeast chromosome and intelligently redesign the genome for our own purposes. And maybe even to add new artificial chromosomes so we can easily add whatever genes we want.
In a new study out in Science, Annaluru and coworkers have taken a major step forward in the Sc2.0 project by replacing all 316,617 base pairs of yeast chromosome III with a 272,871 base pair synthetic version, synIII. That leaves only 15 chromosomes and around 12.2 million base pairs before we have yeast with completely manmade DNA.
Annaluru and coworkers managed to do this with the help of a bunch of undergraduate students and yeast’s love of homologous recombination. The first step was to have undergraduates synthesize around 30,000 base pairs each in the “Build a Genome” class at Johns Hopkins. It took 49 students around 18 months to pull this off for synIII.
Basically they used 60-mer and 79-mer oligonucleotides to PCR up 750 base pair building blocks. These pieces of DNA were designed so that they could be assembled into 2,000-4,000 base pair minichunks. The final step was to transform yeast with an average of twelve of these minichunks and to let the yeast use homologous recombination to replace its native DNA sequence with the added DNA. After 11 rounds of transformation, the yeast now had an artificial chromosome.
As you may have guessed, this chromosome is not exactly the same as the one it replaced. To eventually free up a codon for repurposing later, all 43 of the TAG stop codons were converted to TAA. When this is done with all of the chromosomes, researchers will now have a codon they can use to change this yeast’s fundamental genetic code. This might allow for adding novel amino acids to proteins or even prevent viruses from infecting the new yeast.
Annaluru and coworkers also introduced 98 loxP sites which in the presence of estradiol will cause the yeast to undergo rapid DNA change. The hope is that scientists will be able to harness SCRaMbLE (synthetic chromosome rearrangement and modification by loxP-mediated evolution), as it has been named, to more quickly evolve useful traits in yeast for both study and biotechnological uses.
As a final step, the researchers cleaned up the chromosome by removing 21 retrotransposons and many introns and by moving 11 tRNA genes to a neochromosome. They now had created a leaner, meaner chromosome III.
The next obvious question was whether or not all of these changes affected the yeast. Despite looking very carefully, Annaluru and coworkers could find little that was different between strains carrying natural and synthetic chromosomes. They both grew similarly under 21 different conditions in terms of growth curves, colony size, and cell morphology, and had very similar transcription profiles. But they weren’t identical.
For example, the strain with synIII grew slightly less well in the presence of high sorbitol, and showed differences in expression from wild type in 10 out 6,756 transcripts. Of these ten, eight were intentionally altered in the creation of synIII and so were expected. The two unexpected changes were a ~16-fold decrease in the expression of HSP30 on synIII and a ~16-fold increase in the expression of PCL1 on chromosome XIV.
Since all of these changes had such a small effect on the yeast, it is a green light for plowing ahead with creating yeast with completely manmade DNA. Currently four other chromosomes, II, V, VI, and XII, are nearly done and the design work has been completed for chromosomes I, IV, VII, and XI (see an overview of the project). It will only be a matter of time before we have a strain of yeast with completely synthetic DNA. Scientists are making a powerful tool even better…who knows what this new strain will help us discover.
by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
Categories: Research Spotlight