At first glance, the undergraduate biology curriculum at the Johns Hopkins University doesn't look all that different from other biology programs. All the usual suspects are on the schedule: General Biology and Cell Biology, Virology and Genetics, Immunology and Biochemistry. But down at the bottom of that list is something that you don't see every day — Biology 420: Build-a-Genome.
While the course might sound like the molecular equivalent of a certain mall-based ursine construction boutique, Build-a-Genome is serious science.
The practical portion of the course involves reconstructing a yeast chromosome, base by base, from chemical building blocks. It's part of a larger research effort known as Synthetic Yeast 2.0 (Sc2.0), a project that seeks to reconstruct and redesign the entire yeast genome, all 12 million nucleotides of it.
Sc, or Saccharomyces cerevisiae, “has an ancient industrial relationship with humans which is alive and well today in the form not just of bread and beer but pharmaceuticals like artemisinin, biofuels, and lots of other specialty items,” notes Jef Boeke, a yeast geneticist and director of the High-Throughput Biology Center at the Johns Hopkins University School of Medicine, who leads the Sc2.0 effort.
Boeke and his colleagues, computational biologist Joel Bader and molecular biologist Srinivasan Chandrasegaran, realized that, useful as brewers’ yeast is, it could potentially be made even more so, biotechnologically speaking. Boeke and his team decided to give it a try. Very quickly, though, they realized there was a problem: The yeast genome is big, and DNA synthesis is slow.
“While I was waiting 11 months for my DNA to be delivered by Codon Devices, I was wondering, how am I going to make the other 99% of the genome? At this rate I'll be dead long before I get there,” recalls Boeke. “And then it hit me — we have all these undergrad students across town who were looking for a research experience, and we could give them an excellent one.”
Enter Build-a-Genome. Each semester 15-20 students — mostly undergrads but also the occasional high school student, graduate student, and even professor — take charge of their own 10,000-nucleotide slice of S. cerevisiae chromosome III, building up chunks of synthetic material from smaller 750-base pair fragments.
“Students were remarkably efficient in the synthesis of these building blocks, and the first pass of the entire ∼280 kb synthetic chromosome III sequence was achieved in one academic year (one summer session, two semesters, and one intercession),” wrote Boeke in 2009 (1).
Today, Build-a-Genome students have constructed probably 10-15% of the total genome in pieces of various sizes according to Boeke, as well as making “major progress” on chromosome III.
For students, the results are more intangible, a kind of next-gen independent research experience laced with what the authors call a “cool factor,” though they may ultimately score a publication for their efforts as well. But on a larger scale, the success of Build-a-Genome illustrates just how mature the science of genome engineering has become.
The many roads to genome building
Sc2.0 represents one of two fundamental approaches to genome engineering: the rational design and “writing” of a predefined sequence. In what is inarguably the biggest coup for this approach, researchers at the J. Craig Venter Institute made headlines in 2010 when they chemically synthesized and assembled the 1.08 Mb genome of Mycoplasma mycoides, inserted it into a close relative, and got that relative to shed its own genome in favor of its exogenous counterpart. The resulting strain, M. mycoides JCVI-syn1.0, is for the most part identical to wild-type M. mycoides, save a few genetic “watermarks” carefully inserted in the sequence by the researchers.
JCVI-syn1.0 was assembled using a hierarchical process in yeast where 1078 overlapping 1 kb cassettes were linked by successive rounds of homologous recombination into first 10 kb , then 100 kb, and finally 1 Mb pieces. Boeke's Sc2.0 employs a slightly different strategy. Overlapping 60-mer primers are stitched together into overlapping 750 bp “building blocks.” These blocks are assembled either in vitro or “in yeasto” into 10,000 bp “chunks” (on plasmids) using homologous recombination, and then combined again to build 30-50 kb “megachunks.”