DIY Chromosomes

• This DIY (do-it-yourself) is Actually DIT (do-it-together)!
• A Global Network of Undergraduates are Collectively Assembling Designer Chromosomes for Yeast
• The Inspirational Jolt for this Harnessing of “Student Power” Came in a Caffeinated Coffee Conversation

At a conference held over a decade ago, visionary geneticist Ronald Davis suggested that researchers should synthesize artificial yeast chromosomes and insert them in a living cell. Many of the attendees, including Jef Boeke, didn’t think much of the idea. According to a News Focus article in Science by Elizabeth Pennisi, Davis made the bold prediction that artificial yeast would be the next significant milestone in the then-emerging field of synthetic biology. Boeke didn’t share Davis’s vision, mostly due to the seeming insurmountable task of designing and synthesizing a 12.5-million-base genome comprising 16 chromosomes. According to Pennisi, as Boeke listened to Davis's talk, he says, ‘I remember thinking “Why on Earth would you want to do that?'“

However, Boeke’s opinion about his own rhetorical question completely flip flopped, and has led to an inspiring “collective can do” attitude among scientists in this field. Led by Boeke, hundreds of researchers around the global are now working together as a gigantic team to successfully achieve a goal befitting a “grand challenge,” which would otherwise not be possible.

Here’s the interesting story of what has happened and how it came about.

Boeke (a geneticist who recently moved from Johns Hopkins University to New York University Langone Medical Center) and his colleagues have just finished the first complete synthetic yeast chromosome. They are well on their way to putting together several more—thanks to technological advances in manufacturing DNA oligonucleotides and—importantly—a global army of collaborators who are—amazingly to me—mainly undergraduate students!

This post, which borrows from Pennisi’s article, briefly touches on only a bit of the truly impressive science—which can be read in detail in the original publication by Boeke and his army—and instead emphasizes the enthusiastic undergraduate student participation, along with an equally interesting—I think—odyssey as to how this project progressed to where it is now.

Why Build Completely Customized Yeast?

Good question. For centuries, Saccharomyces cerevisiae (S. cerevisiae), also know as brewer’s, baker’s, or budding yeast, has played a pivotal role in our food and drinks, from baked goods to beer and wine. It’s also a “workhorse” for cellular and molecular biologists. In 1996, it became the first eukaryote to have its genome fully sequenced. Since then yeast geneticists have studied it every which way—facilitated in part by being able to incorporate foreign DNA through a process called homologous recombination. There is even a comprehensive website dedicated to all things yeast.

In contrast, the “ultimate modification” of yeast is designed completely in silico (i.e. using computer programs), and assembled in the lab piece by piece using chemically synthesized DNA oligonucleotides. Each of the ~6,000 genes can thus be pondered with regard to being kept, modified or removed. The same applies to other genetic elements. Moreover, exogenous genes (i.e. not found in natural yeast) or other exogenous functional genetic elements can be inserted to obtain “custom content” genetic variants intended for specific applications, such as production of commercially valuable products.

Caffeinated Coffee Conversation Jolts Inspiration

I’m sure it was more than the caffeine, but its jolt probably helped a little based on Pennisi’s story about Boeke’s “aha moment.” The following anecdote is taken from Pennisi’s article. Boeke traces his change of heart about the synthetic yeast project to 2006, while over coffee, his Johns Hopkins colleague Srinivasan Chandrasegaran was trying to convince him to make a large number of zinc-finger nucleases. These DNA-modifying enzymes are Chandrasegaran's specialty, and a toolkit full of them would make the yeast genome easier to manipulate. Boeke wasn't that interested and, almost as a joke, suggested a more drastic way to take control of the yeast genome: synthesize the whole thing. To his surprise, Chandrasegaran jumped on the idea, and the pair brought Joel Bader, a computer scientist at Johns Hopkins, into the discussion.

Despite Boeke's dismissal of Davis's proposal just 2 years earlier, the trio at Hopkins quickly concluded that making an artificial yeast genome would be worthwhile if it could be a testbed for learning about the genome itself. They decided to start with the 90,000-base "R" arm of chromosome 9—the shortest in the yeast's genome—and spent a year arguing about how the synthetic sequence should differ from the natural one. The trio considered just including the genes they wanted, ‘but we quickly realized it would be very risky to eliminate whole bunches of genes’ without really knowing in advance what the effect of that loss on the whole system would be,’ Boeke recalls.

So the team used the natural sequence of the chromosome 9 arm as the basic building block, and incorporated DNA that would enable them to ‘induce changes at will’. Basically, when activated by a chemical added to cells, it kicks off a chromosomal version of ‘musical chairs.’ By using this process, they were able to generate new yeast strains with different properties. Over time, they have been able to add stability to the yeast genome, helping to ensure integrity of the strain. Readers interested in technical aspects of how they did this—and other genetic edits such as removal destabilizing transposons and non-coding RNA—are referred to the original publication.

From Buy It to DIT—More Inspiration

After months of work and with the help of Bader, Boeke designed a version of the chromosome 9 arm using a software program. He then contracted with a biotech company to synthesize the chromosomal arm. The company was having difficulty synthesizing such a long piece of DNA and they didn’t hear back regarding the project for almost a year. During that time, Boeke began to wonder if he could speed things up himself. He came up the idea of offering a course dedicated to building a synthetic yeast genome. In 2007, the course was offered for the first time as a summer school class, which to me is reminiscent of the

When tasked with painting a fence in front of his house, Tom Sawyer convinced others that doing so is more fun than sitting in the shade watching other people paint. As other children congregate to watch Tom Sawyer paint, he extols the fun of painting, and eventually has the other children clamoring to get to paint the fence (taken from postcrossing.com via Bing Images).

The course proved to be extremely popular and 6 years later it’s one of the most in-demand classes that Hopkins offers. Every section is packed —even on Friday nights! ‘We were overwhelmed with interest’ from biology, engineering, computer science, public health, and other majors,’ said Boeke, who is working with his colleagues to keep the course going at Hopkins despite his move to NYU.

Student-Power Drives Yeast Project Assembly

As a former professor and still active researcher, I have nothing but high praise for Boeke’s Tom Sawyer-like innovation to harness interest and enthusiasm of students through an educational course that achieves a truly grand challenge: a totally synthetic designer-yeast genome. More importantly, here’s what Pennisi reported about a couple of representative undergraduate student genome-builders, James Chuang and Katrina Caravelli, whose “Build A Genome” coursework consisted of sequences of DNA comprised of just four letters: A, C, G, and T in DNA:

For students, the course offers intensive training in synthetic biology and the thrill of taking part in frontline research. ‘I was really fascinated by the potential and by my ability to have an impact,’ recalled Caravelli, who signed up for the course in 2008 and is now Boeke's senior lab coordinator at New York University. During the semester-long course, students learned basic molecular biology procedures such as performing PCR and cloning DNA in bacteria.

Each student committed to completing 10,000 DNA bases during the course. ‘My building blocks went to chromosome 8,’ Chuang, now a graduate student in biomedical engineering at Boston University, proudly told Pennisi. After being supplied with the DNA sequence he needed to synthesize, he started out with 16 DNA oligonucleotides, each about 75 bases long, ordered from a commercial DNA synthesis company. The ends of some pieces overlapped, so when he mixed the pieces together with enzymes, they self-assembled into 750-base spans dubbed building blocks. After making sure the building blocks had the correct sequence, he put them into bacteria to generate many copies of the newly assembled DNA. Now, Johns Hopkins graduate student Jingchuan Luo is putting those bigger DNA sections into yeast and making “minichunks” about 3,000 bases long. If the yeast stays healthy, then she adds the next chunk in line to it, and so on. Her goal: to make a yeast strain with a totally new chromosome 8.

Caravelli recalls that her own bacteria sometimes wouldn't grow with the yeast segment in their midst. She enjoyed figuring out why. ‘The trial-and-error process challenges you to think properly like a scientist.’

Boeke's course has proved such a success that three other U.S. universities are hosting or will soon host their own. Because it's now cheaper to buy 750-base segments than to have students make them, current students start with such DNA blocks and turn them into 3000-base spans, and, ultimately, 10,000-base chunks. Class productivity has soared. Each student's target is now 30,000 to 50,000 bases.

The students have high hopes for their work. ‘I want to see synthetic biology do really useful things for society,’ Chuang told Pennisi. ‘The synthetic yeast genome provides a template for doing [that>

.’

Global Momentum and Sc2.0

After the students in Boeke’s course completed chromosome 9, they moved on to chromosome 3. It took 49 students over 18 months to successfully assemble the 272,871 bases. Their work was detailed in a publication in Science in March of this year. The publication showed that ‘yeast carrying the shortened, modified chromosome grew normally and looked little different from their natural counterparts under almost all of the growing conditions tested.’

You can watch and listen to Jef Boeke talk about this achievement in a 2 minute video.

As they neared completion of chromosome 3, the team continued work on other chromosomes as word was spreading about the novel work coming out of Hopkins. Ying-Jin Yuan of Tianjin University in China heard about the project and was eager to participate. He convinced Huanming Yang from BGI to get involved and Yang helped organize the first synthetic yeast genome meeting, held in Beijing in 2012. Yuan didn’t want to stop there. He forged an international collaboration with Boeke and through his own 2012 "Build-A-Genome" course, he enlisted the help of 60 Chinese students and assembled all the bases needed for chromosome 5.

Popularity of the project continued to grow in other labs around the world. Tom Ellis, a synthetic biologist at Imperial College London, had attended Yang’s meeting in Beijing and was also intrigued by Boeke’s work. He helped organize a second synthetic yeast meeting in July 2013. Soon after, the British government committed £1 million to the project.

The momentum has continued. These like-minded yeast chromosome-builders have created a great website for their project that is called Sc2.0. This site is definitely worth visiting, especially to read the FAQ and learn more about the international Team members. I’ve included a great visual from the website that depicts where in the world each of the 16 chromosomes is being built.

The goal of the yeast genome project is to complete each of the yeast’s chromosomes within 2 years. This will be done by various partners around the global. Once the individual chromosomes are complete, Boeke will lead a team through the largest challenge of all—putting all of the chromosomes together into one organism. ‘It's a little surprising that we can have this grand idea with so many moving parts and multiple organizations making different chromosomes,’ Boeke's senior lab coordinator, Katrina Caravelli told Pennisi. But so far, ‘it's working well.’

The Vision for Custom Yeast

Many yeast biologists are waiting in anticipation for the synthetic yeast genome project to be completed as they are eager to begin testing it themselves. Aside from studying how induced mutations influence function of genes, the way will be open for various applications, such as a new model for human diseases. Boeke, for example, told Pennisi that he ‘would like to install in yeast all of the genes of the molecular pathway that, in humans, is defective in Lesch-Nyhan syndrome, a rare disorder characterized by gout and kidney and neurological problems, including self-mutilating behavior. By modeling the molecular defect in yeast, he hopes to figure out how the mutation affects eukaryotic cells in general.’

On the other side of the equation, Kirsten Benjamin, a synthetic biologist at Amyris Inc., is looking to study what’s wrong with the synthetic strain. ‘We're going to find a bunch of ways where it doesn't work,’ she says. This, she says, ‘will allow us as a scientific community to discover all these unappreciated phenomena.’

While the commercial applications for synthetic genomes are still to be determined due to uncertainty around financial feasibility, it’s not the long term viability that Boeke is after. ‘In a way, you can put it as a kind of milestone, like the first human genome was a milestone for genomics,’ he says. ‘When we finish it, we can really plant a flag in it.’

My opinions on this are that the cost for synthesizing DNA oligonucleotides has continually decreased, while innovative methods for up-scaled mass production and stitching together oligos have continually improved. In concert, these will nullify the aforementioned ‘too costly’ argument and enable custom synthesized genomes.

What do you think?

As always, your comments are welcomed.