NEW YORK (GenomeWeb News) – A team of scientists from the J. Craig Venter Institute has refined its method for building a synthetic genome. In a paper scheduled to appear online this week in the Proceedings of the National Academy of Sciences, the researchers demonstrate that they can assemble dozens of snippets of DNA into a complete Mycoplasma genitalium genome in just one step in yeast.
“Genome assembly in yeast, as we described it, is accomplished not by the addition of overlapping segments one at a time, but rather by co-transformation of 25 different pieces at once,” lead author Daniel Gibson, a JCVI scientist, and his co-workers wrote. “Thus, large DNA molecules can be assembled much more rapidly from synthetic or naturally occurring sub-fragments than with any other system described previously.”
The publication marks the newest milestone in the Venter team’s quest to create a synthetic organism. In the 1990s, the researchers started tinkering with M. genitalium in their effort to create a minimal genome — a feat they accomplished in 1995. By 2003, they had assembled the roughly 5,400 base pair viral genome of a bacteriophage called phi X. And in 2007, the team demonstrated that it could transplant the genome of one microbe into another microbe.
This January, JCVI researchers reported that they had generated and assembled the first complete synthetic genome, called JCVI 1.0, by cloning pieces of the M. genitalium genome in Escherichia coli, joining together bits of the genome in several steps and assembling four quarter genomes in yeast. In the new paper, Gibson and his colleagues improved upon that method by throwing 25 overlapping pieces of the M. genitalium genome into Saccharomyces cerevisiae and generating the entire genome in just one step.
“The paper reports an important breakthrough in DNA construction technology,” Drew Endy, Stanford University researcher and co-founder of the synthetic biology company Codon Devices, told GenomeWeb Daily News in an e-mail message. “They assemble a very long DNA fragment via a single, parallel reaction.”
Endy, who was not involved in the research, explained, “Existing approaches depend on either time-consuming and expensive linear multi-step processes or multi-step geometric methods.”
Gibson and his colleagues synthesized and cloned 25 M. genitalium 17,000 to 35,000 base pair assemblies as bacterial artificial chromosomes in E. coli and then transformed all of them into yeast in one shot. The researchers then screened the yeast colonies, using multiplex PCR and restriction analysis to pinpoint S. cerevisiae colonies that had taken up and correctly assembled all 25 assemblies.
Using this approach, Gibson and his colleagues produced two different versions of the synthetic M. genitalium genome: JCVI 1.1 and JCVI 1.9. The genomes were nearly identical, but contained variant versions of one of the 25 assemblies, each harboring a different vector inserted in the same non-essential gene.
“We continue to be amazed by the capacity of yeast to simultaneously take up so many DNA pieces and assemble them into genome-size molecules,” Gibson said in a statement. “This capacity begs to be further explored and extended and will help accelerate progress in applications of synthetic genomics.”
There are still questions remaining about the overall efficiency of DNA uptake and assembly in yeast. For instance, the authors noted that it’s still unclear how many fragments they can throw into yeast for assembly and just how long a DNA fragment can be assembled in a single reaction.
Endy agreed that these were important outstanding questions. He also suggested that it will be crucial to determine whether the same approach will work for many or even all potential DNA sequences.
Even so, Gibson and his team expressed enthusiasm about applying their approach for assembling other types of synthetic and natural DNA in yeast. Synthetic Genomics, the company that funded the research, is reportedly using the method in its bid to come up with biofuels and other biochemicals using synthetic biology. Venter is the founder and CEO of Synthetic Genomics.
“It remains to be seen how far we can push this yeast assembly platform but the team is hard at work exploring these methods as we work to boot up the synthetic chromosome,” senior author Clyde Hutchison, a distinguished investigator at JCVI, said in a statement.
For his part, Endy said he’s surprised that the technical breakthrough has been reported so quickly. “If the reported method turns out to be reliable, the assembly of genome and chromosome length DNA will be broadly enabling,” he said.