NEW YORK (GenomeWeb News) – A team of researchers from the J. Craig Venter Institute has created the first synthetic organellar genome, using a method called isothermal DNA assembly to construct a synthetic mouse mitochondrial genome from hundreds of overlapping oligonucleotides.
The work, published online yesterday in Nature Methods, is the latest in a series of synthetic biology achievements by the group.
JCVI researchers reported in Science in 2008 that they had put together four Mycoplasma genitalium quarter genomes in Escherichia coli and yeast to create the first synthetic genome, dubbed M. genitalium JCVI-1.0. They later tweaked this process, showing that they could assemble the synthetic M. genitalium genome in a single step in yeast.
And earlier this year the team took another step toward synthetic life when they made a synthetic M. mycoides genome, transplanted it into another bacterial species, M. capricolum, and showed that these transplanted cells were functional and could self-replicate.
For the latest study, the researchers turned their attention to a smaller genome, the 16,299 base mitochondrial genome from mice, which they assembled using the isothermal assembly method.
The strategy relies on a short incubation at one temperature with a trio of enzymes: Phusion polymerase, Taq ligase, and T5 exonuclease, lead author Daniel Gibson, a synthetic biology and bioenergy researcher at the Venter Institute, told GenomeWeb Daily News in an e-mail message.
The method can be used for assembling either single- or double-stranded DNA, he explained, and cuts out many of the steps usually needed for DNA synthesis and assembly.
"Because the method is so simple and robust, and only requires a few steps, it can be automated," Gibson noted. "The method is carried out in vitro and does not necessarily require cloning steps into a host organism."
For the mouse mitochondrial genome, the team put together 600 oligomers, each 60 base pairs long, that had been made by Integrated DNA Technologies.
After joining these bits of DNA together through a series of sub-assembly steps, the researchers created a complete mitochondrial genome sequence that was cloned into E. coli and assessed by sequencing.
The researchers have not yet tested the functionality of the synthetic mouse mitochondrial genome, but Gibson said they intend to do such experiments in the future.
"Cultured mouse cells that contain various mitochondrial genome deficiencies do not undergo aerobic respiration," he said. "We need to find a way to get the synthetic mouse mitochondrial genome into mitochondria so that the deficiencies can be rescued. Selecting for the ability of these cells to now undergo aerobic respiration would be one way to test for the functionality of the synthetic genome."
And since mitochondrial deficiencies are thought to contribute to several important human diseases — from diabetes and cancer to blindness and deafness — such synthetic biology studies could potentially provide hints about engineering related drug or gene therapies, Gibson explained. He noted that the team is also pursuing synthetic biology-based strategies for faster and more efficient biofuel, pharmaceutical, and vaccine production.
"Synthetic biologists are synthesizing and expressing genetic elements to provide a sustainable means for producing desirable products, such as new and improved drugs, vaccines, biosensors, bioremediation tools, food ingredients, cosmetics and industrial compounds," the researchers concluded. "[W]e provide widely applicable procedures for constructing these genetic elements from synthetic DNA oligonucleotides."