Researchers at the J. Craig Venter Institute said they have successfully constructed the first self-replicating, synthetic bacterial cell, a project that is viewed as a breakthrough toward the creation of artificial life forms that could lay the foundation for a new industrial revolution in which man-made life forms are harnessed to produce fuels, drugs and other goods efficiently and inexpensively.

“With this first synthetic bacterial cell and the new tools and technologies we developed to successfully complete this project, we now have the means to dissect the genetic instruction set of a bacterial cell to see and understand how it really works,” says Smith.

The team from the institute synthesized the 1.08 million base pair chromosome of a modified Mycoplasma mycoides genome. The synthetic cell is called Mycoplasma mycoides JCVI-syn1.0 and is the proof of principle that genomes can be designed in the computer, chemically made in the laboratory and transplanted into a recipient cell to produce a new self-replicating cell controlled only by the synthetic genome. The research was published in the online journal Science Express and will appear in an upcoming print issue of Science.

The JCVI scientists hope that the knowledge gained by constructing this first self-replicating synthetic cell, coupled with decreasing costs for DNA synthesis, could lead to wider use of this technology in the development of important applications and products including biofuels, vaccines, pharmaceuticals, clean water, and food products.

At the same time, Dr. Venter and the team at JCVI continue to work with bioethicists, outside policy groups, legislative members and staff, and the public to encourage discussion and understanding about the societal implications of their work and the field of synthetic genomics in general, to ensure that their work will lead to positive outcomes for society.

“We have been consumed by this research, but we have also been equally focused on addressing the societal implications of what we believe will be one of the most powerful technologies and industrial drivers for societal good," says J. Craig Venter, founder and president, JCVI and senior author on the paper. “We look forward to continued review and dialogue about the important applications of this work to ensure that it is used for the benefit of all.”

To complete the final stage in the nearly 15 year process to construct and boot up a synthetic cell, JCVI scientists began with the accurate, digitized genome of the bacterium, M. mycoides. The team designed 1,078 specific cassettes of DNA that were 1,080 base pairs long. These cassettes were designed so that the ends of each DNA cassette overlapped each of its neighbors by 80 base pairs.

In a three stage process they used their previously described yeast assembly system to build the genome using 1,078 DNA cassettes. The first step involved taking 10 DNA cassettes at a time to build a total of 110, 10,000 base pair segments. Then the 10,000 base pair segments were taken 10 at a time to produce eleven 100,000 base pair segments. Finally all 11 100 kb segments were assembled into the complete synthetic genome, which was inserted into yeast cells and grown as a yeast artificial chromosome.

The complete synthetic M. mycoides genome was isolated from the yeast cell and transplanted into Mycoplasma capricolum recipient cells that had the genes for its restriction enzyme removed. The synthetic genome DNA was transcribed into messenger RNA, which in turn was translated into new proteins. The M. capricolum genome was either destroyed by M. mycoides restriction enzymes or was lost during cell replication. After two days viable M. mycoides cells, which contained only synthetic DNA, were clearly visible on petri dishes containing bacterial growth medium.

The initial synthesis of the synthetic genome did not result in any viable cells so the JCVI team developed an error correction method to test that each cassette they constructed was biologically functional. Using a combination of 100 kb natural and synthetic segments of DNA to produce semi-synthetic genomes, they tested each of the 11 synthetic segments in combination with 10 natural segments for their capacity to be transplanted and form new cells. Ten out of 11 synthetic fragments resulted in viable cells; narrowing the issue down to a single 100 kb cassette. DNA sequencing revealed that a single base pair deletion in an essential gene was responsible for the unsuccessful transplants. Once this one base pair error was corrected, the first viable synthetic cell was produced.

“To produce a synthetic cell, our group had to learn how to sequence, synthesize, and transplant genomes. Many hurdles had to be overcome, but we are now able to combine all of these steps to produce synthetic cells in the laboratory,” says Daniel Gibson, a member of the research team and an associate professor in the Synthetic Biology and Bioenergy Department at the JCVI. “We can now begin working on our ultimate objective of synthesizing a minimal cell containing only the genes necessary to sustain life in its simplest form. This will help us better understand how cells work.”

As they did when the successfully synthesized the M. genitalium genome in 2008, the JCVI researchers designed and inserted what they called “watermarks” into the genome, a means to prove that the genome is synthetic and not native, and to identify the laboratory of origin. In a humorous twist, encoded in the watermarks is a new DNA code for writing words, sentences and number that includes a web address to send emails to if you can successfully decode the new code.

It also includes the names of 46 authors and other key contributors and three quotations: “To live, to err, to fall, to triumph, to recreate life out of life” from James Joyce; “See things not as they are, but as they might be” from the book American Prometheus; and “What I cannot build, I cannot understand” from Richard Feynman.