A research team, led by Craig Venter of America’s J. Craig Venter Institute (JVCI), has successfully produced the first self-replicating, synthetic bacterial cell. Called Mycoplasma mycoides JCVI-syn1.0 the synthetic cell 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 resulting bacterium could be regarded as the first truly synthetic organism. The researchers now hope to be able to explore the machinery of life, and to engineer bacteria designed for specific purposes such as producing drugs, biofuels or other useful chemicals.
"This is the first synthetic cell that's been made, and we call it synthetic because the cell is totally derived from a synthetic chromosome, made with four bottles of chemicals on a chemical synthesizer, starting with information in a computer," said Venter. "This becomes a very powerful tool for trying to design what we want biology to do. We have a wide range of applications [in mind].”
The ScienceTo complete this final stage in the nearly 15 year process to construct and boot up a synthetic cell, JCVI scientists modeled the artificial genome after that of the bacterium M. mycoides. Beginning with the accurate, digitized genome of M. mycoides the team designed 1,078 specific cassettes of DNA that were 1,080 base pairs (bp) long. These cassettes were designed so that the ends of each DNA cassette overlapped each of its neighbors by 80bp.
Using the 1,078 cassettes the JCVI team employed a three-stage process to build the genome using a yeast assembly system. The first stage involved taking ten cassettes of DNA at a time to build 110, 10,000 bp segments. In the second stage, these 10,000 bp segments are taken ten at a time to produce eleven, 100,000 bp segments. In the final step, all eleven, 100 kb segments were inserted in yeast cells, the DNA-repair enzymes of which linked the strings together. These medium-sized strings were then transferred into E. coli bacteria, then back into the yeast. After three rounds of this process, they had the complete synthetic genome that was over a million base pairs long.
The complete synthetic M. mycoides genome was isolated from the yeast cell and transplanted into Mycoplasma capricolum recipient cells that have 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.
Because the initial synthesis of the synthetic genome did not result in any viable cells the JCVI team developed an error correction method to test that each cassette they constructed was biologically functional. They did this by using a combination of 100 kb natural and synthetic segments of DNA to produce semi-synthetic genomes. This approach allowed for the testing of each synthetic segment 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; therefore the team narrowed 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.
Dr. Gibson stated, "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." He added, "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."
According to Dr. Hutchison, "To me the most remarkable thing about our synthetic cell is that its genome was designed in the computer and brought to life through chemical synthesis, without using any pieces of natural DNA.”
Genetic WatermarkIn 2008 the same team reported on the construction of the first synthetic bacterial genome by assembling DNA fragments made from the four chemicals of life - ACGT. The final assembly of DNA fragments into the whole genome was performed in yeast by making use of the yeast genetic systems. However, when the team attempted to transplant the synthetic bacterial genome out of yeast and into a recipient bacterial cell, viable transplants could not be recovered.
For that case the team designed and inserted into the genome what they called watermarks. These are specifically designed segments of DNA that use the "alphabet" of genes and proteins that enable the researcher to spell out words and phrases. The watermarks are an essential means to prove that the genome is synthetic and not native, and to identify the laboratory of origin.
The team used the same process to insert watermarks into Mycoplasma mycoides JCVI-syn1.0. Encoded in the watermarks is a new DNA code for writing words, sentences and numbers. In addition to the new code there is a web address to send emails to for anyone who can successfully decode the new code, 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." - JAMES JOYCE; "SEE THINGS NOT AS THEY ARE, BUT AS THEY MIGHT BE." - A quote from the book, "American Prometheus"; "WHAT I CANNOT BUILD, I CANNOT UNDERSTAND." - RICHARD FEYNMAN.
The Ethical Implications The creation of the world’s first truly synthetic organism throws up some serious ethical questions. In designing and creating life, they are “playing God” - something that is sure to concern those that believe such practices should remain the province of their deity of choice. The team’s work also challenges the traditional views of what life is and further blurs the line between living things and machines – the synthetic bacterium possesses features of both.
However, from the very beginning of their quest to build a synthetic genome, Dr. Venter and his team say they have been concerned about the societal issues surrounding the work. In 1995 while the team was doing the research on the minimal genome, the work underwent significant ethical review by an independent panel of experts at the University of Pennsylvania who reached a unanimous decision that there were no strong ethical reasons why the work should not continue as long as the scientists involved continued to engage public discussion.
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 generally. A 20-month study that explored the risks and benefits of this emerging technology, as well as possible safeguards to prevent abuse, including bioterrorism, resulted in a report being published in October 2007 outlining options for the field and its researchers.
Most recently in December of 2008, JCVI received funding from the Alfred P. Sloan Foundation to examine ethical and societal concerns that are associated with the developing science of synthetic genomics. The ongoing research is intended to inform the scientific community as well as educate policymakers and journalists so that they may engage in informed discussions on the topic.
What Next?The JCVI scientists envision that the knowledge gained by constructing this first self-replicating synthetic cell, coupled with decreasing costs for DNA synthesis, will give rise to wider use of this powerful technology. They now plan on designing algae that can capture CO2 and give off hydrocarbons for use in refineries. They also hope to improve vaccine production, create new chemicals and food ingredients, and clean up polluted water.
"This is an important step we think, both scientifically and philosophically. It's certainly changed my views of the definitions of life and how life works," Dr. Venter said. “It's part of an ongoing process that we've been driving, trying to make sure that the science proceeds in an ethical fashion, that we're being thoughtful about what we do and looking forward to the implications to the future.”
The team’s research appears in the journal Science.