Entire Synthetic Genome Created
DNA of the smallest known
Mycoplasma genitalium, stitched together
JOHN ROACH / National Geographic News 25jan2008
Scientists yesterday announced that they have successfully created an entire synthetic genome in the lab by stitching together the DNA of the smallest known free-living bacterium, Mycoplasma genitalium.
Experts are hailing the research as an important breakthrough in genetic manipulation that will one day lead to the "routine" creation of synthetic genomes—possibly including those of mammals.
This is "a striking technical accomplishment," biochemist Leroy Hood, who was not involved in the study, wrote in an email.
"It represents the initial stages of an important new step in studying how genes function together in systems to create complex phenotypes [traits]," added Hood, co-founder of the Institute for Systems Biology in Seattle, Washington.
Step Toward Artificial Life
The new work is an important second step in a three-step process to the creation of synthetic life, said research leader Hamilton Smith, a biologist and Nobel laureate at the J. Craig Venter Institute in Rockville, Maryland.
The first step, reported last year by the same team at Venter's institute, was the successful transplantation of a genome from one species of bacteria into another, effectively switching the bug's identity.
"The third step, which we're working on now, is to take the chemically synthesized DNA, which is in the test tube, and get it into a bacterium where it can take over and produce a synthetic cell," Smith said.
Researchers liken that step to rebooting a computer, because a genome is akin an operating system that makes a cell function.
Successfully completing the final step would create the first synthetic life-form.
The research is also part of a project to create a cell with "the smallest number of genes that can still confer life," Smith added.
The team chose M. genitalium because it contains about 485 genes, the smallest known of any organism capable of surviving on its own. The researchers suspect that about 400 genes are necessary for life.
Once they have created a synthetic copy of the bacteria, scientists can begin to eliminate genes to determine which are essential.
Such an accomplishment would then allow scientists to create synthetic life-forms that may one day produce biofuels, clean up toxic waste, and fight global warming.
So, this is only the beginning," Smith said.
But completing that second step was no easy feat.
While scientists can pretty easily assemble short sequences of DNA—or order them out of a catalog—synthesizing entire genomes is difficult.
That's because as more base pairs of the four building blocks of DNA—adenine, cytosine, guanine, and thymine—are stitched together, the strands tend to weaken and eventually break.
Prior to this research, for instance, the longest synthesized string contained 32,000 base pairs of DNA. The M. genitalium genome is 582,970 base pairs.
So the researchers broke up the genome into 101 segments, called cassettes, each containing between 5,000 and 7,000 base pairs of genetic code.
The researchers also took steps at this stage to address concerns that the technology could be misused to engineer a deadly virus or that an unforeseen innocent error could lead to bacteria run amok.
The researchers added watermarks to the code to differentiate the synthetic DNA from genomes of wild M. genitalium. They also inserted a gene to block the ability of the synthetic genome to infect human or animal hosts.
Much of the cassette assembly work was outsourced to genetics companies.
Back at the lab, meanwhile, Smith and his colleagues devised a process to stitch the 101 cassettes into a full synthetic genome.
They combined increasingly larger sections of the genome together in a test tube with linking and repair enzymes found in the bacterium Escherichia coli until they had four overlapping quarter genome sections.
After unsuccessfully trying to combine the quarters into halves in E. coli, the team switched to brewers' yeast, and the genome came together through a process the yeast uses to repair damaged DNA.
"That was pretty remarkable," Smith said, explaining that scientists had not known that a single yeast cell would pick up all the overlapping pieces and correctly assemble them.
"Yeast will play a big part in the future in assembling large DNA molecules," he added.
Smith and his colleagues report the assembly process in a paper published on the Web site of the journal Science.
"Important Changes" to Genetics
Drew Endy is an assistant professor in the department of biological engineering at the Massachusetts Institute of Technology in Cambridge and an expert in the field of synthetic biology. He was not involved in this study.
In an email, he said "reconstructing a natural bacterial genome from scratch is a great technical feat."
While genomes up to eight million base pairs have previously been assembled from existing DNA fragments, he noted the new accomplishment heralds "important changes in the science of genetics."
By 2012, he added, the technology should exist to routinely design and construct genomes of any bacteria or single-celled organism with a membrane-bound nucleus.
"Which also means," he said, "that it will be possible to construct some mammalian chromosomes."
The Synthetic Genome
Maverick scientist Craig Venter claims he can
create artificial life in the lab.
Is this the dawn of a new era for mankind?
JONATHAN LEAKE The Sunday Times (UK) 27jan2008
Last week that thrilling but unsettling goal appeared to have come a step closer with the announcement by Craig Venter, the maverick scientist, that his laboratory had constructed the world’s first completely synthetic genome.
He described how he had used laboratory chemicals to recreate an almost exact copy of the genetic material found inside a tiny bacterium — and was now attempting to slot it into an empty cell in the hope of creating a new life form.
For the layman, he compared his work with the building of a computer. His breakthrough was the equivalent of creating the software for a computer’s operating system. Now what he had to do was insert it into the computer itself — the empty cell — and “boot it up”.
What’s more, he announced, he was already working on the next stage of his great project. He would build an entirely synthetic organism, which he would then use to save the world from global warming.
For Venter, the showman of the world of science, the result could hardly have been better. Details of the breakthrough went around the world generating positive headlines. The prospect that a painless way of solving the problems of climate change might have been found was particularly attractive.
As the fuss dies down, however, questions remain. Has Venter really come close to creating a new life form? Will the benefits really be so powerful and clear cut? What might the acquisition of such godlike powers actually mean for humanity?
VENTER himself has long been a man of supreme immodesty. Since the 1990s he has scorched his way through the burgeoning science of genomics, leaving a trail of enemies in his path as he set about mapping the human genome.
The feelings he provokes are so intense that one profile in The New Yorker magazine from 2000 began with a quote from a string of fellow scientists, saying: “Craig Venter is an asshole. He’s an idiot. He is a thorn in people’s sides and an egomaniac.”
Venter’s first breakthrough was in developing what is now known as shotgun sequencing, a method for analysing the human genome faster and more cheaply than ever before.
At the time, however, it was unproven and too risky for the government-funded institution where he worked so, after many rows, Venter left and raised the money himself.
An instinctive entrepreneur, he might have expected to feel more at home mixing with fast moving risk-takers like himself, but instead the rows became even more intense. His first business partnership collapsed and his relationship with Celera Genomics, with whom he completed the genome, also proved tempestuous.
Even the publication of the genome itself proved controversial. Fearing that Venter would patent the genome and charge for access, a consortium of scientists launched their own publicly funded rival effort.
The race became so bitter that Bill Clinton, then US president, had to step in to negotiate a truce, with both teams agreeing to publish their findings simultaneously in 2001.
It was supposed to mark the end of hostilities but when Venter held a party his fellow scientists boycotted the event, leaving Venter glowering over a near-empty dance floor.
Soon after he was sacked by Celera. Insiders made clear the firm could no longer sustain such a huge ego.
Again Venter bounced back, using his £100m share of Celera’s stock to found the J Craig Venter Institute. It now has more than 400 scientists and staff based in Rockville, Maryland, and La Jolla, California. For Venter, however, perhaps its most priceless asset is that he controls it.
The years since then have seen Venter repeatedly in the headlines. Last June he announced success in transplanting the entire genome of one bacterium into another, effectively causing the recipient to change species.
Then, in September, he published his own genome, the first time any individual person’s DNA had been sequenced. It was perhaps a mixed blessing, revealing that Venter is at risk of Alzheimer’s, diabetes and hereditary eye disease.
For scientists the benefits of his institute’s synthetic genome are, however, much clearer. Although they have long been able to make synthetic DNA they have only been able to produce it in short lengths. This is because the chemical “bases” that make up the building blocks of DNA – adenine, thymine, cytosine and guanine – are very difficult to work with.
DNA chains are built from pairs of these bases all linked together to form the familiar “twisted ladder” shape. In the test tube, however, the chains become increasingly brittle the longer they get. This means that the largest synthesised DNA chain contained only 32,000 base pairs until now.
Dr Jim Haseloff, a Cambridge University expert in synthetic biology said: “The true breakthrough here is that Venter has built a DNA sequence containing 583,000 base pairs. There is a very good chance that if he can transplant it into a bacterial cell it will start working.”
This event may be far closer even than Venter is saying. The paper published last week was actually written five months ago, since when it has been undergoing peer review by other researchers. In that time the research has intensified.
Dan Gibson, who led the research, and Hamilton Smith, the Nobel prize-winning biologist who worked with him, said: “We are now working towards the ultimate goal of inserting a synthetic chromosome into a cell and booting it up to create the first synthetic organism.”
What it means is that pretty soon we are likely to see the first truly synthetic microbes – and that will be sure to spark fierce debate. Some will accuse Venter of playing God. Others will raise fears of new bioweapons. The simple question is: just what will humanity be able to do with this new technology? ONE thing that is clear is that there is no chance of Venter’s techniques being applied to create synthetic human genomes. Or indeed of it leading to the halting of the human ageing process, as some scientists have speculated.
Mycoplasma genitalium, the bacterium on which Venter’s team worked, was chosen purely because it has a relatively tiny genome. Most bacteria have far more – typically up to 10m base pairs long, while fungi have around 38m and plants 115m. Mammals are thousands of times more complex again with humans having around 3 billion base pairs.
Professor Paul Freemont, head of molecular biosciences at Imperial College, London, said: “There are just 485 genes in Mycoplasma, while humans have 20,000. It is science fiction to think Venter’s work could give scientists control of the human genome.”
There are, however, many other possibilities, some of which were set out by Venter himself in a telling article published last autumn. He described how, in 2003, his team had synthesised the first artificial genome, of an obscure virus called phi-X174.
As news of the breakthrough got out, he was invited to a meeting with John Marburger, the president’s chief scientific adviser. Venter said: “We told him now we had achieved this goal, we could begin to move to creating new types of microorganisms that could be used in numerous ways, as green fuels to replace oil and coal, digest toxic waste or absorb greenhouse gases.”
Alongside these attractive benefits, Venter also set out a more sinister possibility. “We could now probably also synthesise any virus with a genetic code of fewer than 10,000 ‘letters’ of DNA in under a week in the lab, and larger viruses such as the Marburg or Ebola virus [both very unpleasant] in a month or so.”
For Marburger the implications were clear and, soon after, Venter’s research was put under scrutiny by the National Science Advisory Board for Biosecurity which oversees research deemed potentially dangerous.
In public, however, little was said about such fears. Perhaps the only clue came at a press conference when Hamilton Smith blurted out: “We could make the smallpox genome.” Venter later spoke of his relief when only one reporter repeated Smith’s reference to the “possibility of making deadly pathogens”.
It is a worry that plays on people’s minds. Literature and films are littered with the human race being imperilled by biological innovations that have spiralled out of control. Such fears will never go away. Synthetic biology is after all a powerful technology and, just like genetic modification, crossbreeding and every other method for altering the genetic make-up of other living things, can be used for good or evil.
For now, however, the biggest barrier for making any use of such techniques at all lies in our limited understanding of how DNA works.
The researchers who praise Venter’s breakthrough also warn that predicting how a given sequence of synthetic DNA will actually perform is a far harder task.
Jason Chin, who leads a synthetic biology research group in Cambridge, said: “DNA communicates with a cell by prompting it to make proteins, but we have a long way to go in understanding the relationship between a given DNA sequence, the proteins it generates and the final properties of an organism.”
So, for now at least, scientists will be limited to producing synthetic versions of DNA sequences found in nature and tinkering with them.
When will we see the benefits? The history of biotechnology is littered with other reminders that we may have to wait a long time. Stem cells, gene therapy and cloning were all great scientific discoveries but the practical benefits are taking much longer to emerge.
Venter’s ecological claims for his breakthrough have been greeted with cautious optimism by his peers. But they note that there would be significant regulatory hurdles surrounding the release of a new organism into the environment to overcome. The benefits would most likely not been seen within a decade.
For Venter, however, such cautionary notes are simply a challenge. His vision, he told Newsweek magazine last year, is of creating the first “trillion-dollar organisms” — patented bugs that could excrete bio-fuels, generate clean energy in the form of hydrogen and even produce tailor-made foods.
It is a startling vision of a brave new world, but it also sounds like a world that would be largely controlled by J Craig Venter.
Is This The Beginnings Of Artificial Life?
Medical News Today 26jan2008
In what many believe to be a case of creating artificial life, American scientists have found a way of replication a bacterium's 582,970 base pair genome which should allow for the creation of biofuel-manufacturing bacteria 3 in other words, building bacteria from scratch that might produce fuel for things like cars. It is the largest man-made DNA structure ever made. The previous largest one contained only 32,000 base pairs.
You can read about this in Science magazine.
Dr. Hamilton Smith, J Craig Venter Institute, Rockville, USA, and sixteen others built a bacterium's genome by chemically synthesizing DNA blocks. These blocks were then weaved together to create bigger DNA pieces 3 these can be formed to create a synthetic version of Mycoplasma genitalium. The scientists say these tailor-made micro3organisms can be designed to produce hydrogen, or tweaked to absorb surplus carbon dioxide in the air.
The team is not using the term artificial life; they prefer to call it synthetic life. Dr. Smith, in a BBC interview, said "We like to distinguish synthetic life from artificial life. It sets the stage for what we hope is going to be a new approach to engineering organisms."
The J Craig Venter Institute (JCVI) says this is the second of three key steps towards the team's aim of creating a fully synthetic organism. They are currently trying to create a living bacterial cell, based completely on the synthetically made genome.
J. Craig Venter, Ph.D., President and Founder of JCVI, said "This extraordinary accomplishment is a technological marvel that was only made possible because of the unique and accomplished JCVI team. Ham Smith, Clyde Hutchison, Dan Gibson, Gwyn Benders, and the others on this team dedicated the last several years to designing and perfecting new methods and techniques that we believe will become widely used to advance the field of synthetic genomics."
The scientists explain that building blocks of DNA 3 adenine (A), guanine (G), cytosine (C) and thymine (T) are tremendously tricky chemicals to artificially synthesize into chromosomes. The longer the strands become the more brittle they are, making it very hard to work with them. Making the genome of the M. genitalium bacteria with over 580,000 base pairs was an enormous challenge.
Hamilton Smith said "When we started this work several years ago, we knew it was going to be difficult because we were treading into unknown territory. Through dedicated teamwork we have shown that building large genomes is now feasible and scalable so that important applications such as biofuels can be developed."
Ever since the beginning of this project, the team has been concerned with the ethical issues related to their work. The creation of life by humankind is bound to trigger controversy.
About the J. Craig Venter Institute
The JCVI is a not-for-profit research institute dedicated to the advancement of the science of genomics; the understanding of its implications for society; and communication of those results to the scientific community, the public, and policymakers. Founded by J. Craig Venter, Ph.D., the JCVI is home to approximately 400 scientists and staff with expertise in human and evolutionary biology, genetics, bioinformatics/informatics, information technology, high-throughput DNA sequencing, genomic and environmental policy research, and public education in science and science policy. The legacy organizations of the JCVI are: The Institute for Genomic Research (TIGR), The Center for the Advancement of Genomics (TCAG), the Institute for Biological Energy Alternatives (IBEA), the Joint Technology Center (JTC), and the J. Craig Venter Science Foundation. The JCVI is a 501 (c)(3) organization. For additional information, please visit http://www.JCVI.org.
Published Online January 24, 2008 Science DOI: 10.1126/science.1151721 Science Express Index
Research Articles Submitted on October 15, 2007 Accepted on January 11, 2008
Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasma genitalium
Genome Daniel G. Gibson 1, Gwynedd A. Benders 1, Cynthia Andrews-Pfannkoch 1, Evgeniya A. Denisova 1, Holly Baden-Tillson 1, Jayshree Zaveri 1, Timothy B. Stockwell 1, Anushka Brownley 1, David W. Thomas 1, Mikkel A. Algire 1, Chuck Merryman 1, Lei Young 1, Vladimir N. Noskov 1, John I. Glass 1, J. Craig Venter 1, Clyde A. Hutchison III1, Hamilton O. Smith 1* 1 The J. Craig Venter Institute, Rockville, MD 20850, USA.
* To whom correspondence should be addressed. Hamilton O. Smith , E-mail: firstname.lastname@example.org
We have synthesized a 582,970 bp Mycoplasma genitalium genome. This synthetic genome, named M. genitalium JCVI-1.0, contains all the genes of wild-type M. genitalium G37 except MG408, which was disrupted by an antibiotic marker to block pathogenicity and to allow for selection. To identify the genome as synthetic, we inserted "watermarks" at intergenic sites known to tolerate transposon insertions. Overlapping "cassettes" of 5 to 7 kb, assembled from chemically synthesized oligonucleotides, were joined by in vitro recombination to produce intermediate assemblies of approximately 24 kb, 72 kb ("1/8 genome"), and 144 kb ("1/4 genome"), which were all cloned as bacterial artificial chromosomes (BACs) in Escherichia coli. Most of these intermediate clones were sequenced, and clones of all four 1/4 genomes with the correct sequence were identified. The complete synthetic genome was assembled by transformation-associated recombination (TAR) cloning in the yeast Saccharomyces cerevisiae, then isolated and sequenced. A clone with the correct sequence was identified. The methods described here will be generally useful for constructing large DNA molecules from chemically synthesized pieces and also from combinations of natural and synthetic DNA segments.