Now, synthetic cell

A milestone in biology was reached on May 20 when Craig Venter of human genome fame and associates from the J. Craig Venter Institute (JCVI) reported in the online journal Science Express that they had for the first time created a self-replicating cell with a chemically synthesised genome. The actual breakthrough of achieving a blue self-replicating colony of these synthetic cells came in the early morning of March 29 and the paper was submitted for publication on April 9.

Compared with all the unwarranted and, in fact, wrong media hype calling it a creation of synthetic life', the abstract of the paper is matter-of-fact and understated. What Venter's team had done does not amount to creating new life from scratch. It is not even clear if one can call the creation synthetic cells as Venter prefers to call it. But the advancement certainly marks a technical feat that could have major implications for biotechnology in the years to come.

As Venter described it during the press conference at the American Association for the Advancement of Science (AAAS) on May 20, it is a cell made by starting with the digitised information of a naturally occurring bacterial genome; building the chromosome from four bottles of chemicals corresponding to the four basic chemical units of DNA (deoxyribonucleic acid); assembling the chromosome in yeast, transplanting it into a recipient cell of a slightly different bacterium whose replication is now entirely controlled by the new synthetic chromosome; and thus transforming that cell into a new synthetic bacterial species. This is the first self-replicating species that we have on the planet whose parent is a computer, Venter remarked.

Venter's team synthesised the 1.08 million base pair (bp) chromosome of a modified Mycoplasma mycoides genome with 14 of its genes deleted and a watermark' written in another 5,000-odd bp to distinguish it from natural chromosome. The synthetic genome, which is almost identical to that of the natural bacterium, was transplanted into recipient cells of a close microbial relative M. capricolum to produce a new self-replicating cell, but with the characteristics of M. mycoides.

New code

The team has called the new species M. mycoides JCVI-syn 1.0 (or Synthia). The watermark is a new code within the code within the code which allows use of entire English alphabet with punctuations and numbers, according to Venter. It is a code developed by the team for writing and interpreting messages in DNA compared to the ones that have been used for writing messages in genetic code.

The synthetic genome carries in the genetic code language the names of the 47 authors and key contributors to the project and three website addresses. There are also three quotations that have been coded into the genome to give, in Venter's words, a philosophical perspective to this technical advance. The first is from James Joyce's A Portrait of the Artist as a Young Man: To live, to err, to fall, to triumph, and to recreate life out of life. The second is a quote from American Prometheus, the book on J. Robert Oppenheimer: See things not as they are, but as they might be. And the third is the famous Richard Feynman's What I cannot build, I cannot understand.

In December 1967, Arthur Kornberg and colleagues at Stanford University showed that they could copy the DNA of the phage Phi X 174 (a virus that attacks the microbe E. coli) producing an entity with the same infectivity as the wild virus. Though the phage's genome sequence was not known (it was determined 10 years later by Fred Sanger and colleagues), Kornberg had hoped that the technique, besides aiding the study of genetics and the search for the cure of diseases, would reveal the basic processes of life itself. The achievement was hailed as a spectacular breakthrough, which had come the closest yet to creating life.

Modified life

As techniques in biology evolved over the years, we have known that stretches of DNA can be synthesised, and we have also known that these stretches can be introduced into cells of organisms and be expressed. Digitisation of genomic information has increased by more than eight orders of magnitude over the past 25 years.

But what we have here is not just a very long stretch of DNA but the entire genome of a naturally occurring organism synthesised and, more significantly, the synthetic or prosthetic genome transplanted into the cytoplasm of a another organism from which cells are isolated and where the replication is now controlled not by its original chromosome but only by the new synthetic chromosome. That is, cells have now been endowed with a new modified life.

This might seem stunningly close to creating new life' but is still a far cry from it. What has been achieved here is essentially recreation of an existing bacterial form of life. Even if the synthesised genome was substantially different from that of any existing form of life, a conceivable possibility, one can still not call this creation of new life. One can call it that only if the whole cell is synthesised from scratch.

Only a small part of the cell is synthetic as the genome accounts for only 1 per cent of the dry weight of a cell. But, of course, the genome is key to life, without which the cell is dead. The research groups are trying to create fully synthetic cells (also called protocells), but they have met with limited success so far, using chemicals alone.

It took 15 years for Venter's team to arrive at this proof-of-concept experiment. Back in 1995, Hamilton Smith and Clyde Hutchison of his team had sequenced the first two genomes of self-replicating genomes in history Haemophilus influenzae and M. genitalium. The latter, in fact, was a 600,000 bp chromosome, the smallest known genome of a self-replicating organism. However, more than 100 of the 485 protein-coding genes of M. genitalium were apparently found to be dispensable, according to the Science paper.

So the question that Venter and colleagues asked was: If this is supposed to be the smallest genome, could there be even a smaller genome? Could the basis of cellular life be understood at the genetic level? For that two basic steps were required: synthesising a full bacterial genome and getting it to work in a recipient cell.

It's been a 15-year quest just to get to the starting point now to be able to answer those questions, Venter said at the press conference. But there is no cell [yet] and certainly not our synthetic cell where the function of every gene is understood. We don't know yet which genes are essential for life and why. It will be interesting to see how few components are needed to boot up a synthetic chromosome. Perhaps, all it will take is a lipid vesicle and the ability to make messenger RNA [mRNA] and ribosomes, but we don't know, Venter wrote in New Scientist.

In their quest to build a cell with the minimum number of essential genes, they chose the strategy of a synthetic route because it is very difficult to eliminate multiple genes from a cell and you can do only one at a time. [The idea was] if we could synthesise a bacterial chromosome, we could actually vary the gene content to understand the essential genes for life, explained Venter. In 2003, based on a digitised sequence in a computer rather than a copy made by an enzyme, and using new methods for making error-free DNA at a small level, they achieved a synthetic DNA of Phi X 174 of size 5,000 bp.

Once we realised that we could make 5,000 bp viral size pieces, we thought we at least had the means to try and make serially lots of these pieces to be able to eventually assemble them together to make a complete bacterial chromosome, Venter said. It was a long route[and] the team developed methods for synthesising this megabase chromosome (of M. mycoides) substantially larger than we even thought we would go after initially, he added.

Two basic steps

Venter's team had two basic issues to address and solve: the chemistry for making large DNA molecules and the biology of booting up' this new chemical entity in a recipient cell. The phrase booting up' is used perhaps because of the analogy with a computer; what one is doing is similar to changing the operating system of a computer and then rebooting it.

The most important achievement in this quest, according to Venter, was the actual transplanting of a bacterial chromosome from one bacterium to another, a work that was led by Carole Lartigue. This was in 2007, when the naked and intact whole genomic DNA (free of all proteins) from M. mycoides was transplanted into M. capricolum cells and the replicated cell colony were found to contain the complete donor genome and were free of any detectable recipient genome sequences.

I think philosophically, that was one of the most important papers we have ever done, because it showed how dynamic life was. And we knew once that worked we actually had a chance to make the synthetic chromosome do the same. [But] we didn't know that it was going to take several years more to get there, Venter said.

In 2008, the team reported the synthesis of the 500,000 bp M. genitalium genome complete with the watermark signature to tell the cells driven by the synthetic genome apart from the natural ones. However, this watermark, unlike the present one, was a far simpler one that coded merely the names of the authors. But their attempts to boot it up were not successful. This was partly because of the slow growth of M. genitalium and partly because of the various defence mechanisms in the cell machinery that are programmed to reject invading genes. Apparently, the cell that they were transplanting into had a nuclease, an enzyme, which chewed up the synthetic DNA and thus preventing successful transplantation. Because of the slow growth of the small cell, the team switched to working with the much larger M. mycoides genome because the larger cells took only two days to grow as compared with six-week cycles in the case of the small cell.

Last year they demonstrated that they could extract the M. mycoides natural chromosome, place it into yeast, alter the genome, extract it from the yeast and transfer it into M. capricolum. The team had to develop new techniques to actually grow and clone the entire bacterial chromosome in yeast. The essential problem was to find out how to extract a bacterial genome out of the eukaryotic yeast into a form that could be used to transplant into a recipient cell.

Methylation

Transplantation, too, was not a simple task and this had failed even with the simple M. genitalium DNA. The problem actually took more than two years to solve. They discovered that the DNA in the bacterial cell is methylated and methylation protects it from being chewed up by restriction enzymes present in the cell. So the trick was to methylate the chromosome taken out of yeast and then it could be transplanted. Alongside the team also developed techniques to remove the restriction enzyme genes themselves from the M. capricolum cell.

Once that was done, the naked DNA from the yeast itself could be transplanted. The remaining step was to achieve the same with the synthetic copy of the full genome. Having succeeded in this in September 2009, the team, according to Venter, became confident that the next step could be achieved in a few weeks' time. But it wasn't to be because the synthesising technique had one error in the million bp sequence. Just one base pair had been deleted in an essential gene.

Accuracy in the synthesis is essential, points out Venter. There are parts of the genome where even a single error cannot be tolerated and there are parts, like where the watermarks have been put, where blocks of DNA can be inserted and tolerated. This took three months to figure out, and fixing it required developing a new debugging software with which one could test the accuracy compared to the natural or wild type DNA.

The process of synthesis itself, the chemistry part of the game, was quite complex. To begin with, the researchers had the sequence to be synthesised on a computer. The one million bp genome was partitioned into 1,078 overlapping pieces that were 1,080 bp long that could be chemically synthesised. These partitions or cassettes were designed so that a cassette overlapped with its neighbour by 80 bp. These cassettes were synthesised by a DNA synthesis company, Blue Heron Biotechnology, according to the team's specifications.

Three-stage trick

A three-stage process was employed to build the genome based on their earlier work which had shown that yeast had this remarkable capacity to assemble as many as 25 pieces, if not more, at a time. The first stage involved taking 10 cassettes of the DNA at a time and building 110 segments of 10,000 bp each. In the second stage, these 10,000 bp segments are taken 10 at a time to produce 11 segments of 100,000 bp (100 kb) each. In the final stage, all the 11 segments of 100 kb each were used to assemble the complete genome in the yeast cells that grow as an artificial chromosome.

Throughout the process, a piece of yeast DNA is used as a vector to trick the yeast cells into recognising the synthesised pieces as its own DNA. So every time it replicates, it produces another copy of the various intermediate synthetic stretches of DNAs and the complete genome at the very end.

The technique actually produced several versions of the synthetic genome. For months, transplantation experiments were done to isolate these genomes out and put them into M. capricolum cells with their restriction enzyme genes removed. The synthetic genome DNA was transcribed into mRNA, which in turn translated into new proteins. The M. capricolum genome itself was either destroyed by M. mycoides restriction enzymes or was lost during the billion rounds of cell replication that the system underwent, according to Venter.

After two to three days, viable M. mycoides cells, which contained only the synthetic chromosome, were clearly visible as a blue colony in petri dishes containing the bacterial growth medium. But 99 per cent of their attempts had failed to achieve this all because of single base pair deletion.

Ultimate goal

The work, which cost about $40 million, represents the construction of the largest synthetic molecule of a defined structure, the M. mycoides genome being almost double the size of the previously synthesised M. genitalium genome. With this success, Venter's team will now embark on creating the minimal genome, its goal since 1995. We can now begin working on our ultimate objective of synthesising a minimal cell containing only the genes necessary to sustain life in its simplest form, which will help us understand how cells work better, said Dan Gibson, the team member who was chiefly responsible for the synthesis part. This they will do by whittling away at the synthetic genome and repeating transplantation experiments until no more genes can be disrupted and the genome is as small as possible.

Our synthetic cell, wrote Venter, is a small but highly significant step in synthetic genomics. Without this success there would be no future for what has been until now a theoretical field. We have now tools to begin to understand cellular life.

As was mentioned earlier, this demonstration of proof of concept implies that the techniques evolved could easily be applied to produce something that never existed before by, say, introducing new genes or removing many of existing genes. The advantage of synthetic DNA is that it allows even more radical changes than the engineered genome, points out George Church of Harvard Medical School.

Indeed, according to Venter, his team is going to try to make synthetic genomes that carry instructions for bacteria to make flu vaccine. A programme for this has already been launched with funding from Novartis and the National Institutes of Health (NIH). Also Synthetic Genomics (a company founded by Venter and the JCVI) is forming a new vaccine company. And these techniques will make these vaccines in less than 24 hours, instead of weeks and months that current processes take, says Venter.

Synthetic Genomics also has a programme with Exxon-Mobil to develop new strains of algae that can effectively capture CO2 from the atmosphere or from concentrated sources, and make new hydrocarbons that can go into refineries to make normal gasoline and diesel out of CO2. The approach, according to Venter, can be used to design new pharmaceuticals, biofuels, and so on.

Patents & monopoly

The reaction of the biological community has, however, been somewhat mixed. It is cool and has taken a lot of effort. But it doesn't take that much further scientifically, Alistair Elfick of Edinburgh University has been quoted as saying in New Scientist. It is a technical tour-de-force, so it does make a high-profile technical point for people who think about designing such technologies, says Satyajit Rath of the National Institute of Immunology (NII) in similar vein. But it teaches us nothing profound about [biological] mechanisms. It does not make any interesting science more or less feasible; in fact, my guess is that it does not even make new technologies more or less feasible, except that some courts may use the synthetic gene' excuse to provide protection for some gene patents, he adds.

Indeed, John Sulston of the University of Manchester has already voiced concern that efforts to patent the first synthetic cell would give its creator a monopoly on a range of genetic engineering techniques and would inhibit important research. It would be recalled that Sulston had a running battle on intellectual property with Venter on the issue of making genomic data from the Human Genome Project (HGP) openly available. In HGP, both played crucial roles. I have read some of these patents [relating to Synthia] and the claims are very, very broad indeed, Sulston has been quoted as saying. I hope very much these patents will not be accepted because they would bring genetic engineering under the control of the JCVI. The problem has become much worse since I raised the issue 10 years ago, Sulston said.

This is a marvellous advance, but it doesn't immediately open up new studies for the broad community, says James Collins of Boston University. Even if synthetic genomes become dramatically cheaper [than the present $1 per letter], there is still the question of how to write one. We have a long way to go to really develop sufficient understanding to build an operational genome from scratch, he adds. Also, here the booting up was achieved with difficulty even in closely related species. It remains to be seen whether cells will accept the genome of drug making E. coli or biofuel producing or oil-spill eating algae because it would be a far greater challenge to jump between very different species.

From a perspective of scientific potential, very interesting possibilities in the understanding of how large stretches of genetic material are regulated open up, says K. VijayRaghavan, director of the National Centre for Biological Sciences (NCBS), Bangalore, taking a more positive view of the development. One of the greatest challenges in gene-regulation is trying to understand how large stretches of DNA interact with each other, particularly in cells such as ours. Once the Venter synthetic experiment becomes applicable to such contexts, our basic understanding will change dramatically. This will surely have many beneficial applications.

Given the fact that now there is virtually a new species out there, all kinds of ethical questions will arise even though, one must emphasise, no new life has been created in this advance made by Venter and co. For example, there would be worries if there is a synthetic algae that is let loose into the environment. Already some groups have begun to call for moratorium on synthetic biology until international rules governing organisms leaving the lab are put in place.

According to Venter, even before they undertook the first experiments, they had asked Arthur Kaplan's team at the University of Pennsylvania to undertake a review of what were the risks, challenges and ethics of creating new species in laboratory work, and the review took two years, after which the team began its work in 1999.

Safety measures

The ethical difference between creating synthetic cells in research laboratories and those that are released in the environment is significant. Any environmental release would, therefore, occur only under appropriately stringent conditions. Nevertheless, it is possible that unintended environmental release could happen. However, it is not easy to keep synthetic cells alive even under ideal conditions in the laboratory. So, in an accidental environmental release, synthetic cells well die quickly.

There is also active discussion on building in multiple safeguards in synthetic cells. These include giving them a strictly limited lifespan, inserting suicide genes, incorporating an on/off switch, making them dependent on nutrients and conditions that are not present naturally in the environment, and so on.

In addition to safeguards, it is important to build in unique identifying marks, like watermark, so that any damage can be traced back to the origin. The NIH has called for new guidelines on how to regulate DNA synthesis companies to make sure that nobody is creating new pathogens or recreating old pathogens. The United States National Academy of Sciences too has produced a report on the issue.

Significantly, in the wake of this remarkable advance in synthetic biology, U.S. President Barack Obama wrote to his Bioethics Commission, led by the University of Pennsylvania President, Amy Gutmann, to study the implications of this research on the potential medical, environmental, security and other benefits of this field of research as well as any potential health, security or other risks. He has asked the panel to do a six-month study and recommend any actions that the Federal government should take to ensure America reaps the benefits of this developing field of science while identifying appropriate ethical boundaries and minimising identified risks. According to Venter, in 2003, the White House initially considered classifying his work, which was being funded by the U.S. Department of Energy, but later decided to make it open.

Ethical concerns are a matter of continuing debate, but no substantial new ethical questions come from this study yet. While they will surely emerge as the technology becomes more efficient, and these must be watched out for, as of now, the potential positive aspects stand out, says VijayRaghavan.