por admin » Mié May 26, 2010 12:37 pm
Como creamos la primera celula sintetica
How We Created the First Synthetic Cell
The assembled genome is the largest chemically defined structure ever synthesized in the laboratory.
By J. CRAIG VENTER AND DANIEL GIBSON
In 1995, we reported the DNA sequences for the first two cellular genomes. Nowadays genome sequences, which contain the genetic instructions for an organism, are routinely obtained and deposited in computer databases.
Last week, we reported that this process can be reversed. The digitized DNA information of Mycoplasma mycoides, a simple bacterium, can now be brought to life.
To make this happen, our group of 25 researchers had to decipher this bacterium's set of instructions, synthesize them, and then express them in a recipient cell. Many technical hurdles had to be overcome. But 15 years and $40 million worth of research later, we are able to combine all of these steps and produce synthetic cells in the laboratory.
So what is new and unique about what we did? The process of synthesizing a cell began at a computer. We started with the more than one million letters of genetic instructions for Mycoplasma mycoides, and then made slight modifications to its DNA sequence. First, we deleted 4,000 letters, which removed the function of two genes. We then replaced 10 genes with four "watermark" sequences. These watermark sequences are each over 1,000 letters in length and can be decoded to reveal the names of people, famous quotations and a website address. The entire sequence of DNA letters was then partitioned into 1,100 pieces, and each was synthesized using four different bottles of chemicals that make up DNA. These DNA fragments were designed such that adjacent pieces contained an 80-letter overlap, which facilitated the assembly process by providing unique regions where the synthetic pieces could join.
The negatively stained transmission electron micrographs of aggregated M. mycoides.
The synthetic Mycoplasma mycoides genome was assembled by adding the overlapping DNA fragments to yeast. Once inside a yeast cell, the yeast machinery recognized that two DNA fragments had the same sequence and assembled them at this overlapping region. The genome was not assembled from all 1,100 pieces at once but rather in three stages: 1,000 letters to 10,000 letters, 10,000 letters to 100,000 letters, and finally 100,000 letters to complete the 1.08 million letter genome. This assembled genome is the largest chemically defined structure ever synthesized in the laboratory.
The final step in creating a synthetic cell is to activate the chemically synthesized genome in the cytoplasm of a recipient cell. We transplanted the synthetic Mycoplasma mycoides genome into recipient cells of a related bacteria (Mycoplasma capricolum). To make this work, we had to inactivate a restriction enzyme gene in the recipient cells. Otherwise they would have destroyed the incoming synthetic genome. On March 26, the synthetic genome was "booted up," and self-replicating Mycoplasma mycoides JCVI-syn1.0 cells were produced.
We refer to the cell we have created as being a "synthetic" cell because it is controlled only by a synthetic genome assembled from chemically synthesized pieces of DNA. Even though the cytoplasm of the recipient cell is not synthetic, following transplantation and replication on a plate to form a colony, resulting cells will not contain any molecules that were present in the original recipient cell. The DNA software builds its own hardware so that the properties of the cells controlled by the synthetic genome are expected to be the same as if the whole cell had been produced synthetically. The synthetic cell has multiple genetic differences from its nearest relative, including 4,658 letters of DNA sequence that contain the watermark "code within the code."
While there has been widespread media coverage calling our research a fundamental breakthrough in our understanding of life, this is not the first time such headlines have appeared. On Dec. 14, 1967, Arthur Kornberg, along with colleagues Mehran Goulian and Robert Sinsheimer, announced their success in copying the DNA of the phiX174 virus, creating the same infectivity as the wild virus. While this was achieved 11 years before the viral genome sequence was known, they hoped that their achievement would aid future studies of genetics, the search for cures for viral and hereditary diseases, and reveal the most basic processes of life itself. President Lyndon Johnson hailed it as "a very spectacular breakthrough."
Kornberg did not create life in a test tube, nor did we create life from scratch. We transformed existing life into new life. We also did not design and build a new chromosome from nothing. Rather, using only digitized information, we synthesized a modified version of the naturally occurring Mycoplasma mycoides genome. The result is not an "artificial" life form. It is a very real, self-replicating cell that most microbiologists would be unable to readily distinguish from the naturally occurring counterpart without the aid of DNA sequencing.
President Johnson, commenting on Kornberg's work, stated: "Think about the state ordaining life. This is going to be one of the great problems—one of the big decisions. If you think about some of these decisions the present president is making—it is going to be a kindergarten class compared to the decisions some future president is going to have to make."
Johnson was right. In a letter last week to the chair of his new bioethics commission, President Obama wrote: "As you know, scientists have announced a milestone in the emerging field of cellular and genetic research known as synthetic biology. While scientists have used DNA to develop genetically modified cells for many years, for the first time, all of the natural genetic material in a bacterial cell has been replaced with a synthetic set of genes. This development raises the prospect of important benefits, such as the ability to accelerate vaccine development. At the same time, it raises genuine concerns, and so we must consider carefully the implications of this research."
We welcome and encourage such review and dialogue. We requested a bioethical review before we conducted our first experiment and have continued to engage in policy discussions since we're aware that our scientific advancement has many implications. There are approximately 6.8 billion people on our planet, soon to be more than nine billion. We clearly have difficulty providing sufficient food, clean water, health care, and power to our existing residents without degrading the environment. How will it be possible to provide for more than nine billion without some substantial scientific advances? We believe that synthetic genomics can provide one solution.
We are currently working on the design of new cells that can much more efficiently capture carbon dioxide and "fix" (or incorporate) the carbon into new fuel molecules, new food oils, and new biologically derived sources of plastic and chemicals. We already have funding from the National Institutes of Health to use our synthetic DNA tools to build synthetic segments of every known flu virus so that we can rapidly build new vaccine candidates in less than 24 hours. We are also being funded to see if we can take sets of genes out of bacteria to design new synthetic pathways to make antibiotic compounds that are currently too complex for chemists to make.
With such extensive research already underway in this new field of synthetic biology, there will surely be countless developments that we can't imagine today. Moving forward, we need to ensure the safe and responsible use of these technologies so that they can be put to good societal uses.
Mr. Venter, a genomic scientist, is founder and president of the J. Craig Venter Institute and founder and CEO of the biotech company Synthetic Genomics Inc. Mr. Gibson is a molecular biologist and associate professor at the J. Craig Venter Institute.