Team’s next step is to generate the smallest possible viable genome.
Scientists at the J. Craig Venter Institute (JCVI) report on the first ever successful creation of a self-replicating bacterial cell that is driven by a synthetic, computer-designed genome constructed in the laboratory, completely from scratch. The team chemically synthesized small pieces of the 1.08 million base pair chromosome of a modified Mycoplasma mycoides genome, stitched the pieces together, and inserted the full length DNA into a Mycoplasma capricolum recipient cell.
They claim that not only do the cells appear to run perfectly well off the synthetic chromosome, but 30 cell divisions later, dilution effects mean that the daughter cells essentially contain none of the protein molecules that would have been present in the original M. capricolum recipient cell. By this point all their proteins are the result of expression and translation of genes from the synthetic genome.
“The DNA software builds its own hardware,” the researchers write in the published Science paper, titled, “Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome.”
The construction of a synthetic genome capable of supporting a living cell is the culmination of 15 years of research and began when the team successfully sequenced the genome from Mycoplasma genitalium, which has just 485 protein-coding genes. In 2008, the team reported the construction of a completely synthetic M. genitalium genome of about 590,000 base pairs, but they weren’t able to generate viable recipient cells using the transplanted synthetic genome.
For the M. mycoides project the JCVI scientists built on all they had learned over previous years about assembling and error-correcting synthetic genomes, extracting intact chromosomes from yeast, and transplanting and expressing chemically synthesized chromosomes in recipient cells. They started with an accurate, digitized genome of the bacterium and designed 1,078 specific overlapping cassettes of DNA that were 1,080 base pairs long.
A three-stage process using the group’s previously described yeast assembly system was then used to build the genome. The first stage involved taking 10 cassettes of DNA at a time to build 110, 10,000 bp segments. In the second stage, these 10,000 bp segments were taken 10 at a time to produce 11, 100,000 bp segments. In the final step all 11, 100,000 bp segments were assembled into the complete synthetic genome in yeast cells and grown as a yeast artificial chromosome.
The complete synthetic M. mycoides genome was then isolated from the yeast cell and transplanted into Mycoplasma capricolum recipient cells that had the genes for their restriction enzyme removed. 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.
“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,” comments co-author Clyde A. Hutchinson III, Ph.D. “This involved developing many new and useful methods along the way. We have assembled an amazing group of scientists that have made this possible.”
The process was not completely smooth-running, however. “Our success was thwarted for many weeks by a single base pair deletion in the essential gene dnaA,” writes J. Craig Venter, Ph.D. and colleagues. “One wrong base out of over one million in an essential gene rendered the genome inactive, while major genome insertion and deletions in nonessential parts of the genome had no observable impact on viability.”
In a recent interview Venter commented, “This is an important step, we think, both scientifically and philosophically. It’s certainly changed my views of definitions of life. It’s pretty stunning when you just replace the DNA software in the cell, and the cell instantly starts reading that new software, starts making a whole new set of proteins, and within a short while all the characteristics of the first species disappear, and a new species emerges from this software that controls that cell growing forward.”
The published work generated a synthetic gene with only limited modifications in comparison with the naturally occurring M. mycoides genome. However, it paves the way for more significant engineering of synthetic genes. Dr. Venter called it a “new era limited mostly by our imaginations.”
He said that his company, Synthetic Genomics, which funded the research, is already working with oil giant Exxon Mobil to generate either modified versions of existing algae or completely new algal chromosomes for hydrocarbon production. Work is also ongoing with Novartis to speed the production of vaccines. Dr. Venter believes the technology could shorten the process for making flu vaccines each year by some 99%.
Having achieved proof of principle, the group will also focus on creating a minimal genome, which has been their ultimate goal since 1995. By whittling away at the synthetic genome and repeating transplantation experiments, the researchers hope to generate the smallest possible viable genome allowing scientists to analyze the function of every essential gene in a cell.
“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,” Dr. Venter stresses. “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.”
The synthetic chromosome contains four genetic watermarks that essentially tag the genome as synthetic and provide data on its origin. These watermarks even comprise a new DNA code for writing words, sentences, and numbers. Included is a web address that scientists can email if they can successfully decipher the new code, along with the names of 46 authors, 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,”—quote from the book American Prometheus. “What I cannot build, I cannot understand.”—Richard Feynman.
Importantly, the consideration of bioethical implications began well before the experiments even started, Dr. Venter asserts. “This is first incidence in science where the extensive bioethical review took place before the experiments were done, and it’s part of an ongoing process that we’ve been driving, trying to make sure that the science proceeds in an ethical fashion.”
In their published paper the authors say that they will encourage continued discourse. “We have been driving the ethical discussion concerning synthetic life from the earliest stages of this work. We anticipate that this work will continue to raise philosophical issues that have broad societal and ethical implications.”