J. Craig Venter Institute Researchers Clone and Engineer Bacterial Genomes in Yeast and Transplant Genomes Back into Bacterial Cells
New methods allow for the rapid engineering of bacterial chromosomes and the creation of extensively modified bacterial species; should also play key role in boot up of synthetic cell
ROCKVILLE, MD — August 20, 2009 — Researchers at the J. Craig Venter Institute (JCVI), a not-for-profit genomic research organization, published results today describing new methods in which the entire bacterial genome from Mycoplasma mycoides was cloned in a yeast cell by adding yeast centromeric plasmid sequence to the bacterial chromosome and modified it in yeast using yeast genetic systems. This modified bacterial chromosome was then isolated from yeast and transplanted into a related species of bacteria, Mycoplasma capricolum, to create a new type of M. mycoides cell. This is the first time that genomes have been transferred between branches of life — from a prokaryote to eukaryote and back to a prokaryote. The research was published by Carole Lartigue et al in Science Express.
Hamilton Smith, M.D., one of the leaders of the JCVI team said, "I believe this work has important implications in better understanding the fundamentals of biology to enable the final stages of our work in creating and booting up a synthetic genome. This is possibly one of the most important new findings in the field of synthetic genomics."
The research published today was made possible by previous breakthroughs at JCVI. In 2007 the team published results from the transplantation of the native M. mycoides genome into the M. capricolum cell which resulted in the M. capricolum cell being transformed into M. mycoides. This work established the notion that DNA is the software of life and that it is the DNA that dictates the cell phenotype.
In 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 into a recipient bacterial cell, all the experiments failed.
The researchers had previously established that no proteins were required for chromosome transplantations, however they reasoned that DNA methylation (a chemical modification of DNA that bacterial cells use to protect their genome from degradation by restriction enzymes, which are the proteins that cut DNA at specific sites) might be required for transplantation. When the chromosome was isolated direct from the bacterial cells it was likely already methylated and therefore transplantable due to it being protected from the cells restriction enzymes.
In this study, the team began by cloning the native M. mycoides genomeinto yeast by adding a yeast centromere to the bacterial genome. This is the first time a native bacterial genome has been grown successfully in yeast. Specific methylase enzymes were isolated from M. mycoides and used to methylate the M. mycoides genome isolated from yeast. When the DNA was methylated the chromosome was able to be successfully transplanted into a wild type species of M. capricolum. However, if the DNA was not first methylated the transplant experiments were not successful. To prove that the restriction enzymes in the M. capricolum cell were responsible for the destruction of the transplanted genome the team removed the restriction enzyme genes from the M. capricolum genome. When genome transplantations were performed using the restriction enzyme minus recipient cells, all the genome transplantations worked regardless of if the DNA was methylated or not.
"The ability to modify bacterial genomes in yeast is an important advance that extends yeast genetic tools to bacteria. If this is extendable to other bacteria we believe that these methods may be used in general laboratory practice to modify organisms," said Sanjay Vashee, Ph.D., JCVI researcher and corresponding author on the paper.
The team now has a complete cycle of cloning a bacterial genome in yeast, modifying the bacterial genome as though it were a yeast chromosome and transplanting the genome back into a recipient bacterial cell to create a new bacterial strain. These new methods and knowledge should allow the team to now transplant and boot up the synthetic bacterial genome successfully.
The research published today by JCVI researchers was funded by the company Synthetic Genomics Inc., a company co-founded by Drs. Smith and Venter.
Key Milestones/Ethical Issues Background on JCVI's Synthetic Genomics Research
1995: After sequencing the M. genitalium genome (published in 1995), Dr. Venter and colleagues begin work on the minimal genome project. This area of research, trying to understand the minimal genetic components necessary to sustain life, started with M. genitalium because it is a bacterium with the smallest genome known that can be grown in pure culture. This work was published in the journal Science in 1999.
2003: Drs. Venter, Smith and Hutchison (along with JCVI's Cynthia Andrews-Pfannkoch) made the first significant strides in the development of a synthetic genome by assembling the 5,386 base pair genome of bacteriophage φX174 (phi X). They did so using short, single strands of synthetically produced, commercially available DNA (known as oligonucleotides) and using an adaptation of polymerase chain reaction (PCR), known as polymerase cycle assembly (PCA), to build the phi X genome. The team produced the synthetic phi X in just 14 days.
2007: JCVI researchers led by Carole Lartigue, Ph.D., announced the results of work published in the journal Science, which outlined the methods and techniques used to change one bacterial species, Mycoplasma capricolum, into another, Mycoplasma mycoides Large Colony (LC), by replacing one organism's genome with the other one's genome. Genome transplantation was the first essential enabling step in the field of synthetic genomics as it is a key mechanism by which chemically synthesized chromosomes can be activated into viable living cells.
January 2008: The second successful step in the JCVI teams' journey to create a cell controlled by synthetic DNA was completed when Gibson et al published in the journal Science, the synthetic M. genitalium genome.
December 2008: Gibson et al published a paper in Proceedings of the National Academy of Sciences (PNAS) describing a significant advance in genome assembly in which the team was able to assemble in yeast the whole bacterial genome, Mycoplasma genitalium, in one step from 25 fragments of DNA. The work was funded by the company Synthetic Genomics Inc. (SGI). The team is still working to boot up the synthetic cell using all the knowledge gleaned from their previous work.
Ethical Considerations
Since the beginning of the quest to understand and build a synthetic genome, Dr. Venter and his team have been concerned with 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 a panel of experts at the University of Pennsylvania (Cho et al, Science December 1999:Vol. 286. no. 5447, pp. 2087 — 2090). The bioethical group's independent deliberations, published at the same time as the scientific minimal genome research, resulted in 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. As such, the JCVI's policy team, along with the Center for Strategic & International Studies (CSIS), and the Massachusetts Institute of Technology (MIT), were funded by a grant from the Alfred P. Sloan Foundation for a 20-month study that explored the risks and benefits of this emerging technology, as well as possible safeguards to prevent abuse, including bioterrorism. After several workshops and public sessions the group published a report in October 2007 outlining options for the field and its researchers.
About the J. Craig Venter Institute
The JCVI is a not-for-profit research institute in Rockville, MD and La Jolla, CA 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.weizhongli-lab.org.
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JCVI Media Contact: Heather Kowalski, 301-943-8879, [email protected]