One Step Closer to Creating a Synthetic Life Form by Cloning and Engineering Bacterial Genomes

Newly devised techniques allow for the rapid engineering of bacterial chromosomes and the creation of extensively modified bacterial species, and should also play key role in the generation of a synthetic cell.

Researchers from the J. Craig Venter Institute (JCVI; Rockville, MD, USA), a not-for-profit genomic research organization, published results in an article by Carole Lartigue et al 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 genomes have been transferred between branches of life--from a prokaryote to eukaryote and back to a prokaryote. The research was published August 21, 2009, 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 was made possible by earlier 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 hypothesis that DNA is the software of life and that it is the DNA that dictates the cell phenotype.

In 2008, the same investigators reported on the construction of the first synthetic bacterial genome by assembling DNA fragments made from adenine, cytosine, guanine, and thymine. 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 of researchers began by cloning the native M. mycoides genome into 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 effectively transplanted into a wild type species of M. capricolum. However, if the DNA was not first methylated the transplant experiments were not successful. To confirm that the restriction enzymes in the M. capricolum cell were responsible for the destruction of the transplanted genome, the scientists 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 study.

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 investigators to now transplant and boot up the synthetic bacterial genome successfully.

The research was funded by the company Synthetic Genomics, Inc. (La Jolla, CA, USA), a company cofounded by Drs. Smith and Venter.

The JCVI is a not-for-profit research institute in Rockville, MD, USA, and La Jolla, CA, USA, focused on 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.

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J. Craig Venter Institute