Researchers at the J. Craig Venter Institute (JCVI) have synthesized the chromosome of a modified Mycoplasma mycoides bacteria genome and inserted it into a cell stripped of its own DNA. The new cell successfully replicated to form new bacteria, demonstrating that a functional synthetic genome could be used to create a living organism.
The publication of a research paper describing the creation of the first synthetic organism is the result of fifteen years of research at JCVI.
"For nearly 15 years, Ham Smith, Clyde Hutchison, and the rest of our team have been working toward this publication—the successful completion of our work to construct a bacterial cell that is fully controlled by a synthetic genome," J. Craig Venter, founder and president of JCVI, said in a press release. "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.”
How to make a synthetic genome
The researchers synthesized the Mycoplasma mycoides genome, which consists of 1.08 million bp, to form their new genome, M. mycoides JCVI-syn1.0. The genome was designed on a computer, chemically composed in the laboratory, and transplanted into a recipient cell.
Armed with the full sequence of the original genome, the researchers used it to design 1078 DNA cassettes that were each 1080 bp long. The ends of these cassettes were specifically designed to overlap with neighboring strands of DNA by 80 bp.
Then the researchers used a three-step process to build the genome using the DNA cassettes in a yeast cell. In the first step, the researchers assembled the cassettes, connecting 10 overlapping cassettes at a time to build 110 groups of 10,000 bp each. Then those groups were combined, once again, 10 at a time, to create 11 groups of 100,000 bp. The final step was to combine these 11 100,00-bp-long groups into a single genome.
The completed synthetic M. mycoides genome was then isolated from the yeast cell and transplanted into recipient cells of M. capricolum. The researchers used several methods to identify the synthetic genome from M. capricolum's original genome. These methods included locating truncated peptides and evaluating restriction enzyme sites, because the researchers believe the restriction enzymes created by the synthetic genome damage the original genome.
The transfected synthetic DNA was then transcribed and the resulting mRNA sequence was successfully used to create new proteins. After two days, the cells formed new viable M. mycoides cells, which contained only the synthetic genome.
"With this first synthetic bacterial cell and the new tools and technologies we developed to successfully complete this project, we now have the means to dissect the genetic instruction set of a bacterial cell to see and understand how it really works," said Ham Smith, a researcher on the project.
Try, try again
Because initial attempts to synthesize the synthetic genome failed, the researchers developed an error correction method to test each cassette to identify the one that was malfunctioning. The method used a combination of natural and synthetic genome segments to produce semi-synthetic genomes. By testing each synthetic part for function, the researchers were able to isolate the single malfunctioning cassette. Further analysis through DNA sequencing showed that a single base pair deletion in an essential gene caused the failed transplants. When this base-pair error was corrected, the viable synthetic cells were created.
"To produce a synthetic cell, our group had to learn how to sequence, synthesize, and transplant genomes,” said Daniel Gibson, a researcher on the project. “Many hurdles had to be overcome, but we are now able to combine all of these steps to produce synthetic cells in the laboratory."
Synthesizing the future
To date, the JCVI synthetic bacterium is the largest synthetic molecule of a defined structure created.
Buoyed by this success, the researchers intend to take on the ambitious work of creating a minimal genome. To do this, they will remove pieces of the synthetic genome and repeat transplant experiments until the genome is as small as possible while still functional. A minimal genome could help researchers analyze the function of all essential genes in all cells.
Funding for this research was provided by Synthetic Genomics Inc., a company co-founded by Venter and Smith. The paper, “Creation of a bacterial cell controlled by a chemically synthesized genome,” was published online May 20 at Science Express. The article will appear in the next print version of Science.