January 24, 2008
Scientists Close to Creating Artificial Life
Scientists have taken a major step toward creating the first ever artificial life form by synthetically reproducing the DNA of a bacteria, according to a study published Thursday in the journal Science.
A team of 17 researchers at the J. Craig Venter Institute (JCVI) has created the largest man-made DNA structure by synthesizing and assembling the 582,970 base pair genome of a bacterium, Mycoplasma genitalium JCVI-1.0.
The research has important implications for applications such as the development of biofuels.
This work, published online today in the journal Science by Dan Gibson, Ph.D., et al, and represents the second of three key steps toward the team's goal of creating a fully synthetic organism. The next step, which is ongoing at the JCVI, is to create a living bacterial cell based entirely on the synthetically made genome.
The building blocks of DNA"”adenine (A), guanine (G), cytosine (C) and thiamine (T) are not easy chemicals to artificially synthesize into chromosomes. As the strands of DNA get longer they get increasingly brittle, making them more difficult to work with.
Prior to today's publication, the largest synthesized DNA had contained only 32,000 base pairs. Thus, building a synthetic version of the genome that has more than 580,000 base pairs represents a significant achievement in the field of synthetic genomics.
The team began their work by making DNA fragments in the lab, and then developing new methods for the assembly and reproduction of the DNA segments.
The process to synthesize and assemble the synthetic version of the M. genitalium chromosome began first by resequencing the native M. genitalium genome to ensure that the team was starting with an error free sequence. After obtaining this correct version of the native genome, the team specially designed fragments of chemically synthesized DNA to build 101 "cassettes" of 5,000 to 7,000 base pairs of genetic code.
As a measure to differentiate the synthetic genome versus the native genome, the team created "watermarks" in the synthetic genome. These are short inserted or substituted sequences that encode information not typically found in nature. Other changes the team made to the synthetic genome included disrupting a gene to block infectivity. To obtain the cassettes the JCVI team worked primarily with the DNA synthesis company Blue Heron Technology, as well as DNA 2.0 and GENEART.
From here, the team devised a five stage assembly process where the cassettes were joined together in subassemblies to make larger and larger pieces that would eventually be combined to build the whole synthetic M. genitalium genome. In the first step, sets of four cassettes were joined to create 25 subassemblies, each about 24,000 base pairs (24kb). These 24kb fragments were cloned into the bacterium Escherichia coli to produce sufficient DNA for the next steps, and for DNA sequence validation.
The next step involved combining three 24kb fragments together to create 8 assembled blocks, each about 72,000 base pairs. These 1/8th fragments of the whole genome were again cloned into E. coli for DNA production and DNA sequencing. Step three involved combining two 1/8th fragments together to produce large fragments approximately 144,000 base pairs or 1/4th of the whole genome.
At this stage the team could not obtain half genome clones in E. coli, so the team experimented with yeast and found that it tolerated the large foreign DNA molecules well, and that they were able to assemble the fragments together by homologous recombination. This process was used to assemble the last cassettes, from 1/4 genome fragments to the final genome of more than 580,000 base pairs. The final chromosome was again sequenced in order to validate the complete accurate chemical structure.
The synthetic M. genitalium has a molecular weight of 360,110 kilodaltons (kDa). Printed in 10 point font, the letters of the M. genitalium JCVI-1.0 genome span 147 pages.
After several years of work perfecting chemical assembly, the team found they could use a process called homologous recombination (the process cells use to repair damage to their chromosomes) in the yeast Saccharomyces cerevisiae to rapidly build the entire bacterial chromosome from large subassemblies.
"When we started this work several years ago, we knew it was going to be difficult because we were treading into unknown territory," said Hamilton Smith, M.D., senior author on the publication, in a JCVI press release. "Through dedicated teamwork we have shown that building large genomes is now feasible and scalable so that important applications such as biofuels can be developed."
"This is an exciting advance for our team and the field. However, we continue to work toward the ultimate goal of inserting the synthetic chromosome into a cell and booting it up to create the first synthetic organism," said Dan Gibson, lead author.
The research to create the synthetic M. genitalium JCVI-1.0 was funded by Synthetic Genomics, Inc.
The JCVI is a not-for-profit research institute dedicated to the advancement of the science of genomics.
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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