Team Completes Second Step in Three Step Process to Create Synthetic Organism
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ROCKVILLE, Md., Jan. 24 /PRNewswire-USNewswire/ -- 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. This work, published online today in the journal Science by Dan Gibson, Ph.D., et al, is the second of three key steps toward the team's goal of creating a fully synthetic organism. In the next step, which is ongoing at the JCVI, the team will attempt to create a living bacterial cell based entirely on the synthetically made genome.
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The team achieved this technical feat by chemically making DNA fragments in the lab and developing new methods for the assembly and reproduction of the DNA segments. After several years of work perfecting chemical assembly, the team found they could use homologous recombination (a process that cells use to repair damage to their chromosomes) in the yeast Saccharomyces cerevisiae to rapidly build the entire bacterial chromosome from large subassemblies.
"This extraordinary accomplishment is a technological marvel that was only made possible because of the unique and accomplished JCVI team," said J. Craig Venter, Ph.D., President and Founder of JCVI. "Ham Smith, Clyde Hutchison, Dan Gibson, Gwyn Benders, and the others on this team dedicated the last several years to designing and perfecting new methods and techniques that we believe will become widely used to advance the field of synthetic genomics."
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 contained only 32,000 base pairs. Thus, building a synthetic version of the genome of the bacteria M. genitalium genome that has more than 580,000 base pairs presented a formidable challenge. However, the JCVI team has expertise in many technical areas and a keen biological understanding of several species of mycoplasmas.
"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. "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."
Methods for Creating the Synthetic M. genitalium
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 who
