Venter Institute Scientists Create First Synthetic Bacterial Genome

Publication Represents Largest Chemically Defined Structure Synthesized in the
Lab

 Team Completes Second Step in Three Step Process to Create Synthetic Organism

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.

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 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. 

"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.

Background/Key Milestones in JCVI's Synthetic Genomics Research
The work described by Gibson et al. has its genesis in research by Dr. Venter
and colleagues in the mid-1990s after sequencing M. genitalium and beginning
work on the minimal genome project. This area of research, trying to
understand the minimal genetic components necessary to sustain life, began
with M. genitalium because it is a bacterium with the smallest genome that we
know of that can be grown in pure culture. That work was published in the
journal Science in 1995.

In 2003 Drs. Venter, Smith and Hutchison made the first significant strides in
the development of a synthetic genome by their work in assembling the 5,386
base pair 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.

In June 2007 another major advance was achieved when JCVI researchers led by
Carole Lartigue, Ph.D., announced the results of work on genome
transplantation methods allowing them to transform one type of bacteria into
another type dictated by the transplanted chromosome. The work was published
in the journal Science, and 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. Today's announcement of
the successful synthesis of the M. genitalium genome is the second step
leading to the next experiments to transplant a fully synthetic bacterial
chromosome into a living organism and "boot up" the cell.

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 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.JCVI.org. 



Contact:
Heather Kowalski
cell: 202-294-9206
hkowalski@jcvi.org

SOURCE  J. Craig Venter Institute

Heather Kowalski of J. Craig Venter Institute, +1-202-294-9206,
hkowalski@jcvi.org