Artificial Life: First Self-replicating Synthetic Bacterial Cell Created

A fully synthesized genome transforms one species of bacterium into another
Thursday, May 20, 2010

Mycoplasma mycoides JCVI-syn1.0. Credit: J. Craig Venter Institute.

Researchers at the J. Craig Venter Institute (JCVI), a not-for-profit genomic research organization, published results today describing the successful construction of the first self-replicating, synthetic bacterial cell. The team synthesized the 1.08 million base pair chromosome of a modified Mycoplasma mycoides genome. The synthetic cell is called Mycoplasma mycoides JCVI-syn1.0 and is the proof of principle that genomes can be designed in the computer, chemically made in the laboratory and transplanted into a recipient cell to produce a new self-replicating cell controlled only by the synthetic genome.

This research will be published by Daniel Gibson et al in the May 20th edition of Science Express and will appear in an upcoming print issue of Science.

“For nearly 15 years Ham Smith, Clyde Hutchison and the rest of our team have been working toward this publication today – the successful completion of our work to construct a bacterial cell that is fully controlled by a synthetic genome,” said J. Craig Venter, Ph.D., founder and president, JCVI and senior author on the paper.

“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. We look forward to continued review and dialogue about the important applications of this work to ensure that it is used for the benefit of all.”

According to Dr. Smith:

“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."

To complete this final stage in the nearly 15 year process to construct and boot up a synthetic cell, JCVI scientists began with the accurate, digitized genome of the bacterium, M. mycoides. The team designed 1,078 specific cassettes of DNA that were 1,080 base pairs long. These cassettes were designed so that the ends of each DNA cassette overlapped each of its neighbours by 80bp. The cassettes were made according to JCVI’s specifications by the DNA synthesis company, Blue Heron Biotechnology.

The JCVI team employed a three stage process using their previously described yeast assembly system to build the genome using the 1,078 cassettes. The first stage involved taking 10 cassettes of DNA at a time to build 110, 10,000 bp segments. In the second stage, these 10,000 bp segments are taken 10 at a time to produce eleven, 100,000 bp segments. In the final step, all 11, 100 kb segments were assembled into the complete synthetic genome in yeast cells and grown as a yeast artificial chromosome.

The complete synthetic M. mycoides genome was isolated from the yeast cell and transplanted into Mycoplasma capricolum recipient cells that have had the genes for its restriction enzyme removed. The synthetic genome DNA was transcribed into messenger RNA, which in turn was translated into new proteins. The M. capricolum genome was either destroyed by M. mycoides restriction enzymes or was lost during cell replication. After two days viable M. mycoides cells, which contained only synthetic DNA, were clearly visible on Petri dishes containing bacterial growth medium.

The initial synthesis of the synthetic genome did not result in any viable cells so the JCVI team developed an error correction method to test that each cassette they constructed was biologically functional. They did this by using a combination of 100 kb natural and synthetic segments of DNA to produce semi-synthetic genomes. This approach allowed for the testing of each synthetic segment in combination with 10 natural segments for their capacity to be transplanted and form new cells. Ten out of 11 synthetic fragments resulted in viable cells; therefore the team narrowed the issue down to a single 100 kb cassette. DNA sequencing revealed that a single base pair deletion in an essential gene was responsible for the unsuccessful transplants. Once this one base pair error was corrected, the first viable synthetic cell was produced.

Dr. Gibson stated:

“To produce a synthetic cell, our group had to learn how to sequence, synthesize, and transplant genomes. Many hurdles had to be overcome, but we are now able to combine all of these steps to produce synthetic cells in the laboratory.”

“We can now begin working on our ultimate objective of synthesizing a minimal cell containing only the genes necessary to sustain life in its simplest form. This will help us better understand how cells work,” he added.

This publication represents the construction of the largest synthetic molecule of a defined structure; the genome is almost double the size of the previous Mycoplasma genitalium synthesis. With this successful proof of principle, the group will now work on creating a minimal genome, which has been a goal since 1995. They will do this by whittling away at the synthetic genome and repeating transplantation experiments until no more genes can be disrupted and the genome is as small as possible. This minimal cell will be a platform for analyzing the function of every essential gene in a cell.

According to Dr. Hutchison:

“To me the most remarkable thing about our synthetic cell is that its genome was designed in the computer and brought to life through chemical synthesis, without using any pieces of natural DNA. This involved developing many new and useful methods along the way. We have assembled an amazing group of scientists that have made this possible.”

As in the team’s 2008 publication in which they described the successful synthesis of the M. genitalium genome, they designed and inserted into the genome what they called watermarks. These are specifically designed segments of DNA that use the “alphabet” of genes and proteins that enable the researcher to spell out words and phrases. The watermarks are an essential means to prove that the genome is synthetic and not native, and to identify the laboratory of origin. Encoded in the watermarks is a new DNA code for writing words, sentences and numbers. In addition to the new code there is a web address to send emails to if you can successfully decode the new code, the names of 46 authors and other key contributors and three quotations: "To live, To err, To fall, To triumph, To recreate life out of life." – James Joyce; "See things not as they are, but as they might be.” – A quote from the book, “American Prometheus”; "What I cannot build, I cannot understand." – Richard Feynman. 

The JCVI scientists envision that the knowledge gained by constructing this first self-replicating synthetic cell, coupled with decreasing costs for DNA synthesis, will give rise to wider use of this powerful technology. This will undoubtedly lead to the development of many important applications and products including biofuels, vaccines, pharmaceuticals, clean water and food products. The group continues to drive and support ethical discussion and review to ensure a positive outcome for society.

Funding for this research came from Synthetic Genomics Inc., a company co-founded by Drs. Venter and Smith.

Background
The research published today was made possible by previous 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 notion that DNA is the software of life and that DNA dictates the cell phenotype.

In 2008 the same team reported on the construction of the first synthetic bacterial genome by assembling DNA fragments made from the four chemicals of life — ACGT. 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 and into a recipient bacterial cell, viable transplants could not be recovered.

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.

Most recently in December of 2008, JCVI received funding from the Alfred P. Sloan Foundation to examine ethical and societal concerns that are associated with the developing science of synthetic genomics. The ongoing research is intended to inform the scientific community as well as educate our policymakers and journalists so that they may engage in informed discussions on the topic.

About the J. Craig Venter Institute
The JCVI is a not-for-profit research institute in Rockville, MD and La Jolla, CA 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.

Reference:
Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome
Daniel G. Gibson, John I. Glass, Carole Lartigue, Vladimir N. Noskov, Ray-Yuan Chuang, Mikkel A. Algire, Gwynedd A. Benders, Michael G. Montague, Li Ma, Monzia M. Moodie, Chuck Merryman, Sanjay Vashee, Radha Krishnakumar, Nacyra Assad-Garcia, Cynthia Andrews-Pfannkoch, Evgeniya A. Denisova, Lei Young, Zhi-Qing Qi, Thomas H. Segall-Shapiro, Christopher H. Calvey, Prashanth P. Parmar, Clyde A. Hutchison III, Hamilton O. Smith, and J. Craig Venter
Science, Published online May 20 2010; DOI: 10.1126/science.1190719
.........

ZenMaster

---

For more on stem cells and cloning, go to CellNEWS at
http://cellnews-blog.blogspot.com/

---

First Human Female DNA Sequenced

First Human Female DNA Sequenced Monday, 26 May 2008 Geneticists of Leiden University Medical Centre (LUMC) are the first to determine the DNA sequence of a woman. She is also the first European whose DNA sequence has been determined. Following in-depth analysis, the sequence will be made public, except incidental privacy-sensitive findings. The results will contribute to insights into human genetic diversity. DNA of geneticist Marjolein Kriek The first woman in the world to have her complete DNA sequenced is described as a red-haired, 34-year-old Dutch woman. The DNA is that of Dr. Marjolein Kriek, a clinical geneticist at LUMC, scientists at Leiden University Medical Centre announced on Monday. “If anyone could properly consider the ramifications of knowing his or her sequence, it is a clinical geneticist,” says professor Gert-Jan B van Ommen, leader of the LUMC team and director of the Center for Medical Systems Biology (CMSB), a center of the Netherlands Genomics Initiative. Van Ommen continues: “Moreover, while women don’t have a Y-chromosome, they have two X-chromosomes. As the X-chromosome is present as a single copy in half the population, the males, it has undergone a harsher selection in human evolution. This has made it less variable. We considered that sequencing only males, for ‘completeness’, slows insight into X-chromosome variability. So it was time, after sequencing four males, to balance the genders a bit”. He smiled: “And after Watson we also felt that it was okay to do Kriek”. Eight times coverage The DNA sequencing was done with the Illumina 1G equipment. This was installed in January 2007 in the Leiden Genome Technology Center, the genomics facility of LUMC and CMSB. In total, approx. 22 billion base pairs (the ‘letters’ of the DNA language) were read. That is almost eight times the size of the human genome. Dr. Johan den Dunnen, project leader at the Leiden Genome Technology Center: “This high coverage is needed to prevent mistakes, connect the separate reads and reduces the chance of occasional uncovered gaps.” “The sequencing itself took about six months. Partly since it was run as a ‘side operation’ filling the empty positions on the machine while running other projects. Would such a job be done in one go, it would take just ten weeks”. The cost of the project was approximately €40.000. This does not include further in-depth bioinformatics analysis. This is estimated to take another six months. History of human DNA sequencing In 2001, the DNA sequence was published of a combination of persons. The DNA sequences of Jim Watson, discoverer of the DNA’s double helix structure, followed in 2007, and later the DNA of gene hunter Craig Venter. Recently the completion of the sequences of one Han Chinese individual and two Yoruba-Africans was announced. Bessensap The researchers announced their news at the yearly ‘Bessensap’ meeting, bringing together the Dutch scientists and the press. The Netherlands Organization for Scientific Research NWO organizes this event jointly with the Association of Science Writers VWN and Science Center NEMO. In its eight years of existence, Bessensap has had several high-profile news items. It has had a debate with Italian ‘clonedoctor’ Severino Antinori and hosted dino-hunter Jack Horner, who was key in the Jurassic-Park modelling. During Bessensap also the yearly Eureka prize is awarded for the best popular-scientific book and media production. Leiden University Medical Center Leiden University Medical Center (LUMC) is strongly committed to ongoing improvement in health care quality and intends to play a leading role in this field at both national and international level. Its core activities are research, patient care, education and post graduate training. LUMC is part of the Dutch Federation of University Medical Centers (NFU), which promotes the shared interests of the eight University Medical Centers in the Netherlands. See also: The human genome; you gain some, you lose some Kriek, Marjolein 6-Dec-2007 International Human Genome Project Launched CellNEWS - Wednesday, 23 January 2008 ......... ZenMaster

---

For more on stem cells and cloning, go to CellNEWS at http://cellnews-blog.blogspot.com/ and http://www.geocities.com/giantfideli/index.html

---

Synthetic Bacterial Genome

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 Friday, 25 January 2008 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.

---

M. genitalium: Living Organism with the Smallest 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.

---

Circular map of the M. genitalium chromosome. Genes are coloured according to the functional classification of the encoded proteins. The length of each projection is proportional to the number of amino acid residues in the respective protein. The outside ring shows the products of the genes that are transcribed clockwise, and the inside ring shows those transcribed counter clockwise.

---

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 handout photo from the J. Craig Venter Institute shows a single molecule from the synthetic Mycoplasma genitalium bacteria over a period of 0.6 seconds. US scientists have taken a major step toward creating the first ever artificial life form by synthetically reproducing the DNA of a bacteria.

---

“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. 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. Publication: Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasma genitalium Genome Published Online January 24, 2008Science DOI: 10.1126/science.1151721

Links: J. Craig Venter Institute Synthetic biology for energy production Mycoplasma genitalium genome More from CellNEWS: Venter makes synthetic life... or not yet? October 09, 2007 Next Step Towards Artificial Life Whole Genome Transplantation Achieved in Mycobacterium June 28, 2007 Venter attempt at the minimalistic approach of creating ‘artificially-made’ life November24, 2002 ......... ZenMaster

---

For more on stem cells and cloning, go to CellNEWS at http://www.geocities.com/giantfideli/index.html

---

The Latest About Synthetic Life

Read The Latest About Synthetic Life Synthetic DNA on the Brink of Yielding New Life Forms Washington Post - Monday, December 17, 2007 ......... ZenMaster For more on stem cells and cloning, go to CellNEWS at http://www.geocities.com/giantfideli/index.html

Venter makes synthetic life... or not yet?

Venter makes synthetic life... or not yet? Tuesday, 09 October 2007 Rumours of J. Craig Venter's achievements in creating artificial life are again circulating in the press – Ed Pilkington in the Guardian reported this weekend that Venter has successfully made a fully synthetic chromosome, dubbed Mycoplasma laboratorium. The chromosome reportedly consists of 381 genes, and in total contains 580,000 nucleotide base pairs. In a study published this June, Venter and colleagues switched two closely related species of bacteria by transplanting their genomes. This transplantation step would be needed to activate synthetic chromosomes as well. So far, however, the new work is not accompanied by a peer-reviewed publication. Venter "is poised to announce" the discovery in the next couple weeks, according to the Guardian article, and possibly even today, at the J. Craig Venter Institute's annual meeting. Nature's news blog, The Great Beyond points to a report in AFP, in which Heather Kowalski, the J. Craig Venter Institute's media contact, declined to confirm the breakthrough. "We have not achieved what some have speculated we have in synthetic life," Kowalski apparently told AFP. "When we do so there will be a scientific publication and we are likely months away from that." .........

ZenMaster

---

For more on stem cells and cloning, go to CellNEWS at http://www.geocities.com/giantfideli/index.html

---

Read more: Creating life in the laboratory BBC - 2007/10/19 RELATED INTERNET LINKS: J Craig Venter Institute Massachusetts Institute of Technology Vanderbilt University Medical Center Harvard Medical School University of Rome Three Princeton University Los Alamos National Laboratory Southampton University Biobricks

Future of personal genomics

As personal genomics stands poised to go mainstream, researchers urge caution Friday, 21 September 2007 Giving Nobel Laureate James Watson his personal genome and Craig Venter publishing his own complete genome was just the beginning. In a future that promises similar information to much of the population, ethicists, scientists and physicians are only beginning to understand and consider the possibilities. In a commentary in today’s issue of the journal Science, four experts ponder the implications of this new technology and information and ask the crucial questions that should be answered before the era of personal genomics comes to pass. They combined their different perspectives to consider what is possible now and in the future. Along with that, they look at the ethical and legal issues that will inevitably arise with such technology. Imagine this: you visit your clinician, undergo genetic testing, and then you are handed a miniature hard drive containing your personal genome sequence, which is subsequently uploaded onto publicly accessible databases. This may sound like science fiction, but it is scientific fact, and it is already happening. University of Alberta researcher Tim Caulfield and co-authors highlight the need to proceed with caution when it comes to personal genomics projects that represent research milestones but are also fraught with ethical, social and clinical implications. Caulfield, who is the Canada Research Chair in Health Law at the U of A and professor and research director in public health sciences, is recognized as one of the foremost experts in health law research in Canada. Scientists predict that within five years DNA sequencing technologies will be affordable enough that personal genomics will be integrated into routine clinical care. Companies are responding by offering their services for ancestry tracing, forensics, nutritional advice and reproductive assistance. It won’t be long before companies are able to offer Facebook-like social networking services centred around our genomes.

• Once we have our personal genomic information, what will we do with it and how might this information be used outside the medical context?
• How will physicians educate patients about the significance of genetic risk information?
• Will already-strained health-care systems be able to cope with the inevitable influx of “worried well” patients seeking follow-up investigations for genetic risks that are not clinically meaningful?

Caulfield and his colleagues pose these questions and warn that the routine generation of individual genome sequences will pose challenges to our health-care system. They argue that only clinically meaningful genomic test results should be integrated into medical decision-making — however, this will require clear standards, multidisciplinary collaboration and careful consideration of the ethical, social and clinical implications. Drs. Amy L. McGuire of Baylor College of Medicine, Mildred K. Cho of Stanford University in Palo Alto, California; Sean E. McGuire of The University of Texas M.D. Anderson Cancer Center and Timothy Caulfield of the University of Alberta in Edmonton, Canada, participated in this study. ......... ZenMaster

---

For more on stem cells and cloning, go to CellNEWS at http://www.geocities.com/giantfideli/index.html

---

First individual genome sequence published

First individual genome sequence published Tuesday, 04 September 2007 Independent sequence and assembly of the six billion base pairs from the genome of one person ushers in the era of individualized genomics Researchers at the J. Craig Venter Institute (JCVI), along with collaborators at The Hospital for Sick Children in Toronto and the University of California San Diego (UCSD), have published a genome sequence of an individual, Craig Venter, that covers both sets of chromosomes that were inherited from each parent. Two other versions of the human genome currently exist — one published in 2001 by J. Craig Venter, Ph.D., and colleagues at Celera Genomics, and another at the same time by a consortium of government-funded researchers. These genomes were not of any single individual, but, rather, were a melding of DNA from various people. In the case of Celera, it was a consensus assembly from five individuals, while the government-funded version was a haploid genome based on sequencing from a limited number of individuals. Both versions greatly underestimated human genetic diversity. This new genome, known as the “HuRef” version, represents the first time a true diploid genome from one individual — Dr. Venter — has been published. The research is available in the latest issue of the open-access journal PLoS Biology. Researchers at the JCVI have been sequencing and analyzing this version of Dr. Venter’s genome since 2003. Building on reanalyzed data from Dr. Venter’s genome that constituted 60% of the previously published Celera genome, the team had the goal of constructing a true reference human genome based on one individual. Using whole genome shotgun sequencing and highly accurate long reads from Sanger dideoxy automated DNA sequencing, the team produced additional data making the final 32 million sequences. From the combined data set of more than 20 billion base pairs, the researchers were able to assemble the human genome with an overall length of 2.810 billion base pairs. The genome was covered 7.5 times, ensuring that each set of contributing chromosomes was covered over 3.2 times for greater than 96% coverage of the two parental genomes. The team at JCVI compared and contrasted the new HuRef diploid genome sequence to earlier versions of published human genomes and found that the HuRef version improved upon both these early versions by providing more and correctly oriented base pairs. Since the HuRef genome is diploid, each of the parental chromosomes could be directly compared to each other. One of the most surprising and important findings from this research was the high degree of genetic variation that was found between two chromosomes within a single individual. “Each time we peer into the human genome, we uncover more valuable insight into our intricate biology,” said Dr. Venter. “With this publication, we have shown that human-to-human variation is more than seven-fold greater than earlier estimates, proving that we are in fact very unique individuals at the genetic level.” He added, “It is clear, however, that we are still at the earliest stages of discovery about ourselves, and only with continued sequencing of more individual genomes will we be able to garner a full understanding of how our genes influence our lives.” Within the human genome, there are different kinds of DNA variants. The most studied type is single nucleotide polymorphisms, or SNPs. These have long been thought to be the most prevalent and perhaps the most important type of variant implicated in human traits and disease susceptibility. However, in this analysis of Dr. Venter’s genome, the team found a surprising number of other important variants. A total of 4.1 million variants covering 12.3 million base pairs of DNA were uncovered with more than 1.2 million new variants discovered. Of the 4.1 million variations between chromosome sets, 3.2 million were SNPs, while nearly one million were other kinds of variants, such as insertion/deletions (“indels”), copy number variants, block substitutions, and segmental duplications. While the SNPs outnumbered the non-SNP types of variants, the non-SNP variants involved a larger portion of the genome. This suggests that human-to-human variation is much greater than previously thought. The researchers suggest that much more research needs to be done on these non-SNP variants to better understand their role in individual genomics. According to Sam Levy, Ph.D., lead author and senior scientist at JCVI: “The ability to use unbiased, high throughput sequencing methods, coupled with advance computational analytic methods, enables us to characterize more comprehensively the wide variety of individual genetic variation. This offers us an unprecedented opportunity to study the prevalence and impact of these DNA variants on traits and diseases in human populations.” Another important feature that is made possible by having an individual, diploid genome is the ability to begin to do better and more informed haplotype assemblies. Haplotypes are groups of linked variants. Through the government-sponsored HapMap project, many common haplotypes have been identified; however, these are based on averages of large ethnogeographic populations rather than individuals. Having individual haplotypes would enable researchers to understand and find more rare or individual variants that would explain and help predict diseases in that particular person — a truly personalized, individualized genomics paradigm. In the HuRef analysis, the team used the 4.1 million variant set and new algorithms to build haplotype assemblies that, when compared to the HapMap project, represented longer and more complete linkages. The JCVI researchers expect this number to improve significantly as additional sequence coverage is added to HuRef using a variety of new sequencing technologies. Long-range haplotype linkages will enable much more complete analysis of human variation and the genetic association with complex human traits, behaviours’, and diseases. In the near future, the scientists believe that it will be possible to know from which parent various traits were inherited. Already in this analysis, the JCVI team has found more than 300 disease genes and 4,000 genes overall that exhibit different protein forms. This will be an important area for further study and analysis to determine how these altered proteins affect Dr. Venter’s health status. Reference: Levy S, Sutton G, Ng PC, Feuk L, Halpern AL, et al. (2007) The diploid genome sequence of an individual human. PLoS Biol 5(10): e254. doi:10.1371/journal.pbio.0050254. ......... ZenMaster

---

For more on stem cells and cloning, go to CellNEWS at http://www.geocities.com/giantfideli/index.html

---

Next Step Towards Artificial Life

Whole Genome Transplantation Achieved in Mycobacteria June 28, 2007 Researchers at the J. Craig Venter Institute (JCVI) today 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, published online in the journal Science, by JCVI’s Carole Lartigue, Ph.D. and colleagues, outlines 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. “The successful completion of this research is important because it is one of the key proof of principles in synthetic genomics that will allow us to realize the ultimate goal of creating a synthetic organism,” said J. Craig Venter, Ph.D., president and chairman, JCVI. “We are committed to this research as we believe that synthetic genomics holds great promise in helping to solve issues like climate change and in developing new sources of energy.” Methods and techniques The JCVI team devised several key steps to enable the genome transplantation. First, an antibiotic selectable marker gene was added to the M. mycoides LC chromosome to allow for selection of living cells containing the transplanted chromosome. Then the team purified the DNA or chromosome from M. mycoides LC so that it was free from proteins (called naked DNA). This M. mycoides LC chromosome was then transplanted into the M. capricolum cells. After several rounds of cell division, the recipient M. capricolum chromosome disappeared having been replaced by the donor M. mycoides LC chromosome, and the M. capricolum cells took on all the phenotypic characteristics of M. mycoides LC cells. As a test of the success of the genome transplantation, the team used two methods — 2D gel electrophoresis and protein sequencing, to prove that all the expressed proteins were now the ones coded for by the M. mycoides LC chromosome. Two sets of antibodies that bound specifically to cell surface proteins from each cell were reacted with transplant cells, to demonstrate that the membrane proteins switch to those dictated by the transplanted chromosome not the recipient cell chromosome. The new, transformed organisms show up as bright blue colonies in images of blots probed with M. mycoides LC specific antibody. The group chose to work with these species of mycoplasmas for several reasons — the small genomes of these organisms which make them easier to work with, their lack of cell walls, and the team’s experience and expertise with mycoplasmas. The mycoplasmas used in the transplantation experiment are also relatively fast growing, allowing the team to ascertain success of the transplantation sooner than with other species of mycoplasmas. According to Dr. Lartigue, “While we are excited by the results of our research, we are continuing to perfect and refine our techniques and methods as we move to the next phases and prepare to develop a fully synthetic chromosome.”Genome transplantation is an 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. The ability to transfer the naked DNA isolated from one species into a second microbial species paves the way for next experiments to transplant a fully synthetic bacterial chromosome into a living organism and if successful, “boot up” the new entity. There are many important applications of synthetic genomics research including development of new energy sources and as means to produce pharmaceuticals, chemicals or textiles. Background and Ethical Considerations The work described by Lartigue et al. has its genesis in research begun by Dr. Venter and colleagues in the mid-1990’s after sequencing Mycoplasma genitalium and beginning work on the minimal genome project. This area of research, trying to understand the minimal genetic components necessary to sustain life, 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. 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.Dr. Venter and the team at JCVI continue to be concerned with the societal implications of their work and the field of synthetic genomics generally. As such, the Institute’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 15-month study to explore 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 is set to publish a report in summer 2007 outlining options for the field and its researchers. About the J. Craig Venter Institute The J. Craig Venter Institute 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 500 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/. Genome Transplantation in Bacteria - Science, 29/06/2007 Also read: Venter Attempt a Minimalistic Approach of Creating ‘Artificial’ Life ......... ZenMaster

---

For more on stem cells and cloning, go to CellNEWS at http://www.geocities.com/giantfideli/index.html

---