For the first time, the bacterial gene was created - the largest chemical structure synthesized in a laboratory

The team of 17 people from the J. Craig Venter Institute (JCVI) has just created the largest artificial DNA ever by synthesizing and assembling 582,970 base pairs of a bacterium called Mycoplasma genitalium JCVI-1.0. . Their research was published online January 24, 2008 in the journal Science by Dr. Dan Gibson. This is the second important step towards reaching the group's goal of creating a complete artificial agency. Next, the team will continue with the work of producing a bacterial cell that relies entirely on the artificial genome.

The team achieved the feat by creating DNA fragments based on laboratory chemistry and applying new methods for the synthesis and production of DNA fragments . After years of work based solely on chemical synthesis, the team discovered they could use homologous recombination (the process used by cells to repair damage on chromosomes). ) found in Saccharomyces cerevisiae to quickly create a bacterial chromosome that improves from the assembly process on small sections.

Dr. J. Craig Venter, president and founder of JCVI, said: 'This outstanding technological success can only be achieved thanks to the unique and talented JCVI team. Ham Smith, Clyde Hutchison, Dan Gibson, Gwyn Benders and other team members have devoted their final years to creating and refining new methods and techniques that we believe are That method will be widely used to raise the field of artificial genes to a new level. '

The component that makes up DNA - including adenine (A), guanine (G), cyanide (X) and tiamin (T) - are not easily synthesized chemicals to make chromosomes. The longer the DNA circuit is, the more brittle and fragile it is; Therefore, manipulating it becomes more difficult. Before today's achievement, the longest synthetic DNA segment consisted of only 32,000 base pairs. You can imagine how much of an artificial version of the M. genitalium genome contains over 580,000 challenging and challenging base pairs. However, the JCVI team is also knowledgeable in many technical areas and has a profound biological understanding of some species of Mycoplasma bacteria.

Master Hamilton Smith, the lead author of the research report, said: 'A few years ago when we started our research, we knew that work would be really difficult because we were working. foot into an anonymous territory. But thanks to dedication, we have now proven that genome building is feasible, can be achieved step by step. On that basis, we will develop important applications such as biofuels'.

Hamilton Smith Master (Photo: Jcvi.org)

Methods for creating artificial M. genitalium bacteria

The synthesis and assembly of the M. genitalium artificial version of the chromosome begins with the rearrangement of the original genome sequence of M. genitalium to ensure that the team proceeds from a process. Self-gene does not make mistakes. After obtaining the correct version of the gene, the team created synthesized DNA fragments to build 101 coded gene groups that range from 5000 to 7000 base pairs. To distinguish artificial genes and original genes, the team created watermarks on artificial genes. These are sequences of additional or alternative genes that encode information that does not exist in nature. A number of other changes have been carried out with artificial genes, including gene breaking to prevent contagiousness. The JCVI team teamed up with DNA synthesis company Blue Heron Technology, AND 2.0 and GENEART to create coding pieces.

Next, the researchers set up an assembly process consisting of five steps of concentrating genetic groups together in small assemblies that make up larger segments. Finally, these segments combine to form the complete artificial genome of M. genitalium.

In the first step , each of the four gene groups is combined to create 25 small assemblies each consisting of 24,000 base pairs (also called 24kb segments). The 24kb fragments are cloned in the Escherichia coli bacteria to produce a full DNA fragment for the next steps to confirm the validity of the gene sequence.

In the second step , the researchers combined 3 24kb segments together to form 8 assembly blocks, each consisting of 72,000 base pairs of 1/8 length of complete gene. These gene blocks are cloned in E. coli bacteria for production and DNA sequencing.

In step three , the two 72,000 base pairs are linked together to form a larger segment of 144,000 base pairs (equal to 1/4 of the complete gene length). In this step, the team could not clone half of the genome in E. coli so they experimented with yeast and discovered it accepted large DNA molecules originating from the outside. Therefore, the group can assemble the gene segments together by similar recombinant methods. This process is used to assemble the final genome groups, from a complete 1/4-gen length segment to a final gene consisting of more than 580,000 base pairs. The complete chromosome is again sequenced to confirm that this chemical structure is complete and accurate.

The artificial gene segment of M. genitalium has a molecular weight of 360,110 kilodalton (kDa). If the artificial gene structure of M. genitalium is printed on paper with 10 fonts, it will stretch to 147 pages.

Micro images of Mycoplasma genitalium artificial genome over 1 ~ 0.6 seconds (Photo: Jcvi.org)

Dan Gibson said: 'This is indeed an exciting progress for our team and for this field. However, we continue to study to achieve the basic goal of transplanting artificial chromosomes into cells, proceeding to create the first artificial organism '.

The research of artificial genome M. genitalium JCVI-1.0 was created by Synthetic Genomics, Inc. sponsor.

The basics / milestones in JCVI research's artificial gene research

Gibson presented research based on the research of Dr. Venter and his colleagues since the mid-90s after they arranged the genome of the M. genitalim bacteria and began conducting a number of small projects on genetic engineering. transmission. The field of study explores the essential genetic components needed to sustain life conducted on M. genitalium because this bacterium has the smallest genome that can grow in pure culture . Dr. Venter's research report was published in Science in 1995.

In 2003, Dr. Venter, Smith and Hutchison made the first significant advances in the development of artificial genes when they created a 5,386 base bacterium Φ X174 (non X). They also used short and single synthetic gene segments synthesized, commercial DNA (also known as oligonucleotide) and using polymerase chain reaction (PCR) method, also known as polymeraza assembly cycle. (PCA) aims to produce non-X genes. They have successfully created artificial non-X genes in just 14 days.

Dr. Gwynedd A. Benders (Photo: Jcvi.org)

In June 2007, another step was achieved when JCVI researchers led by Dr. Carole Lartique declared success in gene implants; allowing them to transform this form of bacteria into another form of bacteria by implanted chromosomes. Their success was published in Science, and showed the methods and techniques used to transform from this bacterium (eg Mycoplasma capricolum) to other bacteria (Mycoplasma mycoides Large Colony). for example) by replacing this genome with the genome of the other species.

Genetic transplantation is the first essential step in the field of artificial genes because it is a key technique whereby chemical synthetic chromosomes can work in living cells.The success of the artificial M. genitalium gene today is the second step in guiding experiments to transplant a complete artificial bacterial chromosome into a living organism later, then a whole Artificial cells .

Dr. Dan Gibson (Photo: Jcvi.org)

Ethical issues

From the first days of studying and studying artificial genes, Dr. Venter and his colleagues were very interested in social obstacles around their research. In 1995, while his group was working on bacterial genetics, it was met with criticism of ethics from a group of experts at the University of Pennsylvania (Cho et al, Science December 1999: Vol 286, No. 5447, page 2087 - 2090). Thorough discussions of independent ethical groups have helped to make a unified decision that there is no clear moral reason for Venter's research to stop, but scientists participating in the study must let the public comment on their work.

Dr. Venter and his team at JCVI continue to work with bioethics experts, in addition to political groups, legislators, and the public who always support discussions and knowledge. about the social meaning of research in particular and the field of artificial genes in general. Therefore, JCVI's political team, together with the International Center for Strategic and Research (CSIS) and Massachusetts Institute of Technology, was funded by Alfred P. Sloan Foundation for a 20-month study of the dangers and benefits. of promoting this industry. At the same time, the fund also funded research on measures to prevent technology abuse, including bioterrorism. After a number of public seminars and sessions, the team submitted a report in October 2007 highlighting the right to choose in the field as well as the participating researchers.

Dr. Clyde A. Hutchison (Photo: Jcvi.org)