07 December 2010

Minimal genome and new biology

For the first time, American researchers constructed the complete genome of a bacterium "in vitro" and inserted it into the shell of a bacterium of another species, while obtaining a full-fledged living cell capable of reproduction. Now the next step is to create a viable organism with a minimal set of genes. Two articles in the journal "Science and Life" No. 11-2010 are devoted to this achievement

Synthetic genomics: half a step away from the "element of life" Olga Belokoneva 

What I can't create,
I can't understand it.

Richard Feynman, winner of the Nobel Prize in Physics

Chemists who study natural compounds are usually guided by the following logic in their work: first they find a new substance in nature, then determine its functions and structure, and finally they try to synthesize this compound in the laboratory in order to compare the properties of a natural compound and its synthetic analogue. This is the only way to prove that a substance of a given chemical structure has certain properties. But in genetic manipulation, this approach did not work for a long time – the structure of DNA was already known, but no one could solve the inverse problem.

A business that creates science

An American veteran of the Vietnam campaign, Craig Venter, was engaged in biochemistry, received a degree, but did not stay long in the laboratory walls. The young researcher was attracted by business. In 1998, he took part in the creation of the biotech company Celera Genomics. At the time of the company's creation, work was already underway to decode the genome of living beings, including humans. But progress was not great due to the imperfection of DNA sequencing technology (determination of the nucleotide sequence). As part of the research team, Venter took part in the development of the newest sequencing method – the shotgun method. Using this method, the human genome was completely decoded after two years. Venter wanted to sell the results of the company's research, but the scientific community expressed dissatisfaction, and he had to give in. He posted all the results of the genome decoding on the Internet and left Celera Genomics, creating a new institute named after himself.

One of the pioneering initiatives of the Craig Venter Institute in the 2000s was the so-called metagenomic projects. Expeditions organized by the Institute carried out a population analysis of the genome of various organisms living in the Sargasso and other seas. Using genomic technologies, the researchers managed to describe the genetic diversity of the underwater kingdom, while discovering thousands of new genes and new species of living creatures.

Now that the chemical structure of many complex genomes was known, logically, it was necessary to synthesize an artificial genome, which Venter did. Venter's other idea was to create a viable organism with a minimal set of genes. Such a genetic unit could well be called an "element of life" – a "minimal" cell. By analogy, in chemistry, the simplest unit is the hydrogen atom.

There is no "minimal" cell yet, and an organism with a synthetic genome already lives and reproduces in the laboratory of the Craig Venter Institute. This is an ordinary bacterium, which differs from others only in that its DNA is synthesized "in vitro".

An international team of researchers who created synthetic life

From the beginning of the work to the historical publication in May 2010 in the journal "Science" entitled "The creation of a bacterial cell that is controlled by a chemically synthesized genome" took a long 15 years, and the project cost $ 40 million. This major scientific achievement was preceded by another success – in 2003, Venter's team managed to create a virus with an artificial genome.

An international team of successful researchers from two departments of the Institute – in Rockville (Maryland) and in La Jolla (California) – in addition to Venter (pictured on the left) are led by two other outstanding scientists. One of them is the 1978 Nobel laureate Hamilton Smith (right). He received the Nobel Prize for the discovery that marked the beginning of the era of chemical manipulation of the genome: he isolated restrictases – enzymes that cut the DNA molecule into separate fragments. Another leader of the work is an outstanding microbiologist, a representative of the famous scientific dynasty Clyde Hutchison III.

Synthetic DNA, consisting of 1.08 million nucleotides, has become the longest molecule ever synthesized in the laboratory. The first synthetic cell in history contains a completely artificial chromosome synthesized from chemical components using a computer program. These are no longer genetic engineering technologies, when scientists managed to change or supplement the genome of living beings with several genes or a set of genes, this is a complete transplant of the entire genome.

Genome transplantation

The experiment to create artificial life was as follows: scientists synthesized the genome of one bacterium and introduced it into the cell of a bacterium of another species. The resulting organism with the shell of the recipient bacterium Mycoplasma capricolum turned out to be identical to the donor bacterium Mycoplasma mycoides. So for the first time it was reliably shown that DNA really contains complete information about the work of the entire living cell.

The resulting hybrids looked, grew and multiplied in the same way as Mycoplasma mycoides. Another important sign that it was Mycoplasma mycoides – the engineered bacterium synthesized proteins peculiar to this particular species. However, the synthetic bacterium is still different from the natural one. It can live and reproduce so far only in the laboratory, in a special nutrient medium, in natural conditions the bacterium is not viable.

Mycoplasmas are a fairly extensive (about 180 species) group of parasitic bacteria that cause all kinds of diseases in plants, animals and humans. They have a number of properties that make them a convenient object for such studies. Unlike the vast majority of other bacteria with small genomes, mycoplasmas can live outside the host cells, so they can be grown in the laboratory. True, mycoplasmas constantly need intensive nutrition, since they lack the genes necessary for the synthesis of many vital substances. Finally, mycoplasma cells do not have a nucleus, their genetic material is distributed in the cytoplasm. They are surrounded only by a thin and elastic plasma membrane, through which it is quite easy to introduce components of an alien genome.

The parasite bacterium Mycoplasma mycoides was chosen as a donor primarily because it has a very small genome – about a million nucleotides (for comparison, there are 3 billion of them in the human genome). But it is not easy to get such a "short" genome, so DNA was synthesized in parts, which were then joined together. The molecular constructor was assembled in E.coli cells, and then in yeast cells. And only after that, synthetic DNA was injected into the Mycoplasma capricolum cell.

It is often asked why it was impossible to put an artificial genome inside one's own cell? Because the proteins characteristic of it remained inside this cell, which means that the results of the experiment could be explained by their presence. That is, there would be uncertainty in the interpretation of the result.

Why do we need synthetic bacteria?

The reaction to the research in the scientific community is ambiguous. Many believe that it is premature to talk about the practical application of technology: it is one thing to program nuclear–free prokaryotic bacteria, and quite another to create artificial chromosomes of nuclear eukaryotic cells, that is, cells of all plants, animals and humans. When adapting technology to nuclear cells, too many questions arise: how to transfer DNA to the nucleus, how to create and transplant non-nuclear genetic information, etc.

Nevertheless, Venter believes that the research carried out is important for fundamental science, since it opens up new perspectives in the study of the origin of life and the search for an answer to the question of which genes are responsible for the life and reproduction of a living being.

Venter's work promises the prospects of creating organisms with fully specified properties and functions. However, this is a matter of the rather distant future. So far, scientists have managed to "only" implement a genetic program that already exists in nature. But still, the prospects for synthetic genomics are huge. After all, it is so tempting – changing the genetic program at your discretion, to create synthetic bacteria-factories capable of producing medicines, nutritious protein substances, biofuels, purifying water from pollutants and much, much more.

After the successful creation of the first artificial organism, Venter's team, and not only her, concentrated their efforts on implementing another project that logically follows from this achievement. We are talking about creating a cell containing only the genes necessary to maintain life in its simplest form, that is, the "minimal" genome.

The element of life

The definition of a "minimal" genome that provides all the necessary functions that allow a single–celled organism to exist in a certain environment is not an idle question. Solving this problem is necessary to understand the origin of life on Earth, which includes studying the ways of genetic evolution and the mechanism of the origin of genomes as such. In addition, the "minimal" cell will become the basis for the study of all the genes necessary for life.

Work in this direction is carried out mainly with bacteria of the genus Mycoplasma. The genomes of mycoplasmas, as already mentioned, are very small (from 580 to 1400 thousand base pairs) and are well studied. Mycoplasma genitalium has the shortest genome. Its length is about 580 thousand base pairs, which make up 485 genes.

The proposed hypothetical minimum set of genes (according to the latest calculations of the Venter group – from 310 to 388 genes) should include the following vital genetic systems of microorganisms, including: genes for translation, replication, repair, transcription; genes controlling anaerobic metabolism; genes for lipid biosynthesis; genes for protein transport systems; a set of genes that ensure the transport of metabolites; a complete set of nucleotide utilization genes and their biosynthesis genes. Parasitic microorganisms do not need amino acid biosynthesis genes.

By studying the genomes of mycoplasmas, Craig Venter and his colleagues came very close to understanding what the "minimal" genome of future artificial microbes should be. As stated in the patent they have already filed, the "minimal" genome – the main building block or, more precisely, the main "chassis" for creating artificial organisms – consists of less than 400 genes. By introducing a "minimal" genome into a cell and adding other genes to it, the researchers intend to create the simplest organisms with new, predetermined properties.

The "minimal" cell and the paradigm shift in biology D.B.N. Vadim Govorun.
Recorded by Olga Belokoneva.

In June 2010, the 5th International Conference "Genomics, Proteomics, Bioinformatics and Nanobiotechnology for Medicine" was held in St. Petersburg. Well-known Russian scientist Vadim Govorun (left) and Clyde Hutchison III (right), one of the leaders of the synthetic biology group of the Craig Venter Institute, delivered the main plenary reports at the conference. Hutchison believes that all biochemical and molecular processes in a living cell can be described in terms of the laws of physics and chemistry. The scientist is also confident in the possibility of creating a universal "minimal element of life", similar to a hydrogen atom. Talker's point of view differs from Hutchison's.

In a personal conversation with a correspondent of "Science and Life", arguing about the possibility of creating a "minimal" cell, he operates with almost philosophical, ideological concepts.

Brief reference
Govorun Vadim Markovich, Doctor of Biological Sciences, Professor, President of Litech, Deputy Director for Science and Head of the Department of Molecular Biology and Genetics of the Research Institute of Physico-Chemical Medicine of the FMBA of Russia (Moscow). Graduated from the Medical and Biological Faculty of RSMU.
One of the areas of scientific activity of the department, headed by Vadim Govorun, is the development of a platform for obtaining a complete "protein portrait" (proteome) of microorganisms with the smallest genome (mycoplasma, chlamydia and helicobacter), as well as determining the minimum set of genes sufficient for the vital activity of these bacteria. (For more information about Litech and its president, see the article "Vadim Govorun: "My task is to keep the fire going" – VM.)

What is a "minimal" genome is a rather complicated question, because everyone understands their own by its decoding. Until 2002, genome sequencing was understood as a kind of coherent system of efforts and methods that allowed, if not completely, then 80-90% (in the case of the human genome) to obtain fused extended sections of DNA. But the nucleotide sequence is not an alphabet, it's a book. And if you don't read well, then even after reading to the end, you may not understand the meaning of the book.

In genomics, the same thing happens, but only more complicated. Everyone who can read perceives meaningful words. Initially, there are small fragments in the genome, there are "common words", there are even "turns of speech", but this does not mean that they are all meaningful. In fact, the genome is a multidimensional structure...

By "minimal" cell we mean the following. We take some large fragments of genetic material, sometimes even from different sources, close or not very close, and look at how this construct behaves...

It is not evolution that leads to life, but microevolution. Someday intracellular nanorobots will appear, but they will not work with great accuracy. First, they will create some kind of prototype, an information form, and this prototype will be given to approach the necessary characteristics by its own microevolution.

Self–assembly is a property of atoms and molecules. They are capable of self-assembly, self-recognition. Therefore, when we approach the modeling of life, we come to such an interesting question: will life arise immediately or as a result of microevolution? The answer is ambiguous...

The minimal concept is, in fact, an attempt to reach a new level of studying life. As for our knowledge of living matter, we still have a black box. The behavior of a living system is not additive – it is not the result of a simple addition of the action of its parts.

The race in this area has just begun. When scientists begin to manipulate fragments of life safely for themselves and others, there will be a breakthrough. The last 25 years in biology have been stagnating, scientific thought proceeds through the accumulation, identification and analysis of data. There is no paradigm shift. Now, for the first time in the history of mankind, there is an opportunity to embody their ideas about a living cell. Computer modeling is what appears in the construction of life...

Humanity has been going for centuries to prove that life is monovariant, that is, only one combination of genes and proteins breathes life into the cell. In my opinion, life is invariant. The hardest thing is to understand that life arises in different ways, outside of certain chemical reactions...

In fact, modern biology as a science, where a lot of different methods have come, is multidisciplinary, and, consequently, biological thinking is such a set of "noise effects" that it is very difficult to choose the right direction. It just seems that with the help of repeating experiments, statistics, you can calculate something in the science of life. The minimal concept is really a paradigm shift, the thinking of people who are engaged in biology, but in a sense, paradoxically, it is a return to the old biological traditions.

The living is not a synthetic complex consisting of protein molecules. And what it really is, scientists want to find out.

For example, the virus is not alive. It is small and capable of self-assembly. But you will never be able to merge ribosomal proteins, DNA, enzymes, lipids, etc. in one test tube and assemble a bacterium from them, even if it is very small. Therefore, a living cell must be collected in blocks. The study of blocks is not an end in itself, the goal is to create blocks at will. And, gradually understanding how these blocks will act, find methods of their assembly. That's when it will be really artificial life.

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