Synthetic biology in Siberian
The gene factory is a useful thing!
Yu. Alexandrova, "Science in Siberia" No. 15-2012
At one of the March meetings of the Presidium of the SB RAS, the head of the Laboratory of Medical Chemistry of the Institute of Chemical Biology and Fundamental Medicine, Ph.D. A. N. SINYAKOV, presented a scientific report on the topic "Technological platform for synthetic biology". They talked about the opportunities that gene synthesis gives scientists, about biomedical research and the development of biotechnologies, as well as about the work being done at the IHBFM to create a microchip DNA synthesizer. This is our conversation today.
– Alexander Nikolaevich, the first question of an ignorant person is why all this is necessary?
– The installations we work with are needed, first of all, for genetic engineering or molecular biology. Namely – for diagnosis, detection of pathologies, mutations, for the determination of pathogens. Currently, a new interdisciplinary science is being formed, which is based on the creation of artificial living systems using artificial genes – synthetic biology. Its development is carried out on the basis of a revolutionary breakthrough in the field of gene synthesis associated with the development of microchip reactors that will synthesize up to several hundred thousand oligonucleotides simultaneously (it is from them that fragments of genomes are collected).
– Then in a little more detail – what is the synthesis of genes, what is the technology, how does the work work? In a word, it is popular about the actual scientific research.
– If you walk through our laboratories, you will see several automatic DNA synthesizers. In small columns filled with porous silicon oxide, this computer–controlled device synthesizes relatively short, several dozen bases, fragments of DNA - oligonucleotides, which are, so to speak, "bricks" for research in molecular biology.
We make a lot of such oligonucleotides, which are used to create more complex products, in particular, for diagnostics. For example, we produce diagnostic chips – on a small glass "cushion" we place DNA fragments, the so-called oligonucleotide probes, with which we type pathogens.
Work on the synthesis of artificial DNA is carried out on three synthesizers, which in total can give a maximum of 200-300 oligonucleotides of different composition per day. And science is developing rapidly now, and the needs for oligonucleotides of various compositions are increasing dramatically (by the way, the capabilities of researchers are also increasing). And if a modern device synthesizes them in the amount of not tens, but hundreds of thousands, if scientists have a lot of cheap and affordable oligonucleotides, then it will be possible to move on to artificial synthesis of living things, namely, genomes of various organisms. This is exactly the task that researchers are facing today.
– And what has already been done? What is the situation with the synthesis of genomes in the world?
– The simplest genomes have already been synthesized. The first was poliovirus – it was synthesized by Eckard Wimmer and his collaborators in 2002. The genome of the poliovirus is ~7,500 nucleotides. Then a phage parasitizing E.coli was synthesized. And the pinnacle of the synthesis of artificial genomes is the work of Greg Venter, who synthesized the genome of an artificial bacterium: he introduced an artificial genome of one type of bacterium into the shell of another. As a result of the functioning of the artificial genome, the bacterium changed the type of its original shell and became indistinguishable from bacteria with a similar genome. To implement this project, it was necessary to synthesize more than one million pairs of nucleotides. This is an extremely difficult and time-consuming job.
We are trying to create automata – devices that will help researchers design artificial genomes. However, at the first stage, it is not necessary to strive for the synthesis of very complex genomes – it is better to act step by step, for example, to create a factory of genes useful for people. So, a number of important proteins, for example, interferon, which is a non-specific antiviral agent, can be forced to produce special bacteria, in whose genome an artificial gene is embedded. This method is much easier than isolating interferon from donated blood. The spectrum of target genes is very wide – these are pathogen antigens needed for diagnosis, genes encoding mediators of cellular immunity, and genetic constructs necessary for the construction of live vaccines. In general, the gene factory is a very useful thing. So for now we would like to stop at the first stage – the construction of many useful genes.
In order to demonstrate clearly everything he was talking about, Alexander Nikolaevich approaches the laboratory table and returns, holding a Petri dish with some fragments, pieces, plates (and on them - lines, geometric shapes – only a specialist can determine), and continues...
– This is a silicon chip of a microchip DNA synthesizer. Theoretically, 20 thousand oligonucleotides of different compositions can be synthesized on its surface. We are training on this chip, the cells of which you can still see with your eye – there are less than two hundred of them here so far. You need to learn how to deposit silicon oxide into these cells, then "bind" the first link of the future oligonucleotide, and then synthesize oligonucleotides of different composition separately in each cell.
But after the synthesis of oligonucleotides, nothing ends in our business yet. If you want to synthesize something, for example, a fragment of the genome or the genome of a living microorganism itself, you need to learn how to properly stitch these oligonucleotides into the target sequence. The fact is that during synthesis, a certain number of errors inevitably arise, and they will need to be corrected with special enzymes. As soon as you start doing something real, there are a million problems that need to be solved.
– It looks like such jewelry work – it's scary to think. But you probably already have your hand full. How are you coping at all?
– Of course, we are doing this not by our own team, but by joint efforts within the framework of integration projects. Considering the importance of the development of synthetic biology, a consortium of several institutes (IAiE, IFP, IHBFM, NIOH and ITPM) has been organized in the Siberian Branch of the Russian Academy of Sciences to create a microchip synthesizer of oligonucleotides. I am now the coordinator of this project. The work is interdisciplinary in nature, includes the development of chemical reagents, photochemical methods for controlling the reaction of the oligonucleotide chain buildup, the manufacture of a microchip reactor, the development of methods for gene synthesis.
For the successful implementation of the project, physicists need knowledge on the deposition of silicon oxide into the reaction cells of a microchip, photolithography technology, for the formation of supply and discharge channels of microchip reagents – quartz glass and silicon splicing technology, in short, many different large and small tasks. They sometimes do not shock the imagination, but if you make a mistake in at least one, you will not succeed in anything.
Silicon chips themselves are made at the Institute of Semiconductor Physics using the photolithography method. Silicon oxide is also deposited in the cell from the gas phase, channels are treated with plasma so that parallel side synthesis of oligonucleotides does not occur in them. And what is in front of you is a blank, the initial chip for synthesis. To carry out reactions on it, you need to know the chemistry of oligonucleotide synthesis: this is well worked out in the IHBFM, and we transfer almost all the technology here. With one exception.
In our traditional oligonucleotide synthesis machines, acid is added to the reaction columns at a certain stage and the temporary protective group of the growing oligonucleotide is unblocked. The dimensions of the chip cell for the synthesis of oligonucleotides are very small, and in this case we cannot fully use the available technology of traditional synthesizers, so we use photogenerated acids to unlock the protective group during microchip synthesis. Here they are made for us at the Novosibirsk Institute of Organic Chemistry. And not just any one - it is necessary to find the most suitable in terms of parameters. NIOH SB RAS synthesizes a number of photoacid generators that form acids either under the influence of far ultraviolet or near visible light.
The next part is the layout itself, a complex technological device. In each cell of the microchip, its own oligonucleotide of a pre-selected composition is synthesized, for which, at certain moments of synthesis, light rays must be directed to certain cells of the microchip to unlock the protective groups. For these purposes, there are controlled micro-mirrors inside our layout, there are over 800 thousand of them in total. They are very small, about 12 microns, and should illuminate a strictly defined area of the microchip. This is a very delicate work, and it is carried out by the fourth participant of the project – the Institute of Automation and Electrometry.
The work on optimizing the hydrodynamics of the microchip was performed by the fifth participant of our project – ITPM SB RAS.
As a result of the operation of the microchip synthesizer, we get a read number of target oligonucleotide molecules (100-1000 pieces). To get the right gene design, we have such an excellent technology called polymerase chain reaction (PCR). With its help, we can multiply a small amount of the target product many times. PCR also makes it possible to manipulate the material, i.e. to make a variety of designs, embed them in various vectors, to obtain hybrid bacteria, viruses, producers of, say, E. coli. In fact, what is the difference between chemistry and biology – in chemistry, a substance can only be consumed, and in biology, it can also be multiplied. So, by making a fragment of the genome, we can "clone" it, if we don't lose it.
– How long have you been working in the field of microchip gene synthesis?
– We started synthesizing artificial genes 30 years ago, in 2002 we became interested in biological microchips for diagnostics, and about three years ago we received the first integration project to create a microchip DNA synthesizer and started the basics of what the country has not yet had – a new science of synthetic biology.
Now microchip synthesizers exist only in the West, where they also appeared not so long ago. Since such technology opens up the possibility of implementing fundamentally new approaches to the creation of biological weapons, valuable products for industry, medicine and agriculture, restrictions on the export of modern oligonucleotide synthesizers to our country should undoubtedly be expected.
Practical actions to limit the spread of this technology are already being taken. In December 2006, a number of large international companies and research centers in the United States adopted the document "Practical prospects for DNA synthesis and biological hazard", which proposes a practical plan for careful control of chemical DNA synthesis in synthetic biology companies. Companies engaged in DNA synthesis and working in the field of synthetic biology are in close cooperation with government agencies and transmit information about potentially dangerous orders to them.
Under these conditions, it is difficult to expect the preservation of trade secrets and it is impossible to conduct closed research of a defense nature. This concept of work has been approved by the FBI and a number of US government agencies. There are only a few firms in America, England and Germany that accept orders for the manufacture of mixtures of oligonucleotides obtained on microchip synthesizers, and these orders are carefully analyzed using computers. In addition, the customer must explain why he needs these oligonucleotides.
– Does this approach not slow down science?
– Rather than even science, but the technological development of the country. It turns out that some countries can use highly productive equipment that allows them to get some products cheaply and quickly, while others do not have access to this. After all, under such conditions, you will never be able to protect your trade secrets, conduct closed research without the other party knowing what you want to do. In two weeks, high-performance computers will analyze the composition of your order, comparing it with known microorganisms, and then give a conclusion about the true goals of the study.
Well, if we return to our project... The task we are currently working on is very difficult: there is not a single problem that could be solved right away. It is necessary to master modern versions of photolithography, to learn micromechanics, which in the West has already gone far ahead, it is necessary to refine chemistry. Now, since the layout already exists, we would like to fill it with content, "teach" it to work efficiently and correctly, move from the stage of large microchip cells to relatively small ones. This will significantly increase the number of oligonucleotides synthesized on the chip.
– And yet what is more in your works – fundamental or applied science? What will happen then?
– We create tools for fundamental science, in particular, for modern biology, but we do not forget about the applied aspects. You see this glass – it's transparent, slightly yellow; we print chips on such glass substrates. This chip contains 64 dots and can in most cases detect genetically modified foods in your food. We have such a project – the analysis of genetically modified products, which is funded by Ukraine. When we learn how to synthesize not tens, hundreds, but up to twenty thousand oligonucleotides on a chip, we will get a powerful device for diagnostic purposes. In this case, the analyzed sample can be placed directly into a microchip and its composition can be analyzed immediately. If we correctly calculate the composition of the typing probes, the resulting microchip will detect hereditary diseases, genetically modified products, or type any diseases.
Such a chip can serve for two directions – either diagnostic, for the detection of diseases, or for the synthesis of mixtures of oligonucleotides and the construction of genome fragments. Thus, we are trying to "teach" our microchip synthesizer to produce sets of a large number of oligonucleotides for the needs of synthetic biology, as well as to use microchips with non-cleaved oligonucleotides directly for the diagnosis of diseases and pathologies.
We are also trying to adjust the consumption of chips that we will synthesize – here, too, not everything is simple. Unfortunately, in our country, if you do not implement what you have invented, invented, created, no one will do it for you. Now, at the suggestion of Academician V. V. Vlasov, we are preparing a conference on synthetic biology, we are going to invite scientists who know how to make genes from microchip oligonucleotides – in order to establish connections, save time and money for research. And all this is a natural way of developing research.
Portal "Eternal youth" http://vechnayamolodost.ru12.04.2012