08 October 2015

Synthetic life

In this they resemble engineers, and their work is the result of many millions of years of evolution. Synthetic biology is an area where the science that studies living things becomes the science that creates it, trying not only to understand the fundamental principles of the organization and operation of living systems, but also to solve applied problems – from the treatment of diseases to biotechnologies of the future.

The behavior of a cell – irritation, division, growth, differentiation – is subject to chemical reactions, the components of which – proteins – are encoded in its genome. The genome* is the totality of the entire DNA of a cell, while resembling an "instruction" or "program", which contains all the hereditary information in the form of a sequence of four symbols (bases) – A, T, G and C – necessary for the construction and maintenance of life. 

* – Read about the large-scale project "Human Genome", its results and prospects of the post-genomic era: "Human Genome: how it was and how it will be" [1]. – Ed.

To execute commands, this code must be interpreted (read), and information from the sequence of bases is implemented (via RNA and proteins) in a set of chemical reactions in the cytoplasm (Fig. 1). 

Figure 1. Schematic representation of the cell. The DNA of a cell is a "program code" that contains all the information necessary for life, and the cytoplasm (the internal contents of the cell) is a "way of implementing" the code (an environment that interprets information encrypted in DNA). Green dots represent ribosomes; red indicates the channels through which the exchange with the environment takes place. The scale is not observed. Drawing of the author of the article.The latter ultimately determine all the properties of living things, and this is true for all organisms – from bacteria and archaea to higher eukaryotes, which emphasizes the fundamental nature of such a mechanism.

Over many millions of years of evolution, organisms acquired (and still do) various genes that helped them better adapt to conditions in their niche. Therefore, different living beings have different genomes that are adapted to the appropriate living conditions, although some elements are similar (conservative) in the most phylogenetically distant organisms.

To "read" the information encoded in DNA, scientists resort to various sequencing methods*, reading each character (base) and eventually obtaining a sequence (on a computer) similar to the genome of a cell (Fig. 2).

* – New methods of reading DNA are described in the articles "454-sequencing (high–performance DNA pyrosequencing)" [2], "Sequencing of single cells (version - Metazoa)" [3] and "A method for analyzing gene expression at the level of individual cells has been developed" [4], and about the problems associated with increasing the pace of sequencing of complete human genomes (including in Russia), – in the review "The Code of life: to read does not mean to understand" [5]. – Ed.

Figure 2. An example of displaying a "read" DNA sequence. The letters mean nitrogenous bases (a – adenine, t – thymine, g – guanine, c – cytosine). A fragment of the human hemoglobin gene sequence was taken from GenBank (number DQ659148.1).The concept of synthetic biology

Synthetic biology as a science appeared at the beginning of this century [6], when artificial simple gene-regulatory systems were obtained.

So, in 2000, a genetic trigger was constructed that switches between two stable states in the E. coli bacterium under the action of thermal or chemical stimuli (Fig. 3) [7]. It resembles an electrical circuit with several switches (genes and proteins) and an output signal (a "light bulb"), which can be a change in the state of the cell – for example, the synthesis of a fluorescent protein*. The successful experiment marked new opportunities in molecular and cellular biology and became one of the first steps in the creation of "artificial" life.

* – Articles about the diversity of fluorescent proteins and their use in biological research are: "The Fluorescent Nobel Prize in Chemistry" [8], "Fluorescent proteins: more diverse than you thought!" [9], "Let's draw a "living cell" [10]. – Ed.

Figure 3. Switch of two genes and a reporter gene. Both genes (red and blue) mutually repress each other (blunt arrow), and when the expression of one is suppressed, the second gene is activated. Chemical agent and heat shock (heating) are used as expression inactivators in this model. The second gene activates the third (green), expressing a fluorescent protein, the glow of which can be registered. Drawing by the author of the article.Currently, synthetic biology is understood as the creation of new biological constructs and systems, as well as the modification of natural living systems in order to obtain an organism with the desired qualities [11]; mathematical models serve as a support for this (for example, the Jacob–Mono trigger).

The first implies the synthesis of the genome de novo, the second – purposeful modification of an existing one (introduction or removal of its elements).

Minimal genomeThe minimal genome is an important concept in the context of synthetic biology.

It represents the smallest number of genetic elements that are necessary for the existence of a free-living cellular organism [12]. This concept is closely related to species specificity and external factors (unlimited resources, optimal physical conditions, lack of competition must be provided) and depends on the ultimate goal [13]; without taking this into account, such a concept loses its value due to the inability to support life (at least of the modern type). The prerequisite for this was the search for the minimum required number of genes [14], which is sufficient for the cell (under the above conditions). To do this, the shortest genome among free–living organisms was compared – Mycoplasma genitalium (0.58 million bp (million pairs of nucleotides), about 524 genes) - with the genome of Haemophilus influenza (1.83 million bp, about 1788 genes), after which 256 candidate genes were found sufficient to support modern-type life. In addition to the predictive model, experimental approaches were used to search for the necessary genes (under ideal conditions): transposon mutagenesis, sequential shutdown of genes and suppression of gene expression by antisense RNAs.

The first approach is based on the transformation (transfection) of cells by a vector containing a transposon (mobile, that is, an element capable of moving in the genome) and a selective marker (for example, a marker of antibiotic resistance), according to which it will be possible to select bacterial colonies into which the vector has fallen. The transposon is embedded in the genes of the host cell, "tearing" and inactivating them (Fig. 4). Figure 4. Schematic representation of transposon mutagenesis.

Drawing by the author of the article.If the insertion occurred in a vital gene (genes), then the cell dies, leaving no descendants, and the colony is not formed; at the same time, the cell is tolerant to inserts in non-vital genes (the main principle: if the gene can be "torn", then the cell can do without it under these conditions).

Further selection on a selective medium and the study of the geno-/phenotype make it possible to identify inactivated genes. However, some necessary genes may be tolerant to transposition, and a decrease in the growth rate caused by some mutated "secondary" genes may form a false idea of their significance – the experimenter risks mistaking one for the other [15].

Directed synthesis of antisense RNAs (RNAs that complementarily bind to the mRNA of the target gene, forming a double-stranded untranslated complex) in cells suppresses gene expression at the post-transcriptional level, which is used for its targeted inactivation (Fig. 5) [16]. 

Figure 5. The mechanism of blocking the translation of antisense RNA. Forming a double-stranded complex (on the principle of complementarity) with matrix RNA, antisense RNA prevents translation (synthesis of the protein product of the gene). Drawing from the website www.scq.ubc.ca .The problem is that it is not always possible to achieve adequate expression of antisense RNA, and this imposes its limitations on the use of such a method.

Based on the above (and using some other methods), a later study [17] characterized 206 genes necessary to maintain the life of a cell with a minimum genome size. The genetic apparatus of such a cell encodes an almost complete set of proteins responsible for replication (replicome proteins, auxiliary proteins), transcription (polymerases, etc.) and a less complete complex of translation proteins (ribosome subunits, translation factors), as well as highly reduced DNA repair and transcription regulation systems. There are no pathways of amino acid biosynthesis in this cell, the biosynthesis of lipids and carbohydrates is reduced, and substrate phosphorylation acts as an energy source – a primitive way of synthesizing energy–rich compounds (Fig. 6, for more details, see [17]).Figure 6. Metabolism of a cell with a minimal genome.

The white boxes contain the classification numbers of enzymes and the names of the genes encoding them. In green – intermediate and final products of metabolism, and in pink – sources of chemical energy. Figure from [17].The importance of the minimal genome model lies in the ability to better understand the fundamental principles of life, guided by a simpler system; the initial simplicity also helps to better control such a system when adding new, complicating functional constructs.

Having sets of vital genes, it is possible to build more complex modules for subsequent systems (synthetic genomes), as well as predict the "vulnerabilities" of cells that can be affected for their own purposes (for example, targeted production of new antibiotics against pathogenic organisms).

Synthesized lifeOne of the triumphs in synthetic biology was the de novo synthesis of the Mycoplasma genitalium genome [18, 19] (Fig. 7). 

Figure 7. General scheme of de novo organism synthesis. Drawing from the website www.jcvi.org .A bacterium with a synthesized genome with a length of more than 580 t.p.n. (more precisely, 582970 bp.n.) was named Mycoplasma genitalium JCVI–syn1.0 (on the right is an electronic micrograph of these cells from the site www.jcvi.org ) in honor of the institute where the work was carried out (J. Craig Venter Institute). 

The use of bacteria as a model system is convenient because of the small size of the genome, the rapid growth of bacterial colonies and the ease of transplantation of the synthesized chromosome into the recipient cell. 

Initially, the entire genome was modeled on a computer, where in intergenic regions (DNA sequences between genes) tolerant to transposition, DNA fragments were hierarchically assembled into larger and larger parts. 

At the first stage, sections of DNA ("cassettes", 101 pieces) were collected 5-7 t.n. long with overlapping sequences to connect with each other (the last cassette completely overlaps with the first one to close into a ring – a typical form of DNA existence in bacterial cells) (Fig. 8). 

Figure 8. The scheme of the M. genitalium JCVI-syn1.0 genome assembly. The figure of the author of the article.Then, by in vitro recombination methods (a complex of genetic engineering methods based on in vitro manipulations leading to the formation of new hybrid DNA molecules), such cassettes were assembled four by four and as part of a BAC vector (bacterial artificial chromosome, one of the forms of transmission of large DNA fragments into the cell, in our case - 10-20 t.p.n.) were transferred to E. coli for their further development.

(The fact is that without the stage of multiple "reproduction" in bacteria, it is impossible to obtain normal copies of fragments "in vitro" for a number of reasons.)

Sequences were assembled from such modules in several stages up to inserts of 1/4 of the M. genitalium genome, after which difficulties arose with assembling large-sized DNA fragments. This problem was solved by using a TAR vector (a special vector for cloning into yeast cells, an "artificial chromosome") and copying constructs in S. cerevisiae yeast. The selection of the vector with the insertion of the synthesized genome and the subsequent annotation of the latter showed the identity of the computer-modeled and obtained sequences.

After that, the Mycoplasma mycoides* gene was synthesized in a similar way (M. mycoides JCVI- syn1.0, Fig. 9) [20], but, unlike the previous experiment, it was successfully transplanted into a recipient cell of M. capricolum to produce a viable bacterium. 

* – This is described in detail in the articles "The first living being with a synthetic genome was created" [21] and "Life in the era of synthetic life" [22]. – Ed.

Figure 9. M. mycoides JCVI-syn1.0 genome assembly scheme. Figure from [20].Despite the natural origin of the plasma membrane and cytoplasm, such bacteria showed phenotypic signs of M. mycoides cells.

This experiment shows the functional relevance of the artificial chromosome and the process of transition from the "program" to its "implementation".

Currently, "synthetic" experimenters are working to minimize the size of the M. mycoides JCVI-syn1.0 genome, guided by the approaches described earlier ("switching off" genes in the whole genome or predicting vital elements and then de novo synthesis). Perhaps soon the number of "synthetic" bacteria will increase by one more, but this time it will be a non–naturally occurring organism - a simplified version of its natural counterpart.

The "programming language" of lifeCreating a controlled and debugged system requires strict typing of actions and a clear set of rules and instructions; moreover, the presence of a formalized language significantly increases the reproducibility of results.

So, in 2011, a "language" appeared in which it is possible to "communicate" in the context of synthetic biology – SBOL (Synthetic Biology Open Language) [23], somewhat resembling a programming language. It is a set of standard components (building blocks, building blocks) that reflect various genetic elements (Fig. 10). 

Figure 10. Graphic elements of SBOL. Drawing from the website www.sbolstandard.org .Their combination makes up genetic constructs with certain functions stored in the form of a library, so that you can use ready-made templates (or constructs) or refine them for your needs due to the hierarchy of the language.

Thus, from specifying the sequence of nucleotides in the genome, we move to a higher level of assembly from a complex of sequences associated with functional significance.

Stumbling blocksDue to their simplicity, synthetic organisms are very demanding of growth conditions, losing flexibility to their natural counterparts.

In addition, significantly slower growth causes inconvenience during their study and practical use (there is also a reverse effect [24]). The need for large resources to obtain and study synthetic organisms also provokes certain difficulties. However, such systems provide a detailed understanding of the interaction of genes and much more control when adding different biochemical pathways. Unfortunately, so far all the studies have been carried out on bacteria (due to the complexity of eukaryotic organisms: intricate epigenetic regulation*, the large length of the genome and the peculiarities of its organization), although now a project is being implemented jointly by many scientists to synthesize the genome of yeast Saccharomyces cerevisiae (Synthetic Yeast 2.0) [25]. 

* – This term refers to the effects on gene expression that do not affect the sequence of DNA nucleotides. Epigenetic modifications of chromatin simply determine how hereditary information should be "read" – which genes should be silent and which ones should work: "Development and epigenetics, or the story of the minotaur" [26], "Histone rolls, rolls to DNA" [27]. These modifications are sometimes no less significant for human health than mutations that violate the primary structure of DNA: "Epigenetic clock: how old is your methylome?" [28], "Epigenetics of behavior: how does grandma's experience affect your genes?" [29], "Pills for the epigenome" [30]. – Ed.

If the goal is achieved, it will definitely become a new breakthrough in the art of creating synthetic life – there will be a transition from prokaryotic organisms to eukaryotic, and in the future, perhaps, to multicellular creatures.

Applied valueHaving obtained a model system with pre-known properties, it can be improved depending on the goals that the researcher faces.

For example, adding sets of genes responsible for the desired biochemical pathways, including combining them so as to obtain new substances not found in nature [31]. Such organisms can become potential energy sources and fuel producers, as well as a means of bioremediation (cleaning the environment with the help of biological objects) [32, 33]. They can find their application in gene therapy, and knowledge about the genes vital for the cell can form the basis for the production of fundamentally new antibiotics.

ConclusionSince ancient times, people have been trying to get the living from the inanimate (see Self-generation), and if such methods look more like a figment of fantasy, then with the development of the living sciences and the advent of molecular biology, more and more true data about the functioning of a living organism and the role of the genome in it began to accumulate.

Having entered the era of genomic technologies and DNA decoding, scientists have received a key tool for the artificial creation of genomes, which became a prerequisite for the birth of synthetic biology. But... for the final assembly of the genome and obtaining a working system, another living organism is still needed, that is, "omnis cellula e cellula" ("a cell comes only from another cell", Rudolf Virchow) is still valid. However, the rapid development of DNA technologies* and the study of artificial cell systems [34] capable of simulating cell division [35] give hope that a worthy alternative will be offered to this postulate in the future.

* – Now genetic engineers can operate not only "the old-fashioned way" – in vitro, but also directly in living objects. The editing systems ZFN, TALEN and CRISPR/Cas9, based on the site-specific action of nucleases in vivo, have become particularly popular: "And should we take a swing at... genome modification?" [36], "CRISPR systems: immunization of prokaryotes" [37], "Mutagenic chain reaction: genome editing on the verge of fiction" [38]. However, this does not mean that the apocalypse is just around the corner – control, control and many more times control (and at the legislative level) has not been canceled. But the victory over many severe ailments (and old age?) it can promise. – Ed.

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