The first artificial bacterial genome

In the summer of 2007, the Institute of the famous Craig Venter completed work on the creation of the world's first "man–made" bacterium, Mycoplasma laboratory - or rather, not the bacterium itself, but its DNA, and filed a patent application for a set of genes sufficient to create a free-living, capable of independent growth and reproduction of a microorganism with a minimal set of genetic material.

Yesterday, the media again remembered Cynthia (Synthia – so dubbed the future achievement of synthetic biology): The on-line version of the journal Science published an article by 17 researchers from the J. Craig Venter Institute (JCVI) describing the technology of manufacturing an artificial bacterial chromosome.

This work, conducted under the guidance of Dr. Carole Lartigue, is the second of three key steps towards the main goal of the authors – the creation of a completely artificial microorganism. Now scientists have already started the last stage – an attempt to create a man-made bacterium based on the genome synthesized by them. They intend to introduce an artificial genome into a living bacterial cell and thus turn it into the first human-created bacterium.

Venter believes that engineered genomes have enormous potential. Synthetic bacteria may be more suitable for microbiological synthesis than the microorganisms currently used, bred through selection and mutations or genetic engineering. On the other hand, there are scientists who consider his claims to be overly dramatized and more calculated for the public, and the goals set by Venter (synthesis of various substances, including obtaining biofuels, absorption of carbon dioxide from the atmosphere) can be achieved by traditional methods of gene manipulation, without resorting to the loud label "Life 2.0" and synthesized DNA. But the grandiose scientific value of this work does not cause doubts among specialists.

The largest artificial DNA to date (several viruses were previously obtained synthetically; one of them was also made in JCVI) is exactly 582970 pairs of nucleotide bases that make up 381 genes – the minimum set sufficient for life "in vitro".

The researchers brilliantly solved their task by chemically synthesizing DNA fragments and developing new methods for their assembly and reproduction. Experts believe that over time, the authors' developments will find wide application in the developing field of synthetic genomics.

It is quite difficult to synthesize a chromosome from the building blocks of DNA – nucleotide bases: adenine (A), guanine (G), cytosine (C) and thymine (T) by chemical methods. As the DNA strand lengthens during synthesis, its fragility simultaneously increases, which significantly complicates the work. Compared with the latest record in the field of DNA synthesis – a DNA strand consisting of 32,000 pairs of nucleotides – a synthetic genome comprising more than 580 t.p.n is a truly grandiose achievement.

Methods used in the creation of an artificial genome

Before starting the work, in order to make sure that they have an error-free sequence of the bacterial chromosome as a template, the scientists re-sequenced the genome of the usual Mycoplasma genitalium strain. After receiving the final version of the genome, the authors synthesized DNA fragments by chemical methods, from which 101 "modules" of 5-7 t.n. were subsequently built. In order for the synthetic genome to be easily distinguished from the natural one, a kind of "watermarks" were embedded in it – short DNA sequences encoding information not found in nature. Another difference between the synthetic genome and the natural one is the absence of genes that ensure the infectivity of the microorganism. When creating the modules, the authors collaborated mainly with the DNA synthesis company Blue Heron Technology, as well as the companies DNA 2.0 and GENEART.

After that, the researchers developed a 5-step process of sequentially assembling modules into larger fragments. At the first stage, combining the four modules made it possible to obtain 25 DNA fragments, each of which consisted of about 24 t.n. These fragments were cloned in Escherichia coli bacteria (Escherichia coli, one of the main workhorses of molecular biologists) to obtain enough DNA to carry out subsequent stages and control the correctness of the sequence.

The next stage consisted in combining the obtained fragments into 8 blocks of 72 t.p.n. These larger fragments were also cloned using E.coli, after which they were combined in pairs. The result was 4 large fragments, each of which contained about 144,000 pairs of nucleotide bases and represented a fourth part of the mycoplasma genome.

At this stage of the work, the authors found that E.coli cells cannot synthesize DNA fragments of the size they need and, in search of a replacement, found that yeast is able not only to clone large DNA fragments, but also to combine them with each other using a homologous recombination mechanism. As a result, the quarters of the genome were successfully combined into a single whole containing more than 580,000 pairs of nucleotide bases. The resulting chromosome was sequenced again to confirm the accuracy of the chemical structure.

The synthetic chromosome of M.genitalium JCVI-1.0 has a molecular weight of 360.110 kilodaltons (kDa). Printed on paper in a 10-point font, the sequence of a synthetic chromosome occupies 147 pages.

Key stages of work on the creation of an artificial genome

The roots of the idea of synthesizing an artificial organism go back to the mid-90s, when, after sequencing the genome of M.genitalium, Venter and his colleagues launched the project "Minimal Genome". The aim of the work was to identify the minimum number of genes necessary to ensure the vital activity of an independent organism. The object of the study naturally became M.genitalium, which has the smallest genome among the organisms known to man, suitable for cultivation in culture. The results of the work were published in 1995 in the journal Science.

In 2003, a group of researchers took the first significant steps towards creating an artificial genome. Scientists have managed to synthesize the DNA sequence of the bacteriophage ΦX174 (phi X), consisting of 5386 pairs of nucleotide bases. To do this, they used commercially available artificial single-stranded DNA strands (oligonucleotides), which were combined using a modification of the polymerase chain reaction (PCR) method, called polymerase cycle assembly (PCA). It took the researchers only 14 days to create an artificial bacteriophage.

Ethical issues

From the very beginning of their work in the field of synthetic biology, Venter and his colleagues realized that their actions would cause a lot of questions from the public. But a group of bioethics experts from the University of Pennsylvania, as a result of an anonymous vote, decided that as long as researchers submit their actions to the public, there are no reasons to stop working in their chosen direction.

Evgeniya Ryabtseva
Portal "Eternal youth" www.vechnayamolodost.ru based on the materials of ScienceDaily.

25.01.2008