A bacterium without extra codons

E. coli's genetic code was rewritten

Alexander Ershov, N+1

Biologists from Harvard, MIT and several other American institutes spoke about the work on the genome-wide replacement of the genetic code in E. coli. In the future, this will allow creating a special "artificial" bacterium that is unable to reproduce outside the laboratory and at the same time is completely resistant to all existing viruses. The work of the group led by George Church is far from complete, but has already been published in Science (Ostrov et al., Design, synthesis, and testing towards a 57-codon genome).

The genetic code determines the correspondence between the sequence of nucleotides in genes and those amino acids from which proteins are built based on these genes. Every three nucleotides (codon) encode one amino acid. Since there are 64 variants of nucleotide triplets, and there are only 20 basic amino acids in proteins, many of the codons are synonymous, that is, they encode the same amino acid.

The idea of the authors was to replace some (mostly rare) codons with their synonyms, and to do this in the entire genome of a full-fledged bacterium. Such a bacterium would have some interesting properties. With a sufficiently large number of discarded codons (at least more than three), for example, it would be completely resistant to any existing viruses, since the viral genetic information in it would simply become "unreadable". Accordingly, she could not receive useful, but not "adapted" genetic information from the external environment (antibiotic resistance genes, etc.), which is important for biotechnological applications. In addition, artificial amino acids and chemical groups that do not occur in nature can be introduced into the proteins of such a bacterium, for example, for the synthesis of some new drugs or industrial substances. Moreover, this can be done using standard molecular biological methods, without developing the system anew every time.

The difficulty of creating such a bacterium is that it is necessary to replace the selected DNA triplets in the entire genome, and to do this not for one gene, but for the entire genome. Previously, similar codon replacement work had already been carried out (including by Church's group — scientists removed one of the stop codons from the Escherichia coli genome), but their scale was significantly smaller. However, when it comes to replacing seven codons in the entire genome, as planned in this paper, this not only requires the introduction of a very large number of changes (148955 nucleotide substitutions), but also threatens to cause a variety of unforeseen negative effects, which do not happen when working only with stop codons.

The work was carried out as follows. Scientists have selected seven fairly rare codons that could be removed along with the tRNA that recognizes them. Then the authors created a program that allows, based on a known genome sequence (a minimized, "purified" version of the Escherichia coli genome was already used), to replace the discarded codons with their synonyms.

A scheme for replacing the genetic code of a protein fragment
from natural type to "recoded".
Nili Ostrov et al., Science, 2016

When choosing a replacement, many factors were taken into account that could potentially affect the viability of the organism: scientists tried to predict and preserve as much as possible the important properties of the original DNA. For example, the program corrected the proportion of GC nucleotides (it affects the mechanical rigidity of DNA and much more), preserved the landing sites of ribosomes and those places in the matrix RNA where it forms a secondary structure.

The growth rate of bacteria with a partially replaced genome.
Strains with separate synthetic segments are shown:
the higher the point, the "more broken" the segment is.
Nili Ostrov et al., Science, 2016

As a result, the program produced several thousand edited fragments that had already been chemically synthesized into chains 2-4 thousand bases long. These fragments were placed in yeast, where 55 large segments of the new genome were collected from them by recombination. Each of the large segments was then individually tested in a "normal" E. coli — scientists checked how much the growth of the bacterium slows down if a natural piece of the genome is replaced with a synthetic one with a modified genetic code. Moreover, as expected, the effectiveness of computer predictions in some cases was not enough: many segments "did not work" in bacteria due to the replacement of important sites that could not be found in silico. We had to work with them "manually".

As a result, at the time of publication, scientists tested the operability of 63 percent of the synthetic genome (it was called rE.coli-57), and 91 percent of the tested genes retained their operability. However, the work on the synthetic genome is not finished yet (perhaps due to financial difficulties). The authors estimate the total cost of the project at one million dollars.

The improvement of natural microorganisms at the deep molecular biological level belongs to synthetic biology and is now going in different ways. In addition to replacing codons (which began in the late 80s with Schultz's work), scientists, for example, are trying to embed new, fully synthetic base pairs into the genome, which expand the information capacity of DNA and increase the space of the genetic code. Another way is, for example, the Craig Venter Institute, which recently reported on the creation of a bacterium with a fully synthetic minimal genome. You can read more about this work here.

Portal "Eternal youth" http://vechnayamolodost.ru  26.08.2016