Pure synthetics
Synthetic E. coli had its genetic code cut
Daria Spasskaya, N+1
Biologists have created a strain of Escherichia coli, whose genome has been completely synthesized anew. At the same time, three codons in her genome were completely replaced with synonymous ones, thus reducing the genetic code from 64 to 61 positions. If the genome of the first organism with a synthetic genome contained "only" a million base pairs, this time this figure was exceeded four times, the authors of the article in Nature state (Fredens et al., Total synthesis of Escherichia coli with a recoded genome).
In 2010 , Craig Venter 's team stated about the creation of the first organism with a fully synthetic genome. The organism was a strain of the bacterium Mycoplasma mycoides, which was obtained by introducing a chromosome fully synthesized in vitro into the recipient cell. The size of the synthetic chromosome was slightly more than one million base pairs. After this experiment, biologists were also able to synthesize and assemble several artificial yeast chromosomes, comparable in size.
A team of scientists from the Cambridge Molecular Biology Laboratory led by Thomas Elliott and Jason Chin has created a strain of E. coli with a de novo synthesized chromosome of 4 million base pairs. It was possible to assemble such a huge genome only in parts directly in the cells of the "host", thanks to the ability of E. coli to recombine DNA with high efficiency.
For the synthesis and assembly of fragments, the genome of the Escherichia coli strain MDS42 was divided into 37 overlapping fragments, each slightly larger than 100 thousand base pairs. Each such piece was assembled from smaller pieces using recombination in yeast cells (just as it was done in Venter's work). The obtained synthetic fragments were introduced into MDS42 cells as part of plasmids and, using a modified REXER method based on homologous recombination, the host cells were forced to exchange a fragment of their chromosome for a fragment of a synthetic one.
Scheme of sequential replacement of E.coli's own chromosome (gray) with a synthetic one (pink). Drawings from an article in Nature.
After several rounds of recombination, the scientists obtained seven different strains containing seven large parts of a synthetic chromosome as part of their genome. Then the conjugation process was induced in the cells — an analog of the sexual process in bacteria, during which the exchange of genetic information is possible. This allowed the researchers to sequentially combine individual fragments of the synthetic chromosome in one strain. The resulting "synthetic" strain of E. coli was named Syn61.
According to the authors' idea, its main distinguishing feature was a non-standard genetic code. During the synthesis of parts of the genome, the TCG and TCA codons encoding the amino acid serine were excluded from the genetic code and replaced with synonymous codons also encoding serine. This became possible due to the degeneracy of the genetic code — since each amino acid corresponds to a codon of three letters, and there are only four letters, 20 amino acids are encoded using 64 codons (this also includes three stop codons indicating the end of the protein sequence). At the same time, as many as six codons are allocated for serine.
Scheme of codon replacement in the Syn61 genome/
In addition to the extra serine codons, scientists removed one of the stop codons from the Escherichia coli code - all TAG codons were replaced with TAA. The genetic code of the organism Syn61, therefore, consists of 61 codons instead of 64.
In total, 18214 codons were replaced in the synthetic chromosome. Previously, researchers were able to exclude one of the stop codons from the code by recombination with mutant oligonucleotides, but for this they had to change a maximum of 321 codons.
Tests have shown that the Syn61 strain feels quite normal and grows only 1.6 times slower than the ancestral strain MDS42. As it turned out, the change in the genetic code made some genes unnecessary, for example, the gene of one of the tRNAs for serine, which was previously vital for E. coli.
As the researchers explain, this work demonstrates the fundamental possibility of the existence of life with a truncated genetic code. In addition, proven genome assembly technologies bring scientists closer to creating organisms with specified properties, for example, including amino acids that are not naturally occurring in proteins. We have already talked about similar experiments where bacteria were forced to synthesize new proteins by, on the contrary, expanding the genetic code by introducing new letters into the genetic alphabet.
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