Gene mutations are essential for innovation and evolution, however, having too many of them – or the wrong mutations – can be fatal. So researchers at Cambridge created an artificial system for 'orthogonal' DNA replication coli bacteria They can use it as a risk-free way to generate and study such mutations. It is orthogonal because it is completely separate from the system in which it is coli bacteria It is used to copy the actual genome containing the genes coli bacteria He needs to stay.
Genes in the orthologous system are transcribed using an unusually error-prone DNA replication enzyme, which stimulates rapid evolution by generating many random mutations. This continues for a while coli bacterias genes are replicated by the natural DNA replication enzyme HD. The two enzymes work side by side, each doing its own job but not interfering with the other's genes.
Rapid surge engineering
This is a great idea, isn't it? Scientists stole it from nature. Yeast already has a system like this, with a set of genes copied by a dedicated enzyme that does not replicate the rest of the genome. but coli bacteria It's much easier to work with than yeast, and it can double in 20 minutes, so you can do a lot of iterations and evolve quickly.
The researchers created the system by hijacking phages, a virus that infects humans coli bacteria. They removed all of the phage genes that allow the phage to grow uncontrollably until it explodes coli bacteria The infected cell is open. The engineering left only a strand containing the genes responsible for copying the phage genome. Once this cassette is inserted in coli bacteria Genome, it can simultaneously replicate at least three different sequences of genes placed next to it in the DNA, maintaining them for more than a hundred generations – all while leaving the rest of the genes behind. coli bacteria Genome to be copied by other enzymes.
The scientists then tweaked the mutation rate of the orthologous DNA replication enzyme, ultimately boosting it by 1,000-fold. To test whether the system could be used to develop new functions, they introduced a gene for resistance to one antibiotic and observed how long it took for that gene to mutate into a gene that confers resistance to a different antibiotic. Within twelve days, they became 150 times more resistant to the new antibiotic. They also inserted the gene that encodes green fluorescent protein, and increased its fluorescence by more than 1,000-fold in five days.
Evolution of detoxification
Less than 20 pages later, in the same issue of Science, Frances Arnold's lab had a paper providing evidence of the power of this approach. This team directed enzyme evolution the old-fashioned way: through successive rounds of random mutations and selection of the desired trait. Arnold won the 2018 Nobel Prize in Chemistry for directed evolution of enzymes, so she knows what she's talking about. In this latest work, her lab has produced an enzyme that can degrade volatile methylsiloxane. We make megatons of these compounds every year that stick in cleaning products, shampoos, lotions, and industrial products, but they remain in the environment. They contain carbon-silicon bonds, which never existed until humans made them about 80 years ago; Since nature never made these bonds, there is no natural way to break them either.
“Directed evolution using siloxane has been particularly challenging,” the authors note in their introduction, for various technical reasons. “We started from an enzyme we had previously designed for other chemistry on siloxanes — this enzyme, unlike the natural enzyme, showed a small amount of activity in cleaving the Si-C siloxane bond. The whole project, from the initial discovery to figuring out how to measure what We wanted it, it took several years.” This is only the first step in making siloxanes biodegradable. It is hoped that the continued, accelerated development allowed by the new orthosteric system will facilitate the development of enzymes and other proteins like these that will have applications in research, medicine and industry.
We don't (yet) have machines that can efficiently assemble long stretches of DNA or make proteins. But cells do these things very efficiently, and coli bacteria Cells have long been those used in the laboratory as mini-factories, producing whatever genes or proteins researchers program into them. now coli bacteria They can be used for another molecular task: they can be small hotspots for evolution.
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