\u201cSloppy\u201d by design<\/p>\n
Directed evolution is a method scientists use to \u201cspeed up\u201d natural selection in the lab. They make random changes (mutations) to a gene, then test and keep the versions that work best, repeating this process many times.<\/p>\n
Another method, continuous evolution, such as phage-assisted continuous evolution (PACE), can do these mutation-and-selection cycles very quickly, but they have two main problems: they can only handle small pieces of DNA (about 8,000 DNA letters long), and they can get \u201ccheaters\u201d where the bacteria mutate their own DNA in a way that tricks the test and helps them survive, without actually improving the target gene.<\/p>\n
\u201cLySE sidesteps those two problems by exploiting bacteriophage T7, a virus that infects E. coli bacteria,\u201d explained PhD candidate, Shujian Ong, who conducted much of the research. \u201cT7 replicates rapidly and breaks the bacterial cell open within minutes. We have engineered the virus so that, when it makes new virus particles, it also packs in an extra small ring of DNA called a phagemid which carries the group of genes they want to improve.\u201d<\/p>\n
To make a lot of the new versions of those genes, the phagemid is copied by a specially engineered DNA-copying enzyme (T7 DNA polymerase) that is intentionally error-prone. Think of a normal DNA polymerase as a precise photocopier: this engineered variant is deliberately sloppy, making many \u201ctypos\u201d (mutations), \u2014 about 160,000 times more than the bacterium\u2019s own DNA copying system.<\/p>\n
Paradoxically, the high error rate is what makes the system controllable. Because the polymerase is so \u201csloppy\u201d, it also messes up the virus\u2019s own DNA. As a result, the phage becomes weaker, losing the ability to spread uncontrollably; it can only destroy the bacteria when added in large numbers.<\/p>\n
By adjusting the ratio of phage to bacteria, the researchers toggle between a mutation phase in which the target genes get a lot of new mutations and these mutated genes are packed into new phage particles, and a selection phase, in which mutated genes are put into fresh, normal bacteria and tested for improved function.<\/p>\n
From antibiotic resistance to plastic digestion<\/p>\n
The NUS team validated the LySE method in two ways. In an antibiotic-resistance check, the improved trait persisted after the genes were moved into new bacteria, confirming the changes were built into the target gene cluster.<\/p>\n
Second, the researchers tried improving a whole \u201cmini-factory\u201d in cells: a five-gene pathway that lets bacteria use ethylene glycol for growth and energy. After five rounds with less glucose each time, the best-performing strain produced 50.9 per cent more biomass using ethylene glycol as its sole food source.<\/p>\n
Sequencing showed LySE changed both regulatory regions (switches that control how much a gene is turned on or off) and protein-coding genes, and each helpful mutation was confirmed by adding it back one at a time into a fresh host.<\/p>\n
\u201cWithout LySE, a bacterium\u2019s instinct is to mutate its own entire genome to find ways to eat more plastic, but it struggles to find optimal solutions that way,\u201d added Asst Prof Fredens. \u201cLySE improves the target gene cluster tremendously without accumulating unwanted mutations in the rest of the bacterium\u2019s DNA. Because all the improvements are strictly contained within our specific gene cluster, we can easily transfer this highly optimised pathway into entirely different bacteria.\u201d<\/p>\n
Engineering new-to-nature biology<\/p>\n
The platform\u2019s capacity to handle gene clusters of up to 40 kilobases in length \u2014 five times the limit of the most\u00a0 commonly used phage-based evolution method \u2014 opens the door to applications that were previously impractical. These include optimising biosynthetic pathways for pharmaceuticals, engineering microbes that break down environmental pollutants and evolving entirely synthetic metabolic routes for carbon capture. The workflow requires only standard laboratory equipment and the mixing of phage lysates with cell cultures, making the technology accessible to laboratories without specialist phage biology expertise.<\/p>\n
A patent has been filed for the LySE technology. Looking ahead, the team plans to apply LySE to systems that are entirely synthetic and new to nature.<\/p>\n
\u201cA key target is engineering synthetic CO2-fixing metabolic pathways, taking computationally designed routes that have never existed in the real world and optimising them so they actually function efficiently inside living cells,\u201d said Asst Prof Fredens. \u201cWith LySE, we can take AI-designed enzymes and metabolic pathways and rapidly optimise them to work in practice. That is where massive potential lies.\u201d<\/p>\n","protected":false},"excerpt":{"rendered":"\u201cSloppy\u201d by design Directed evolution is a method scientists use to \u201cspeed up\u201d natural selection in the lab.…\n","protected":false},"author":2,"featured_media":410998,"comment_status":"","ping_status":"","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[7],"tags":[97146,111,2055,139,69,211447,147],"class_list":["post-410997","post","type-post","status-publish","format-standard","has-post-thumbnail","category-science","tag-national-university-of-singapore-nus","tag-new-zealand","tag-newswise","tag-newzealand","tag-nz","tag-phagephage-biologyphage-researchphagemidmicrobiology","tag-science"],"_links":{"self":[{"href":"https:\/\/www.newsbeep.com\/nz\/wp-json\/wp\/v2\/posts\/410997","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.newsbeep.com\/nz\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/www.newsbeep.com\/nz\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/www.newsbeep.com\/nz\/wp-json\/wp\/v2\/users\/2"}],"replies":[{"embeddable":true,"href":"https:\/\/www.newsbeep.com\/nz\/wp-json\/wp\/v2\/comments?post=410997"}],"version-history":[{"count":0,"href":"https:\/\/www.newsbeep.com\/nz\/wp-json\/wp\/v2\/posts\/410997\/revisions"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/www.newsbeep.com\/nz\/wp-json\/wp\/v2\/media\/410998"}],"wp:attachment":[{"href":"https:\/\/www.newsbeep.com\/nz\/wp-json\/wp\/v2\/media?parent=410997"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.newsbeep.com\/nz\/wp-json\/wp\/v2\/categories?post=410997"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.newsbeep.com\/nz\/wp-json\/wp\/v2\/tags?post=410997"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}