Researchers have developed an engineered bacteriophage T7 replisome – a multi-protein complex that carries out DNA replication – that can introduce high rates of mutation into specific plasmids in Escherichia coli while leaving the rest of the genome unchanged. The study, published in Science, shows how this targeted approach could accelerate laboratory evolution of proteins and support research into antimicrobial resistance.
The system, called T7-ORACLE, uses a modified T7 DNA polymerase that mutates DNA at rates around 100,000 times higher than normal. It only replicates circular plasmids, allowing scientists to focus mutations on selected genes without affecting other parts of the bacterial genome.
To demonstrate the method, the team applied T7-ORACLE to the TEM-1 β-lactamase gene, which provides resistance to certain antibiotics. Within a week, they generated enzyme variants with greatly increased activity against some monobactam and cephalosporin drugs.
Although primarily a research tool, the system could be useful in diagnostics. By rapidly evolving resistance genes in the lab, scientists could anticipate changes that may appear in clinical settings. This information could help update molecular tests to detect new variants and ensure assays remain accurate.
It may also be used to improve enzymes used in diagnostic kits, making them more efficient or better suited to different testing conditions.
Because the mutations are limited to plasmid DNA, the system allows for safe, controlled testing under appropriate biosafety protocols. The approach could be adapted to other bacterial hosts or used with larger plasmids to target multiple genes.
By enabling fast, targeted mutation without harming the host genome, T7-ORACLE provides a way to explore genetic changes that affect enzyme performance, antibiotic resistance, and diagnostic accuracy.
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