A new platform developed at Scripps Research enables fast, scalable protein evolution. This potentially opens the door to new therapies and diagnostics. In addition, the technology may aid scientists in predicting resistance mutations across for cancer, neurodegeneration and many other disease areas.
The invention is termed T7-ORACLE. This is a powerful new tool that, essentially, speeds up evolution. The net effect enables scientists to design and improve proteins thousands of times faster than any process that might occur in nature.
Designing systems for continuous evolution is complex. The objective is to create conditions where proteins evolve inside living cells without manual intervention. The aim of technological advancement in this field is to streamline this process by enabling simultaneous mutation and selection with each round of cell division (roughly 20 minutes for bacteria). Previous approaches have been limited by technical complexity or modest mutation rates.
The T7-ORACLE circumvents these bottlenecks by engineering Escherichia coli bacteria to host a second, artificial DNA replication system derived from bacteriophage T7, a virus that infects bacteria.
Bacteriophages (phages) are viruses that exclusively infect bacteria, replicate within them, and often cause the lysis of the bacterial cells during the release of progeny phage particles.
T7-ORACLE enables continuous hypermutation and accelerated evolution of biomacromolecules, and is designed to be broadly applicable to many protein targets and biological challenges.
Conceptually, the T7-ORACLE builds on and extends efforts on existing orthogonal replication systems. This means they operate separately from the cell’s own machinery. The new technology benefits from the combination of high mutagenesis, fast growth, high transformation efficiency, and the ease with which both the E. coli host and the circular replicon plasmid can be integrated into standard molecular biology workflows.
The T-7 ORACLE orthogonal system targets plasmid DNA (these are small, circular pieces of genetic material), leaving the cell’s host genome untouched. By engineering T7 DNA polymerase (a viral enzyme that replicates DNA) to be error-prone, the researchers introduced mutations into target genes at a rate 100,000 times higher than normal without damaging the host cells.
Using engineered bacteria and a modified viral replication system, the novel method can create new protein versions in days instead of months. In tests, the T7-ORACLE rapidly produced enzymes that could survive extreme doses of antibiotics.
To demonstrate the T7-ORACLE system, the research team inserted a common antibiotic resistance gene, TEM-1 β-lactamase, into the system and exposed the E. coli cells to escalating doses of various antibiotics.
Beyond this, the broader potential of T7-ORACLE lies in its adaptability as a platform for protein engineering. Although the system is built into E. coli, the bacterium serves primarily as a vessel for continuous evolution. Scientists can insert genes from humans, viruses or other sources into plasmids, which are then introduced into E. coli cells. T7-ORACLE mutates these genes, generating variant proteins that can be screened or selected for improved function. Because E. coli is easy to grow and widely used in labs, it provides a convenient, scalable system for evolving virtually any protein of interest.
In less than a week, the system evolved versions of the enzyme that could resist antibiotic levels up to 5,000 times higher than the original. This proof-of-concept demonstrated not only T7-ORACLE’s speed and precision, but also its real-world relevance by replicating how resistance develops in response to antibiotics.
The research appears in the journal Science, titled “An orthogonal T7 replisome for continuous hypermutation and accelerated evolution in E. coli.”
