Highly mutagenic continuous evolution in E. coli using a Φ29-based orthogonal replication system
A phi29-based orthogonal replication system was implemented in Escherichia coli to enable continuous, highly mutagenic in vivo evolution. The synthetic replicon requires TP and DNAP for maintenance, is editable in cells using lambda Red recombination, and remains stable across hundreds of generations. An optimized replication operon increased replicon copy number and engineering efficiency.
Why it mattersElectroporation-enabled replacement of tetracycline with kanamycin at ~10^-4 frequency enables iterative in-cell replicon editing for continuous.