A system for continuous directed evolution of biomolecules

This paper represents a milestone in protein engineering. Phage Assisted Continuous Evolution (PACE) of T7 RNA polymerase allowed hundreds of rounds of evolution in a few days and resulted in variants that recognize new promoters or initiate transcripts with ATP or CTP instead of GTP.

PACE is pretty complicated. Basically, media containing E. coli was pumped to flow through a flask with a residence time shorter than E. coli replication time. The bacteria contain two plasmids, a mutagenesis plasmid and an auxilary plasmid containing gene pIII with an unnatural promoter. M13 bacteriophages containing a plasmid with the T7 RNA polymerase gene (RNAP) attempt to infect the bacterial cells. Their ability to infect the cells is dependent on pIII. Only phage containing RNAP variants that can express the pIII gene will propagate. The mutagenesis plasmid provides the continuous high mutation frequency.

The ability to do directed evolution at this speed (24-30 rounds per day) is phenomenal. Unfortunately, in order to use PACE to evolve an enzyme, the desired phenotype must result in increased expression of pIII. This does not provide any clear way to extend this technique to enzymes other than those which modify DNA or RNA.