Figure 1. Schematic of phage-assisted continuous evolution. Phage require protein III to effectively infect host cells, and viral particles lacking this protein are about 100 million times less infectious than wild-type particles.2

Esevelt et al. exploit this property for a directed evolution approach they call phage-assisted continuous evolution (PACE). In this method, researchers link the production of protein III in the M13 filamentous bacteriophage to an activity of interest by deleting the associated gene from the DNA of the phage vector and inserting a copy of the gene into an accessory plasmid (orange circle; AP) in Escherichia coli host cells. The accessory plasmid is designed such that molecules with higher levels of the activity of interest increase protein III production. The E. coli host cells also carry a mutagenesis plasmid (green circle; MP) that researchers can use to modulate the error rate during DNA replication.

Host cells that flow into a chamber are infected with selection phage from a library [a]. Selection phage encoding genes for molecules that have higher levels of the activity of interest (red circle; SP) will induce greater expression of protein III production from the bacterial cell's accessory plasmid than phage encoding genes for less active or nonfunctional molecules (blue circle; SP) [b].

Infected cells that produce more protein III will generate a larger number of infectious phage progeny that also encode the gene for the more active molecule [c]. These phage will propagate in larger numbers and infect more cells than those encoding molecules with lower levels of activity.

While inside the host cell, phage DNA also can acquire mutations that affect the activity of the encoded molecule [d]. These mutations result in the generation of phage encoding gene variants that are not found in the original library. Researchers control the rate of mutagenesis via the mutagenesis plasmid.

The constant outflow from the system means that phage must propagate in sufficient numbers to maintain their presence. Thus the system continuously selects for phage encoding molecules with higher levels of the desired activity.

Figure adapted from Esevelt et al.1