Phage-assisted continuous and non-continuous evolution

Abstract

Directed evolution, which applies the principles of Darwinian evolution to a laboratory setting, is a powerful strategy for generating biomolecules with diverse and tailored properties. This technique can be implemented in a highly efficient manner using continuous evolution, which enables the steps of directed evolution to proceed seamlessly over many successive generations with minimal researcher intervention. Phage-assisted continuous evolution (PACE) enables continuous directed evolution in bacteria by mapping the steps of Darwinian evolution onto the bacteriophage life cycle and allows directed evolution to occur on much faster timescales compared to conventional methods. This protocol provides detailed instructions on evolving proteins using PACE and phage-assisted non-continuous evolution (PANCE) and includes information on the preparation of selection phage and host cells, the assembly of a continuous flow apparatus and the performance and analysis of evolution experiments. This protocol can be performed in as little as 2 weeks to complete more than 100 rounds of evolution (complete cycles of mutation, selection and replication) in a single PACE experiment.

Figure 1.. Overview of phage-assisted continuous evolution (PACE).

(a) In a fixed-volume vessel, the “lagoon”, selection phage encoding an evolving protein of interest (POI) trigger gIII expression from an accessory plasmid in host E. coli cells, resulting in the production of the essential phage protein, pIII. The lagoon is continuously diluted with a culture of fresh host cells. All replicating DNA within the lagoon is mutagenized via an engineered mutagenesis plasmid to provide genetic diversity. Only the SP containing genes encoding functional POI variants are capable of replicating faster than the rate of dilution, allowing them to persist in the lagoon. (b) Gene variants on the SP that encode active POIs trigger the expression of gIII from the AP, typically through the activation or recruitment of an RNA polymerase (RNAP). The MP expresses mutagenic genes under the control of the arabinose promoter; induction of mutagenesis occurs upon addition of arabinose to the growth media. (c) Established PACE selection strategies for various POI (blue) activities, including RNA polymerase activity (upper left), protein:DNA binding (upper middle), protein solubility (upper right), proteolysis of specific amino acid sequences (middle left), protein:protein binding( middle right), base editing (lower left), and incorporation of non-canonical amino acids by orthogonal aminoacyl-tRNA synthetases (lower right), and base editing (lower right). (d) Negative selections can be performed in PACE by linking the expression of a dominant-negative version of gIII, gIII-neg, to undesired activity, such that phage propagation requires SP encoding POI variants with the desired activity and lacking the undesired activity.

Figure 2.. Assembly of a PACE apparatus.

(a) List of parts required for assembly of a PACE apparatus. (b) Schematic for preparation of PACE tubing segments using parts listed in (a). (c) Construction of PACE tubing using parts shown in (b). (d) An example of a media carboy suitable for supporting the host-cell chemostat during PACE. (e) Assembly of the full PACE apparatus from parts depicted in (a-d).

Figure 3.. Monitoring the PACE experiment.

(a) Simulated lagoon phage titers (pfu/mL) from a theoretical PACE experiment. In general, titers range from 10⁵ to 10⁸ pfu/mL. Flow rates can be increased when titers increase or remain stable to exert greater selection pressure. (b) Simulated lagoon phage titers (pfu/mL) from a theoretical PACE experiment incorporating drift through use of a drift plasmid (DP). [aTc] = anhydrotetracycline concentration. (c) Representative examples of plaque assays using wild-type M13 phage (top), ΔgIII selection phage containing a ~2-kb gene encoding the protein of interest (middle), and ΔgIII SP with a ~4-kb insert (bottom) using S2208 cells. From the top left quadrant, movement clockwise along the plate represents a 100-fold dilution of phage relative to the previous quadrant.