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Phage-assisted continuous evolution (PACE)

Phage-assisted continuous evolution (PACE)

Phage-Assisted Continuous Evolution (PACE) is a form of directed evolution, a laboratory process that uses the phage virus bacterial infection cycle to generate multiple rounds of DNA sequence changes and selection for DNA changes in a target gene that result in a desired structure or activity in the encoded protein.

PACE is an advancement in directed evolution techniques which serve both industrial applications and researchers seeking to address unanswered questions about the principles of evolution in nature. Directed evolution is a laboratory process where multiple rounds of genetic mutations are generated and screened for desired changes in structure or activity of the protein encoded by the gene. The process is iterative so that the best mutants from each round are used as templates for the next rounds of mutation and selection and the process is repeated until the determined functional improvement to the protein is attained . The goal can be to produce a protein that is more compatible with industrial processing conditions, like increased stability or more active biocatalyst activity. The aim can also be to modify protein pathways to create novel whole cell biocatalysts that synthesize chemicals, biofuels or pharmaceuticals. Directed evolution may seek to improve on an existing protein or protein pathway may be improved upon or derive completely novel functions not found in nature .

PACE is much faster and requires less human labor than other forms of directed evolution. The speed of the system comes from the use of M13 bacteriophage, a bacteria infecting phage virus with a life cycle of 10 minutes . While traditional directed evolution methods create a library of genes with slightly different DNA sequences that code for proteins with small differences in function, M13 bacteriophages automatically generate such libraries upon replicating inside Eschericia coli (E. coli) cells . The E. coli in this system carry a mutagenesis plasmid, engineered using recombinant DNA technology, to be induced by addition of arabinose. Upon induction the mutagenesis plasmid elevates error rate during DNA replication by expressing proteins that disrupt proofreading and repair. Full induction increases mutation rate by 300,000 fold .

Using recombinant DNA technology the gene required for phage infection, gene III is removed and the gene encoding the target protein to be evolved is introduced into a "selection phage" vector. Gene III is inserted into plasmid DNA in the phage host bacteria E. coli where it is engineered to express the gene and the encoded protein pIII at levels proportional to the evolved activity of the target protein. The phages compete to optimize their gene/protein, because it maximizes their fitness, by allowing them to better infect and replicate in their host. An important part of the PACE design is that bacterial cells flow through an evolution vessel, called a “lagoon” faster than they can divide, which prevents the bacterial genome from interfering with the experiment by evolving at the same time. In addition, the PACE system allows the mutation rate to be higher than the bacteria can tolerate. Only phage that induce sufficient pIII from the E. coli plasmid will be able to remain in the lagoon, going on to infect more E. coli. Unlike other directed evolution methods PACE is continuous because there is no need to stop and screen a library for successful variants after each round .

The PACE system was developed by David R. Liu with his team at Harvard University and Howard Hughes Medical Institute and described in his 2011 Nature paper. In it the gene III expression vector was engineered to actively express the gene III under conditions where there is a desired protein-protein interaction or when recombinase activity flips the gene to the correct orientation . The team also evolved T7 RNA polymerases which normally do not work on T3 promoters to do so in less than one week . In 2017 Liu’s team reported used PACE to evolve Tobacco Etch Virus (TEV) protease over 2500 generations to cleave a target sequence in humans that could work as a therapeutic by having anti-inflammatory activity . In this case PACE was set up so that RNase polymerase needs to be cleaved by the evolved protease in order to be active and transcribe gene III .

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David R. Liu

Inventor of PACE

Kevin Esvelt

Inventor of PACE

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A system for the continuous directed evolution of biomolecules

Baker, K., Wanner, B. L., Friedman, S. M., Keenan, R., Kevin M. Esvelt, Riechmann, L., Raskin, C. A., Steinbach, P. A., Cheetham, G. M., Kauffman, S., van den Pol, A. N., Jeruzalmi, D., Das, A. T., Padilla, R., Kuzmine, I., Coleman, J. E., Mills, D. R., Murli, S., Gibson, D. G., Johnson, B. P., McAllister, W. T., Jackson, W. C., Imburgio, D., Vidal, M., Camps, M.

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