Directed evolution: an evolving and enabling synthetic biology tool

Abstract

Synthetic biology, with its goal of designing biological entities for wide-ranging purposes, remains a field of intensive research interest. However, the vast complexity of biological systems has heretofore rendered rational design prohibitively difficult. As a result, directed evolution remains a valuable tool for synthetic biology, enabling the identification of desired functionalities from large libraries of variants. This review highlights the most recent advances in the use of directed evolution in synthetic biology, focusing on new techniques and applications at the pathway and genome scale.

Figure 1

A PACE experiment. E. coli cells continuously flow through a ‘lagoon’ containing replicating phage, which carry the target gene of interest (1). Following phage infection of an E. coli cell (2), functionality of the target protein is linked to production of pIII, a protein that the phage requires to produce infectious progeny. Thus, when a phage encoding a protein with the desired function infects E. coli, pIII can be synthesized, and so further infectious phages are produced harboring the functional target gene (3). When a phage encoding a nonfunctional target protein infects E. coli, pIII is not synthesized, and so that cell does not produce any infectious phage with the nonfunctional target gene (4). The result is a phage population in the lagoon encoding functional target genes that can re-infect host cells (5), while nonfunctional genes are washed out with the noninfectious progeny (6).

Figure 2

Recent targets for the evolution of biosynthetic pathways include (a) promoters, (b) ribosome binding sites, (c) intergenic regions, and (d) gene order.

Figure 3

MAGE can achieve targeted multiplex modification of a genome by introducing synthetic oligonucleotides recursively to an evolving cell population.