Directed Evolution Through DNA Shuffling for the Improvement and Understanding of Genes and Promoters

Unlike rational protein engineering, directed evolution provides an a priori approach toward the engineering of improved proteins and novel promoters. This minimally recursive technique builds upon small improvements by selecting and combining the best changes. Protein–protein/DNA interactions, catalytic efficiency, or resilience to inhibitors can be improved by thousands of times. By working within a subspace of homologous sequences, DNA shuffling recombines that subspace. Individuals are screened for a particular trait or two and selected for when they meet a set threshold. Here we explain basic principles to follow and provide procedures for the preparation, fragmentation, efficient size fractionation, and purification of parental material, as well as for the reassembly and rescue polymerase chain reactions (PCRs).