Abstract

Directed Protein Evolution in a Single Step

Chee Yang-Lee , Field Applications Scientist, Agilent

The ability to design and synthesize almost any DNA sequence you desire is fueling the explosion of advancements being made in Synthetic Biology in recent years. With this increase in availability of novel and previously unattainable DNA sequences the need for new tools that either utilize these novel sequences or allow the end-user to implement existing methodologies on a much larger scale are in high demand. To address this need Agilent has been working on several new technologies that can fulfill this need to help drive innovation. One such technology is Agilent’s DNA Oligo Library based Site Directed Mutagenesis. Previous methods, to simultaneously mutate every codon within a gene sequence relied upon random mutagenesis using either error prone PCR or the incorporation of degenerate oligos. Although both methods were relatively simple to perform the user had very little control over where or what mutations were generated requiring many thousands of mutants to be screened before the most advantageous mutations could be identified. Agilent has combined its unique ability to generate hundreds of thousands of unique DNA oligo sequences with the powerful QuikChange mutagenesis methodology to develop a simple and rapid mutagenesis method whereby every codon within a gene sequence can be simultaneous interrogated via a targeted mutagenic oligo design scenario. QuikChange HT enables Alanine (or single amino acid) Scanning and Codon Saturation Scanning at up to 10-20 different sites (in one plasmid or different plasmids), where each target region spans up to 50 amino acids. Researchers can survey the impact of substitutions at up to 1,000 different amino acids, or test up to a hundred thousand combinations of key mutations across 20-50 contiguous amino acids. Thus, the QuikChange HT Protein Engineering System enables rapid resolution of structure-function questions, with the ability to create rational variant libraries of up to 120,000 recombinant mu