(518b) Pfunkel: Efficient, Expansive, User-Defined Mutagenesis

An efficient and high-throughput mutagenesis strategy is an integral part of protein studies, directed evolution and biological studies. The mutagenesis toolbox currently lacks a method for creating extensive DNA libraries with a researcher-defined mutational composition spanning across an entire gene.  For example, until now there has been no efficient method to make a library consisting of all 18,900 possible single codon substitutions of a 300 amino acid long protein, nor is there a method to make a researcher-prescribed subset of only 2000 of these 18900 mutations. We have developed a method termed PFunkel mutagenesis by which such libraries are readily created.  Additionally, the protocol has been adapted for the introduction of multiple defined mutations per gene with very high efficiency. The method is rapid, taking a single day and performed in a single tube. In short, PFunkel is a versatile and efficient mutagenesis technique that enables creation of gene libraries or individual constructs where each member receives one or multiple user-defined mutations at any position.

Using PFunkel we constructed a comprehensive codon substitution library of nearly all of the ~18,000 possible codon substitutions in the TEM-1 gene, which encodes the TEM-1 b-lactamase protein. TEM-1 has high activity for degrading the beta-lactam antibiotic ampicillin, but negligible activity on the antibiotic cefotaxime. By subjecting this library to a selection for increased cefotaxime resistance we have identified nearly all single-codon mutations that provide increased activity on cefotaxime. This extensive set of improved variants of a single gene revealed interesting patterns in the nature of gain-of-function mutations.  In a second experiment, we constructed a TEM-1 library in which four distal positions that are known to be important for activity on cefotaxime were simultaneously randomized to all possible codons. From this library we discovered new variants that confer greater resistance to cefotaxime than any of those identified in seven previous directed evolution studies.