Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

The overall goal of this protocol is to show how to construct and screen mutant libraries in Saccharomyces Cerevisiae for directed evolution experiments. This method can help answer key questions in laboratory creation use for focused directed evolution experiments such as how to assign overlapping areas for in the assembly and cloning. The main advantage of this technique is its simplicity and robustness since in just one transformation step it's something levels with good quality can be assigned.

This protocol for creating and screening mutant libraries will be demonstrated for a fungal aryl-alcohol oxidase or AAO. The regions for focused directed evolution are first chosen with the help of computational algorithms based on the available crystal structure or homology models of the enzyme. Two regions of AAO will be targeted for random mutagenesis and recombination:the M1 region and the M2 region.