In Vitro Directed Evolution of Enzymes Expressed by E. coli in Microtiter Plates

A method is described for using 96-well plates to prepare libraries of Escherichia coli cultures for screening a library of gene variants. This approach bypasses colony-picking to allow standard molecular biology laboratories to carry out directed evolution efficiently with a 96-well plate-reader and multichannel pipettes. Initial screens are applied to cultures that are rapidly prepared by diluting transformed cells so that an average of four cells starts each culture. Subsequent screens are used to isolate individual enzyme-expressing clones that exhibit activity higher than the parental clone. The outlined method also includes guidelines for preparing a library of gene variants and for optimizing a screening method.