Evaluating a Screen and Analysis of Mutant Libraries

Directed evolution by sequential cycles of random mutagenesis and screening has proven to be useful for producing new or improved enzyme properties (1 ,2 ). The first step is construction of a mutant library, usually accomplished by random point mutagenesis with error-prone PCR (3 ) or by DNA shuffling (recombination) (4 ,5 ). The second, and most critical step is finding the desired mutants by screening or selection of the libraries. Screens for enzyme activity usually operate via detection of optical absorption or fluorescence. Some screens are performed directly in agar plates, where changes of color are observed by direct visualization (6 ,7 ) or by digital image analysis (8 ). However, most screens involve transferring individual colonies to multi-well microplates containing culture medium, growing the cells until stationary phase, and inducing protein expression. If the target protein is intracellular, a cell lysis step has to be carried out to release the cellular contents. Finally, enzymatic activity is assayed with a microplate reader spectrophotometer. For many enzymes, thermal stability (9 ) is conveniently assayed by making a replica of the library and measuring the ratio of the activity after heating at defined temperature and time (residual activity, R_A ) to the activity measured before the heating (initial activity, I_A ) (see Chapter 11 ).