E. coli alkaline phosphatase has been thoroughly mutated in order to demonstrate the relationship between structure and function. The objective of this research project is to initiate a directed evolution approach in order to explore the relationship between structure and function of alkaline phosphatase mutants.

In order to begin the directed evolution, a particular mutant was chosen, which demonstrated a large decrease in catalytic activity. This mutant, when subjected to sequential rounds of random mutagenesis, will hopefully evolve to exhibit a higher catalytic activity than the wild type enzyme. In order to do so, a selected region of the phoA gene, which does not encode the mutant residue, will then be subjected to a random PCR mutagenesis. Thus, random base substitutions will occur at a particular rate. The resulting PCR products, will then be subcloned into a vector, containing the mutant residue. Through a screening method, it will then be determined which clones demonstrate increased alkaline phosphatase activity.

Concurrently, the same approach of directed evolution will be applied for the b-lactamase mutants. These mutants, which are ampicillin sensitive, will be tested for an increase in ampicillin resistance. An effective screening method to confer this type of activity must be developed and tested.