1. Shake out the bacterial, buffer, and substrate from each microplate down the sink drain. 2. Dunk the plate in 1% Alconox detergent. The wells should fill up without additional agitation. 3. Dunk the microplate into a bucket of water. Shake the inverted microplates up and down several times. Shake the water out into the sink drain. 4. Dunk the microplate in 95% ethanol. Again, the wells should fill up without agitation. Shake the ethanol back into the reservoir. 5. Stack the microplates into a rack. Heat in a drying oven at 50  until the liquid has evaporated. Overheating (or autoclaving) will cause the polystyrene microplates to warp. Acknowledgments We thank the National Science Foundation (MCB0109668) and the Emory University Research Committee (URC 00–01 Matsumura) for support. We are grateful to Monal Patel for her helpful comments on the manuscript. [13] Periplasmic Expression as a Basis for Whole Cell Kinetic Screening of Unnatural Enzyme Reactivities By Grazyna E. Sroga and Jonathan S. Dordick Introduction The application of evolutionary and combinatorial techniques to gen- erate improved enzymes, achieve novel enzyme selectivities, and probe enzyme mechanisms has become one of the most dynamic tools of chemis- try and biology. Two principally different strategies can be applied to alter enzyme properties by targeting the enzyme gene(s)—rational design, which has been applied extensively, and directed evolution. While the former has seen some specific successes, 1–6 the lack of detailed structural 1 T. Yano, S. Oue, and H. Kagamiyama, Proc. Natl. Acad. Sci. USA 95, 5511 (1998). 2 G. MacBeath, P. Kast, and D. Hilvert, Science 279, 1958 (1998). 3 B. Van den Burg, G. Vriend, O. R. Veltman, G. Venema, and V. G. H. Eijsink, Proc. Natl. Acad. Sci. USA 95, 2056 (1998). 4 J. R. Cherry, M. H. Lamsa, P. Schneider, J. Vind, A. Svendsen, A. Jones, and A. N. Pedersen, Nature Biotechnol. 17, 379 (1999). [13] whole cell kinetic screening of enzyme variants 145 Copyright 2004, Elsevier Inc. All rights reserved. METHODS IN ENZYMOLOGY, VOL. 388 0076-6879/04 $35.00