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Screening and Selection Systems for Directed Evolution of Enzymes
The concept of "directed evolution" is dependent on the availability of systems that allow the identification of interesting enzyme variants within a large, artificially generated diversity. Thus, one prerequisite is the availability of systems that allow the detection of specific enzyme features such as activity, selectivity, stability etc. at smallest culture scales and at high-throughput conditions. Therefore, focus is put on the development of methods witch are rapid, sensitive and cost-effective and preferably work at the microwell-plate, single colony or even single cell level.
Surrogate substrate analogues which would allow easy detection of reaction products by e.g. fluorescence techniques are not feasible due to the "First Law of Directed Evolution" which says "you get what you screen for". The "real" substrates should be converted or at least derivatives that are very close to these substrates have to be used in such systems. Therefore, general analytical methods which allow following the enzyme-catalyzed reaction of any desired substrate are developed.
Another important prerequisite is the availability of methods that allow a high degree of automation. Such methods include high throughput detection systems based on image analysis and software for accurately recognizing hits. In addition, statistical methods and systems for data management are developed to properly set up and evaluate the results of screening programs and to handle large data volumes.