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Directed Evolution in Microreactors

A. Hübner, H. Leemhuis and F. Hollfelder
University of Cambridge, GB

Keywords:
directed evolution, microreactor, in vitro translation/transcription, human acetyltransferase

Abstract:
We apply directed evolution (i.e. the generation of genetic diversity followed by selection of improved variants of the starting protein) to improve the activity of a human Histone Acetyltransferase (HAT). Genetic diversity is created by error-prone PCR and semi rational design (=saturation mutagenesis of selected active site residues) followed by reshuffling of those libraries of mutated hPCAF genes.
In different rounds of directed evolution we select for specificity as well as for activity (=kcat). Linking the genotype/phenotype via a bead we transcribe and translate single genes in microdroplet microreactors (size ~ 5 femtoliter). By using antibodies targeting acetylated lysine residues we can analyze 10 high 9 mutants per day by FACS. Current high-throughput methods (e.g. in 96-well microtiter plates) allow analysis of only 500 mutants per day (unpublished results). With this new method we might be able to define the role of individual histone lysine residues that are crucial for gene silencing or activation in living cells.

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