Directed evolution of β‐galactosidase from Escherichia coli by mutator strains defective in the 3′→5′ exonuclease activity of DNA polymerase III
- 30 March 2001
- journal article
- Published by Wiley in FEBS Letters
- Vol. 493 (2-3), 139-143
- https://doi.org/10.1016/s0014-5793(01)02293-1
Abstract
Directed evolution of Escherichia coli β-galactosidase into variants featuring β-glucosidase activity was challenged. To this end, mutagenesis of lacZ was performed by replication in E. coli CC954, a mutator strain containing a DNA polymerase III defective in 3′→5′ exonuclease activity. β-Galactosidase variants can be isolated upon mutagenesis of lacZ hosted into the self-transmissible episome F′128. Optimal evolution of lacZ can be achieved by propagation of E. coli CC954/F′128 cultures for 15 generations; further growth of mutator cultures for 37 or 55 generations imposes a high mutational load on lacZ and hinders the selection of efficiently evolved clonesKeywords
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