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pShuffle: A Plasmid for in vitro Evolution

Dirk Tischler, P. Zwicker, R. Schwabe, J.A.D. Gröning, S.R. Kaschabek, M. Schlömann

Abstract


Multi-gene shuffling is a powerful method used to combine and optimize attributes of various proteins. Here we report on the design and construction of the plasmid “pShuffle” which is suited for a variety of in vitro DNA-recombination techniques. The multiple cloning site (MCS) of pShuffle was designed to allow for the cloning of genes as well as their expression under control of either a lac- or a T7-promoter. As a specific feature, this MCS allows for the fusion of special linker sequences to both ends of cloned genes. After subsequent DNA-recombination steps, these linkers facilitate reamplification of generated gene variants, and thus may be used to construct clone libraries for activity screenings. The suitability of pShuffle for multi-gene shuffling applications was further shown with a set of styrene monooxygenase genes originating from proteo- and actinobacteria.

Keywords


DNA-shuffling; Enzyme evolution; Flavin monooxygenase; Multi-gene shuffling; DNA-recombination

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References


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