Sunday, July 29, 2007
P9
Random Insertion of Tags in Biocatalytic Enzymes
Brigitte M. Hoeller1, Birgit Reiter1, Sandra Abad1, Ina Graze1, and Anton Glieder2. (1) Research Centre Applied Biocatalysis, Petersgasse 14/3, 8010 Graz, Austria, (2) Institute of Molecular Biotechnology, Research Centre Applied Biocatalysis, Graz University of Technology, Petersgasse 14/2, 8010 Graz, Austria
Nature mainly uses point mutations, recombination and insertions/deletions to generate diversity in evolutionary processes. Here we present a simple and general method for in vitro mutagenesis of polynucleotide sequences by random deletions, insertions or replacement. TIM (Transposon Integration mediated Mutagenesis) is based on random integration of modified bacteriophage Mu transposons and subsequent excision employing class IIS restriction endonucleases. TIM enables deletion and/or insertion of an arbitrary number of bases and insertion of functional sequence tags at random positions. In addition, also the random replacement of bases by a specific codon (e.g scannings) and simple gene site-saturation mutagenesis are possible. In fact, one of the most favourable characteristics for the use of bacteriophage Mu as tool in molecular biology is its ability to insert with very low site preference into a target DNA. A variety of transposon tools for sequencing and mutagenesis based on modified mini-Mu transposons are commercially available.
We combined the mini-Mu transposon integration with type IIS restriction endonucleases, which allow a complete and customizable removal of the transposon DNA. Therefore, the mini-Mu transposon from the GeneJumper™ Primer Insertion Kit for Sequencing from Invitrogen was engineered to serve as carrier to deliver specific ENase IIS restriction sites into the target gene.
We combined the mini-Mu transposon integration with type IIS restriction endonucleases, which allow a complete and customizable removal of the transposon DNA. Therefore, the mini-Mu transposon from the GeneJumper™ Primer Insertion Kit for Sequencing from Invitrogen was engineered to serve as carrier to deliver specific ENase IIS restriction sites into the target gene.
We used an esterase gene as a model for insertion and deletion mutagenesis and screened a library for random integration of 6xHis tags.
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