Abstract
The Mu-transposon system is one of the best characterized transposition systems. Under minimal in vitro set-up, Mu transposition requires only a simple reaction buffer, MuA transposase protein, mini-Mu transposon DNA (donor) and target DNA. The reaction proceeds via initial assembly of the transposition complex that directs transposon integration into target DNA with high efficiency and relatively low target site selectivity. These characteristics make the Mu in vitro transposition technology ideal for the generation of comprehensive mutant DNA libraries usable in a variety of molecular biology applications. This technology has successfully been used for DNA sequencing, functional analyses of plasmid DNA and virus genomes, protein engineering for structure/function and protein-protein interaction studies and generation of gene targeting constructions. When electroporated, the in vitro–assembled Mu transposition complexes can also be used for efficient gene delivery in bacteria, yeasts and mammalian cells. Using this protocol we have identified several mutants where Cat-Mu insertion has interrupted genes involved in lipopolysaccharide (LPS) biosynthesis (Pinta et al., 2012).
Keywords: Yersinia enterocolitica, Mu transposase, Transposon insertion library, Lipopolysaccharide, Bacteriophage
Materials and Reagents
Equipment
Procedure
Recipes
Acknowledgments
The protocol was adapted from our previously published paper Pinta et al. (2012). This work was supported by the Academy of Finland (projects 114075, 104361 and 50441) to M.S. and Finnish Glycoscience Graduate School to E.P. We thank Dr Eckhard Strauch for providing the enterocoliticin used in the work.
References
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