Production of combinatorial libraries of fused genes by sequential transposition reactions

The use of in vivo and in vitro transposition reac- tions to perform non-combinatorial manipulation of DNAs in molecular biology is widespread. In this work we describe a technique that utilizes two sequential, directed transposition reactions in order to carry out combinatorial DNA manipulations. The methodology relies on the use of two different mutant Tn5 transposase proteins that have different transposon end recognition specificities. We demonstrate that the technique can be used to create large libraries of random fusions between two genes. These transpositional fusions are defined by insertion of a 32 bp linker sequence. We applied the technique to a model system, chloram- phenicol acetyl transferase, to create functional fusions from N- and C-terminally truncated, non- functional genes. Comparative structural analysis suggests that both sides of the linker are inserted into disordered regions in functional proteins.