Generation of deletions and duplications using transposons as portable regions of homology with emphasis on mud and Tn10 transposons.

In bacteria complementation and dominance testing requires the establishment of a diploid state for the gene of interest. In addition, it is often desirable to characterize reporter fusion constructs in strains with both the reporter fusion and an intact gene copy present in single copy. Transposons provide portable regions of homology to facilitate construction of targeted chromosomal rearrangements such as deletions and duplications. The properties of the large Mud transposons, MudA and MudB allow for the direct duplication and deletion of virtually any region of the Salmonella enterica chromosome between the points of two Mud insertions in a simple bacteriophage P22 transductional cross. Furthermore, duplication construction will be described for the generation of strains with a lac operon transcriptional fusion or lacZ gene translational fusion to any gene of interest at the join-point of the duplication with a second intact copy of the gene of interest located in tandem single copy in the same chromosome. In addition, methods for generation of tandem chromosomal duplications using transposon Tn10 as portable regions of homology are presented. These allow construction of strains duplicated for any gene of interest in tandem, single copy on the chromosome to allow for complementation and dominance testing for alleles for virtually any gene.