Strategy for recursive deletion and coupled deletion/plasmid formation systems. The strategy for deletion formation can be used after integration of the transposon into the host's genome. The internal transposon ends (MEs) are used in the second transposition event. Two deletions result from this transposition event in vivo, the first leading to the removal of the internal part of the transposon, and the second resulting in the deletion of a portion of the chromosome. TnpEK/LP binds to the MEs, resulting in blunt-end cleavage and loss of the donor DNA. Tnp-EK/LP then facilitates intra molecular strand transfer into the chromosome. (A) Not all events during strand transfer will result in deletion. Inversions may also result in this mechanism. Deletions may happen to the left or to theright, defining the loss of the corresponding part of the transposon. Deletions to the right will result in loss of all transposon DNA, with the exception of a neutral linker. This event can be detected by replica plating (under conditions of high transposition frequency). Traditional transposition by use of the external pair of transposon ends (IEs) does not occur because the expressed Tnp does not interact with the external ends. (B) The addition of a conditional origin of replication allows for the capture of the deleted chromosomal material into a complementary self-replicating plasmid. The presence of IPTG in the medium results in the capture of these circular DNAs as plasmids that are complementary to the chromosome.
