Progress Towards an Inducible, Replication-proficient Transposon Delivery Vector for

Genetic systems have been developed for but the extremely low transformation frequency remains a significant bottleneck.  Our goal is to develop a self-replicating transposon delivery vector for which can be expanded prior to transposase induction. We made / shuttle vectors bearing the C9  transposase under control of the promoter and a novel rearrangement of the transposon with the β-lactamase gene.  Activity of the transposase was monitored by immunoblot and by DNA sequencing. We constructed pSW2-mCh-C9, a plasmid designed to act as a self-replicating vector carrying both the C9  transposase under promoter control and its transposon.  However, we were unable to recover this plasmid in following multiple attempts at transformation. Therefore, we assembled two new deletion plasmids pSW2-mCh-C9-ΔTpon carrying only the C9  transposase (under promoter control) and a sister vector (same sequence backbone) pSW2-mCh-C9-ΔTpase carrying its cognate transposon.  We demonstrated that the biological components that make up both pSW2-mCh-C9-ΔTpon and pSW2-mCh-C9-ΔTpase are active in Both these plasmids could be independently recovered in We attempted to perform lateral gene transfer by transformation and mixed infection with strains bearing pSW2-mCh-C9-ΔTpon and pSW2-RSGFP-Tpon (a green fluorescent version of pSW2-mCh-C9-ΔTpase).  Despite success in achieving mixed infections, it was not possible to recover progeny bearing both versions of these plasmids. We have designed a self-replicating plasmid vector pSW2-mCh-C9 for carrying the C9  transposase under promoter control.  Whilst this can be transformed into it cannot be recovered in  Based on selected deletions and phenotypic analyses we conclude that low level expression from the inducible promoter is responsible for premature transposition and hence plasmid loss early on in the transformation process.