Progress towards an inducible, replication-proficient transposon delivery vector for

DOI

10.12688/wellcomeopenres.16665.1

Authors

Rachel J. Skilton, Molecular Microbiology Group, Faculty of Medicine, University of Southampton, Southampton, Hants, SO16 6YD, UK.
Colette O'Neill, Molecular Microbiology Group, Faculty of Medicine, University of Southampton, Southampton, Hants, SO16 6YD, UK.
Nicholas R. Thomson, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambs, CB10 1RQ, UK.
David J. Lampe, Department of Biological Sciences, Duquesne University, 600 Forbes Ave., Pittsburgh, Pennsylvania, 15116, USA.
Ian N. Clarke, Molecular Microbiology Group, Faculty of Medicine, University of Southampton, Southampton, Hants, SO16 6YD, UK.

Document Type

Journal Article

Publication Date

1-1-2021

Publication Title

Wellcome open research

Keywords

Chlamydia, Himar1, transformation, transposon

Abstract

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.

Open Access

OA

Repository Citation

Skilton, R. J., O'Neill, C., Thomson, N. R., Lampe, D. J., & Clarke, I. N. (2021). Progress towards an inducible, replication-proficient transposon delivery vector for. Wellcome open research. https://doi.org/10.12688/wellcomeopenres.16665.1