Skilton Rachel J, O'Neill Colette, Thomson Nicholas R, Lampe David J, Clarke Ian N
Molecular Microbiology Group, Faculty of Medicine, University of Southampton, Southampton, Hants, SO16 6YD, UK.
Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambs, CB10 1RQ, UK.
Wellcome Open Res. 2021 Apr 13;6:82. doi: 10.12688/wellcomeopenres.16665.1. eCollection 2021.
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.
已经为……开发了遗传系统，但极低的转化频率仍然是一个重大瓶颈。我们的目标是开发一种自我复制的转座子递送载体用于……，该载体可以在转座酶诱导之前进行扩增。我们构建了/穿梭载体，其携带在……启动子控制下的……C9转座酶以及转座子与β-内酰胺酶基因的新型重排。通过免疫印迹和DNA测序监测转座酶的活性。我们构建了pSW2-mCh-C9，一种……质粒，设计用作自我复制载体，携带在……启动子控制下的……C9转座酶及其转座子。然而，经过多次转化尝试后，我们无法在……中回收该质粒。因此，我们组装了两个新的缺失质粒pSW2-mCh-C9-ΔTpon，其仅携带（在……启动子控制下的）……C9转座酶，以及一个姊妹载体（相同序列骨架）pSW2-mCh-C9-ΔTpase，其携带其同源转座子。我们证明，构成pSW2-mCh-C9-ΔTpon和pSW2-mCh-C9-ΔTpase的生物成分在……中具有活性。这两种质粒都可以在……中独立回收。我们试图通过转化以及与携带pSW2-mCh-C9-ΔTpon和pSW2-RSGFP-Tpon（pSW2-mCh-C9-ΔTpase的绿色荧光版本）的……菌株进行混合感染来进行横向基因转移。尽管成功实现了混合感染，但无法回收携带这两种质粒版本的后代。我们设计了一种自我复制质粒载体pSW2-mCh-C9用于……，其携带在……启动子控制下的……C9转座酶。虽然它可以转化到……中，但无法在……中回收。基于选定的缺失和表型分析，我们得出结论，诱导型启动子的低水平表达导致过早转座，从而在转化过程早期导致质粒丢失。
