An efficient stress- free strategy to displace stable bacterial plasmids
An efficient stress- free strategy to displace stable bacterial plasmids
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DOI：
10.2144/000113366
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发表时间：
2010-03-01
期刊：
影响因子：
2.7
通讯作者：
Thomas, Christopher M.
中科院分区：
文献类型：
--
作者：
Hale, Lisa;Lazos, Orestis;Thomas, Christopher M.
确定质粒赋予的表型的关键阶段是其置换或“固化”，以产生无质粒菌株。然而，许多质粒是非常稳定的，不仅因为它们含有多个复制子，而且因为它们可以编码分离后杀伤系统，该系统降低无质粒分离体的活力。我们已经开发了一种有效的治疗策略，包括将复制子的关键区域和分离后的杀伤位点结合到携带sacB的不稳定克隆载体中，sacB赋予对蔗糖的敏感性。针对大肠杆菌F家族和广泛宿主范围的IncP-1家族的质粒，我们证明了来自所有测试克隆的易感常驻质粒的置换。在蔗糖上生长允许分离出许多没有质粒的克隆。这种策略是高效的，并且避免了诱导分离后杀伤系统或其他致死基因产物的影响并使其存活的应激。
A key stage in determining the phenotype(s) conferred by a plasmid is its displacement, or 'curing,' to create a plasmid-free strain. However, many plasmids are very stable, not only because they contain multiple replicons, but also because they can encode post-segregational killing systems that reduce the viability of plasmid-free segregants. We have developed an efficient curing strategy that involves combining key regions of the replicons and the post-segregational killing loci into an unstable cloning vector carrying sacB, which confers sensitivity to sucrose. Targeting plasmids of both the F family of Escherichia coli and the broad-host-range IncP-1 family, we demonstrated displacement of susceptible resident plasmids from all clones tested. Growth on sucrose allowed the isolation of many clones without either plasmid. This strategy is highly efficient and avoids the stress of inducing and surviving the effects of post-segregational killing systems or other lethal gene products.