Engineering bacterial genomes or foreign DNA cloned as bacterial artificial chromosomes (BACs) relies on usage of helper plasmids, which deliver the desired tools transiently into the bacteria to be modified. After the anticipated action is completed the helper plasmids need to be cured. To make this efficient, plasmids are used that are maintained by conditional amplicons or carry a counter-selection marker. Here, we describe new conditional plasmids that can be maintained or cured by using chemical induction or repression. Our method is based on the dependency of plasmids carrying ori6Kγ origin of replication on the presence of protein Π. Ori6Kγ based plasmids are tightly regulated conditional constructs, but they require usually special E. coli strains to operate. To avoid this, we placed the Π protein expression under the control of a co-expressed conditional repressor. Regulating the maintenance of plasmids with administration or removal of chemicals is fully compatible with any other conditional amplicons applied to date. Here, we describe methods for inducing sites specific recombination of BACs as an example. However, the same strategy might be used to construct appropriate helper plasmids for any other transient components of genome editing methodologies such as λred recombinases or CRISPR/Cas components.

工程细菌基因组或克隆为细菌人工染色体（BAC）的外源DNA依赖于辅助质粒的使用，这些辅助质粒可将所需工具瞬时传递到待修饰的细菌中。在完成预期的操作后，需要对辅助质粒进行固化。为了使这种方法有效，使用了由条件扩增子维持或带有反选择标记的质粒。在这里，我们描述了可以通过使用化学诱导或抑制来维持或治愈的新条件质粒。我们的方法是基于携带ori6Kγ复制起点的质粒对蛋白质Π的依赖性。基于Ori6Kγ的质粒是严格调节的条件构建物，但它们通常需要特殊的大肠杆菌应变操作。为避免这种情况，我们将Π蛋白表达置于共表达的条件阻遏物的控制之下。通过施用或去除化学物质来调节质粒的维持与迄今为止应用的任何其他条件扩增子完全兼容。在此，我们以诱导BAC的位点特异性重组的方法为例进行说明。然而，可以使用相同的策略为基因组编辑方法的任何其他瞬时组件（如λred重组酶或CRISPR / Cas组件）构建合适的辅助质粒。