KU Leuven, Department of Pharmaceutical and Pharmacological Sciences, Rega Institute for Medical Research, Herestraat 49, 3000 Belgium.
University College London, Institute of Structural and Molecular Biology, University College London, Gower Street, London WC1E 6BT, UK.
Nucleic Acids Res. 2022 Sep 9;50(16):9568-9579. doi: 10.1093/nar/gkac682.
Plasmids of the ColE1 family are among the most frequently used in molecular biology. They were adopted early for many biotechnology applications, and as models to study plasmid biology. Their mechanism of replication is well understood, involving specific interactions between a plasmid encoded sense-antisense gene pair (RNAI and RNAII). Due to such mechanism, two plasmids with the same origin cannot be stably maintained in cells-a process known as incompatibility. While mutations in RNAI and RNAII can make colE1 more compatible, there has been no systematic effort to engineer new compatible colE1 origins, which could bypass technical design constraints for multi-plasmid applications. Here, we show that by diversifying loop regions in RNAI (and RNAII), it is possible to select new viable colE1 origins compatible with the wild-type one. We demonstrate that sequence divergence is not sufficient to enable compatibility and pairwise interactions are not an accurate guide for higher order interactions. We identify potential principles to engineer plasmid copy number independently from other regulatory strategies and we propose plasmid compatibility as a tractable model to study biological orthogonality.
ColE1 家族的质粒是分子生物学中最常使用的质粒之一。它们很早就被用于许多生物技术应用，并被用作研究质粒生物学的模型。它们的复制机制已经被很好地理解，涉及到质粒编码的 sense-antisense 基因对（RNAI 和 RNAII）之间的特异性相互作用。由于这种机制，两个具有相同起源的质粒不能在细胞中稳定共存——这一过程被称为不相容性。虽然 RNAI 和 RNAII 的突变可以使 colE1 更加相容，但还没有人系统地努力设计新的相容 colE1 起源，以绕过多质粒应用的技术设计限制。在这里，我们展示了通过多样化 RNAI（和 RNAII）中的环区，可以选择与野生型相容的新的可行的 colE1 起源。我们证明序列的差异不足以实现相容性，并且成对相互作用并不是更高阶相互作用的准确指南。我们确定了一些可以独立于其他调控策略来设计质粒拷贝数的原则，并提出了质粒相容性作为研究生物学正交性的一个可行模型。
