Chaillou, Santiago; Stamou, Pinelopi-Eleftheria; Torres, Leticia L; Riesco, Ana B; Hazelton, Warren; Pinheiro, Vitor B; (2022) Directed evolution of colE1 plasmid replication compatibility: a fast tractable tunable model for investigating biological orthogonality. Nucleic Acids Research , 50 (16) pp. 9568-9579. 10.1093/nar/gkac682.

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Abstract

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.

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