| Popis: |
Plasmids of the ColE1 family are among the most frequently used plasmids in molecular biology. They were adopted early in the field for many biotechnology applications, and as model systems to study plasmid biology. The mechanism of replication of ColE1 plasmids is well understood, involving the interaction between a plasmid-encoded sense-antisense gene pair (RNAI and RNAII). Because of its mechanism of replication, bacterial cells cannot maintain two different plasmids with the same origin, with one being rapidly lost from the population – a process known as plasmid incompatibility. While mutations in the regulatory genes RNAI and RNAII have been reported to make colE1 plasmids more compatible, there has been no attempt to engineer compatible colE1 origins, which can be used for multi-plasmid applications and that can bypass design constrains created by the current limited plasmid origin repertoire available. Here, we show that by targeting sequence diversity to the loop regions of RNAI (and RNAII), it is possible to select new viable colE1 origins that are compatible with the wild-type one. We demonstrate origin compatibility is not simply determined by sequence divergence in the loops, and that pairwise compatibility is 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. New characterised plasmid origins increase flexibility and accessible complexity of design for challenging synthetic biology applications where biological circuits can be dispersed between multiple independent genetic elements. |
