Resolving replication incompatibility between chloroplast and conjugative plasmids in E. coli

Chloroplast genomes present a promising chassis for engineering photosynthetic eukaryotes, but efficient delivery of large DNA constructs back into the organelle remains a major technical barrier. Conventional transformation methods rely on purified DNA and physical force to drive uptake into the chloroplast, often resulting in DNA shearing and thus low transfer efficiency for large constructs. Bacterial conjugation offers an attractive alternative as this is an entirely in vivo process, enabling DNA transfer without any physical manipulation. To assess the feasibility of this approach, we attempted to generate an Escherichia coli donor strain carrying both the broad-host-range conjugative plasmid pTA-Mob (52.7 kb) and the cloned Phaeodactylum tricornutum chloroplast genome (pPt_Cp, 132.9 kb). Unexpectedly, pTA-Mob and pPt_Cp proved incompatible: co-maintenance could not be achieved via electroporation, and conjugation with a self-transmissible pTA-Mob variant resulted in a ∼10□-fold decrease in transfer efficiency. Systematic testing of pTA-Mob and pPt_Cp plasmid variants as well as sequence analysis of evolved transconjugants revealed that the incompatibility arose from the pBBR1 replicon present in pTA-Mob. Guided by these insights, we identified an alternative conjugative plasmid, pRL443, that was compatible with pPt_Cp. Together, these findings provide a framework for dissecting plasmid incompatibility when working with large constructs and establish a functional conjugative system capable of mobilizing the P. tricornutum chloroplast genome.