✅ CIFR (Clone-Integrate-Flip-out-Repeat): a toolset for iterative genome and pathway engineering of Gram-negative bacteria

science

Advances in genome engineering enable precise and customizable modifications of bacterial species. Toolsets for metabolic engineering that exhibit broad-host compatibility are particularly valued owing to their portability. Tn5 transposon vectors have been widely used to establish random integrations of desired DNA sequences into bacterial genomes. However, the iteration of the procedure remains challenging because of the limited availability of selection markers. Here, we present CIFR, a mini-Tn5 integration system for iterative genome engineering. The pCIFR vectors incorporate attP and attB sites flanking an antibiotic resistance marker used to select for the insertion. Subsequent removal of these antibiotic determinants is facilitated by the Bxb1 integrase, and a user-friendly counter-selection marker, both encoded in auxiliary plasmids.

BioRxiv     Nov 07, 2024