Fig. 7

Diagram for the CRIPSR–Cas9-assisted genome editing in P. putida KT2440. Day 1: Introduce the pCAS-RK2K plasmid into P. putida KT2440, and then inoculate the transformants in LB medium overnight; Day 2: Transfer the cultivated cells into fresh LB medium and add arabinose to trigger expression of λ-Red proteins. Next, cells were prepared as elecompetent cells and the pSEVA-gRNAT plasmids were transferred to KT2440 harboring pCAS-RK2K. Day 3: Screen out the mutants by colony PCR. The mutants were inoculated in LB medium containing antibiotic and rhamnose in the morning; Streak the cultivated cells on LB agar in the evening; Day 4: Screen out the mutants that have lost the pSEVA-gRNAT plasmids, and inoculate the mutants in LB medium containing glucose and sucrose. Next, in the evening, the cultivated cells were streak on plate containing glucose and sucrose, and then cultivated overnight. The mutants that have been cured of pSEVA-gRNAT can be used for the next round of genome editing; Day 5: Identify the mutants from the selection plate