Plasmid pK18UMP was integrated
into the chromosome of P. putida KT2440 via single crossover. Positive
clones should be Kan resistant and 5-FU sensitive. One clone selected
on LBKan platewas incubated in LB at 30 °C for 24 h to promote the double
crossover. Further selectionwas performed on an LB5-FU plate to select
for clones that had lost the vector. Ten positive clones that were
resistant to 5-FU and sensitive to kanamycin were obtained.