[Construction of luxAB-labelled Pseudomonas aeruginosa]

To construct a genetically engineered Pseudomonas aeruginosa expressing luxAB luciferase gene.

Use the Bgl II enzyme to cut the plasmid pUC-luxAB, then recovered luxAB segment, and concatenate the plasmid carrier cut by the enzyme BamH I to form the reconstructed plasmid pBBR-luxAB, then transform E. coli DH5alpha competent cells. The positive clones that contain the reconstructed plasmid pBBR-luxAB experience the multiple screening by gentamycin (Gm), chloromycin (Cm) and luminescence detection, and at the same time, set up the control bacteria. Extract the plasmid pBBR-luxAB, then cut by enzyme, and then gel electrophoresis, and validate the rightness of the plasmid construction. By the mean of 2-generation mating, introduce the plasmid pBBR-luxAB into Pseudomonas aeruginosa, construct the gene engineered bacteria P. aeruginosa (pBBR-luxAB), and then measure the reproductive stableness of the plasmid, bioluminescence kinetic curves and relationship between luminescence and plate counting.

Successfully construct the pBBR-luxAB reconstructed plasmid, and surely, it is successfully introduced to P. aeruginosa. After 4 successively transfer, the pBBR-luxAB retaining rate still can keep at 93%. After the addition of substrates for 20 min, the luminous intensity of the reconstructed bacteria tends to be stable (1.32 mV/ml). There is a significantly positive correlation between luminous intensity and viable cell counting (r = 0.96, P < 0.05).

This research successfully constructed luxAB labelled P. aeruginosa, providing a useful means to study the survival, migration and damage of P. aeruginosa.