This study aimed to identify the function of polyphosphate kinase gene (ppk) in phosphorus removal. With the Red system, the target DNA with the homologous short arms was amplified in the plasmid pKD4. Then the target DNA was transformed into E. coli ATCC25922 which already had the suicide plasmid pKD46 by electroporation. The plasmid pCP20 was transformed into the recombinant strains to delete the kanamycin resistance gene. With the screening by negative resistance, together with verification using positive and negative primers, the construction of ppk gene deletion strain E. coli/ppk- Kan- was confirmed. The growth characteristics of both the wild-type strain and the mutant strain were determined, and the phosphate accumulating characteristics were compared when cultured in the phosphate luxuriant medium after induced in the phosphate lacking medium. Also the phosphate accumulating characteristics of the two strains were compared after cultured in the anaerobic and aerobic alternating conditions for 5 times. The results showed that the ppk deletion strain E. coli/ppk- Kan- was successfully constructed. There was no growth difference between the mutant strain and the wild-type strain. But in the first 4 hours of log phase, the mutant strain grew faster than the wild-type strain. And 8h later, when both strains were in stationary phase, the mutant strain grew slower than the wild type, indicating that ppk affected the growth of the bacteria. Cultured in the phosphate lacking medium and the phosphate luxuriant medium, the mutant strain's ability of phosphate accumulating didn't decrease in spite of having no ppk gene. After 5 times induction, the amounts of phosphorus in both strains were about 1%-2%. The phosphate amounts in the cells did not increase with increasing inducing times. Polyphosphate or PHB was detected neither at anaerobic phase nor at the aerobic phase. It indicated that the deletion of ppk did not affect the phosphorus removal in wastewater treatment process, and the ppk gene did not show the function of phosphorus removal.
Keywords: E coli.; biological phosphate removal in waste water; phosphate removal characteristics; polyphosphate kinase (PPK); scarless gene deletion.