[Integration and expression of polyphosphate kinase gene in Pseudomonas putida]
Hong-wei Du1, Jun Wu, Lin Xiao
1State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing 210093, China. hongwd@gmail.com
Abstract:
In order to construct high accumulating-phosphate microorganism, the ppk gene from E. coli was inserted to the broad-host-range plasmid pBBR1MCS-2 to form plasmid pBBR1MCS-2-ppk. The complete ppk gene with promoter and terminator sequences from pBBR1MCS-2-ppk was then cloned and inserted to suicide plasmid pUTmini-Tn5 to form plasmid pUTmini-Tn5-ppk, which was transformed into Pseudomonas putida KT2440 by triparental conjugation. Finally, ppk gene was integrated into the chromosomal DNA of KT2440. The results of RT-PCR showed that the selected genetically engineered bacterium KT2440-PPK expressed ppk efficiently, while KT2440 as control expressed weakly. The results of artificial wastewater treatment showed after 1h inoculation, the concentration of poly-phosphate in KT2440-PPK came to the maximum approximately 3.05 mg/g, which was 15 times higher than that in KT2440 at the same experimental condition. And KT2440-PPK can remove more than 90% phosphate in artificial wastewater.
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