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(P346) Quantification of the toluene dioxygenase of Psudomonas putida F1 by Real-Time TaqMan PCR.
Narasimmalu, Rajendran1, Aoyagi, Reiji1, Kanazawa, Nobuhiro2, Urushigawa, Yoshikuni*,2, Ito, Kiminori1, 1 Yokohama National University, Yokohama, Japan2 Akita Prefectural University, Honjo, Akita Pref., Japan
ABSTRACT- Real-time PCR assay was used to quantify toluene dioxygenase (tod) from Pseudomonas putida F1. Cells were harvested from medium grown with one of the substrates (toluene, benzene, glucose, peptone, yeast extract and succinate) and were analyzed for DNA and RNA. DNase treatment was carried out to remove contaminating DNA from the extracted RNA. RNA samples that showed no amplification of contaminating DNA by conventional PCR were used for quantitative analysis of tod gene (RT-PCR), contaminated DNA, if present (PCR) and 16SrRNA (PCR) by real-time TaqMan PCR. Concentrations of extracted DNA and RNA in P. putida were lower in toluene and benzene added culture than that observed in other substrates. The copies of tod gene were higher in cultures grown with toluene and benzene (105 to 106 copies/ul ng RNA) than that observed in other substrates. The proportions of tod gene in 16SrRNA in P. putida showed a wide variation ranging from 0.01 (glucose) to 100 (benzene), revealing that toluene dioxygenase was successfully inducted by P. putida exposed to benzene and toluene, whereas, no such induction was observed for other substrates. Although conventional PCR assay did not reveal the presence of contaminating DNA in extracted RNA, real time PCR showed amplification of the contaminated DNA in the range of 102 to 103 copies/ul of RNA indicating that the contaminating DNA was present in RNA samples, even after the DNase treatment. From the results, it can be concluded that real-time PCR allows to quantitatively determining the number of gene, contaminating DNA and total number of bacteria in a sample. Such a precise and accurate estimation would be useful for efficiently monitoring bioremediation processes.
Key words: Toluene dioxygenase, Real-time PCR, Substrate, gene expression
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