Cloning and studing the function of opd gene encoding organophosphorus hydrolase in Escherichia coli

Document Type : Research Paper

Authors

1 Ph. D. Candidate, Department of Plant Protection, University College of Agriculture & Natural Resoucrs, University of Tehran, Karaj, Iran

2 Professor, Department of Plant Protection, University College of Agriculture & Natural Resoucrs, University of Tehran, Karaj, Iran

3 Postdoc Fellowship, Department of Biology, McMaster University, Hamilton, Canada

Abstract

Organophosphorus compounds are widely used, and their presence in different components of the ecosystem has led to harmful effects on the environment. The use of microorganisms in detoxification of xenobiotic compounds and measurement of pesticide residues is considered as an environmental-friendly and appropriate method. Organophosphorus hydrolase (opd), is a phosphotriester hydrolase enzyme which is discovered in some soil microorganisms such as Flavobacterium sp. and has a wide range of substrates and is able to hydrolyze many organophosphorus compounds. In the present study, codon optimization was done to express the protein in Escherichia coli. Following omission of the signal peptide sequence and substitution of serine by methionine as the start codon, this fragment was cloned under the control of lac promoter in a promoter-probe vector (pTH1705) as a transcriptional fusion and was used for transformation in two Escherichia coli strains DH5α and XL1-blue. To measure the performance of the transgenic bacteria expressing opd gene (as in trans), they were cultured in M9 mineral medium containing 10 and 50 mg/L diazinon and appropriate available carbon and nitrogen sources. Studying the growth curve of V103 strain revealed that bacterial growth in the presence of 50 mg/L diazinon had been affected while this effect in the presence of 10 mg/L diazinon was minor. Results obtained from high-performance liquid chromatography as a standard method to compare with the performance of obtained transgenic strain showed that after 24 hours, diazinon concentration (initially 10mg/L ) in the bacterial culture medium of V100, V101, V102 and V103 strains reached to 6.38, 7.19, 7.09 and 5.74, respectively. These results reflected the successful transfer and expression of target gene in the bacterium and this genetically engineered bacterium was able to degrade organophosphorus compounds directly.

Keywords

References

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Iranian Journal of Plant Protection Science
Volume 48, Issue 2 - Serial Number 2
February 2018
Pages 253-269

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History

  • Receive Date: 07 May 2017
  • Revise Date: 24 July 2017
  • Accept Date: 23 January 2018

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