Construction of Novel Phytochelatins by Overlap Oligonucleotides

Authors

  • Behnaz Saffar Genetic Department, Faculty of Science and Research Institute of Biotechnology, Shahrkord University, Shahrkord, Iran.
  • Mohsen Mobini Genetic Department, Faculty of Science and Research Institute of Biotechnology, Shahrkord University, Shahrkord, Iran.
  • Azadeh Mohamadi Genetic Department, Faculty of Science and Research Institute of Biotechnology, Shahrkord University, Shahrkord, Iran.

Keywords:

Design of oligonucleotide, Hexahistidine, Synthetic phytochelatin

Abstract

Synthetic phytochelatins are protein analogs of phytochelatin with similar heavy metal binding affinities that can be easily produced from a synthetic DNA template. We design synthetic phytochelatin [(Glu-Cys)n Gly] linked to hexahistidine by viral linker peptide and then followed by gene synthesis and cloning of it. Then peptide coding gene (synthetic phytochelatin with linker and hexahistidine) was designed exactly and constructed with step by step methods by overlapping oligonucleotides using T4 DNA Ligase. Finally, synthesized gene amplified by PCR, cloned in pTZ57R/T and transformed to Escherichia coli (DH5α). The results of sequencing show that some types of synthetic phytochelatin (EC4, EC12, and EC20) with linker and hexahistidine were constructed and cloned in vector.

Downloads

Download data is not yet available.

References

Bae, W., Mulchandani, A. & Chen, W. (2002). Cell surface display of synthetic phytochelatins using ice nucleation protein for enhanced heavy metal bioaccumulation. J. Inorg. Biochem., 88(2): 223–227. https://doi.org/10.1016/S0162-0134(01)00392-0.

Bontidean, I., Ahlqvist, J., Mulchandani, A., Chen, W., Bae, W., Mehra, R.K., Mortari, A. & Csöregi, E. (2003). Novel synthetic phytochelatin-based capacitive biosensor for heavy metal ion detection. Biosens. Bioelectron., 18(5-6): 547–553. https://doi.org/10.1016/s0956-5663(03)00026-5.

Cobbett, C.S. (2000). Phytochelatins and their Roles in Heavy Metal Detoxification. Plant Physiol., 123(3): 825–832. https://doi.org/10.1104/pp.123.3.825.

Bae, W., Mehra, R.K., Mulchandani, A. & Chen, W. (2001). Genetic Engineering of Escherichia coli for Enhanced Uptake and Bioaccumulation of Mercury. Appl. Environ. Microbiol., 67(11): 5335–5338. https://doi.org/10.1128/AEM.67.11.5335-5338.2001.

Bae, W. & Mehra, R.K. (1997). Metal-binding characteristics of a phytochelatin analog (Glu-Cys)2Gly. J. Inorg. Biochem., 68(3): 201–210. https://doi.org/10.1016/S0162-0134(97)00099-8.

Bae, W., Chen, W., Mehra, R. & Mulchandani A. (2000). Heavy metal removal using bacteria displaying synthetic phytochelatins. Acs Division of Environmental Chemistry, Preprints. 40: 793-794.

Cobbett, C. & Goldsbrough, P. (2002). Phytochelatins and metallothioneins: roles in heavy metal detoxification and homeostasis. Annu. Rev. Plant Biol., 53: 159–182. https://doi.org/10.1146/annurev.arplant.53.100301.135154.

Xue, F., Gu, Z. & Feng, J.A. (2004). LINKER: a web server to generate peptide sequences with extended conformation. Nucleic Acids Res., 32: W562–W565. https://doi.org/10.1093/nar/gkh422.

Zhang, J., Yun, J., Shang, Z., Zhang, X. & Pan, B. (2009). Design and optimization of a linker for fusion protein construction. Prog. Nat. Sci., 19(10): 1197–1200. https://doi.org/10.1016/j.pnsc.2008.12.007.

George, R.A. & Heringa, J. (2002). An analysis of protein domain linkers: their classification and role in protein folding. Protein Eng. Des. Sel., 15(11): 871–879. https://doi.org/10.1093/protein/15.11.871.

Mahnam, K., Saffar, B., Moboni, M. & Mohamadi, A. Design and construction of a novel metal binding peptide for sequestering of heavy metals by molecular dynamics simulation. Unpublished.

Borodina, T.A., Lehrach, H. & Soldatov, A.V. (2003). Ligation-based synthesis of oligonucleotides with block structure. Anal Biochem., 318(2): 309–313. https://doi.org/10.1016/s0003-2697(03)00250-1.

Rouillard, J.M., Lee, W., Truan, G., Gao, X., Zhou, X. & Gulari, E. (2004). Gene2Oligo: oligonucleotide design for in vitro gene synthesis. Nucleic Acids Res., 32: W176–W180. https://doi.org/10.1093/nar/gkh401.

Hoover, D.M. & Lubkowski, J. (2002). DNAWorks: an automated method for designing oligonucleotides for PCR-based gene synthesis. Nucleic Acids Res., 30(10): e43. https://doi.org/10.1093/nar/30.10.e43.

Au, L.C., Yang, F.Y., Yang, W.J., Lo, S.H. & Kao, C.F. (1998). Gene synthesis by a LCR-based approach: high-level production of leptin-L54 using synthetic gene in Escherichia coli. Biochem. Biophys. Res. Commun., 248(1): 200–203. https://doi.org/10.1006/bbrc.1998.8929.

Bae, W., Chen, W., Mulchandani, A. & Mehra, R.K. (2000). Enhanced bioaccumulation of heavy metals by bacterial cells displaying synthetic phytochelatins. Biotechnol. Bioeng., 70(5): 518–524. https://doi.org/10.1002/1097-0290(20001205)70:5<518::aid-bit6>3.0.co;2-5.

Saffar, B., Yakhchali, B. & Arbabi, M. (2007). Development of a bacterial surface display of hexahistidine peptide using CS3 pili for bioaccumulation of heavy metals. Curr. Microbiol., 55(4): 273–277. https://doi.org/10.1007/s00284-005-0511-2.

Downloads

Abstract views: 26 / PDF downloads: 15

Published

2012-07-01

How to Cite

Saffar, B., Mobini, M., & Mohamadi, A. (2012). Construction of Novel Phytochelatins by Overlap Oligonucleotides. Advances in BioScience, 3(3), 135–138. Retrieved from https://journals.sospublication.co.in/ab/article/view/99

Issue

Vol. 3 No. 3 (2012): July

Section

Articles

License

Copyright (c) 2012 The author(s) retains the copyright of this article.

Creative Commons License

This work is licensed under a Creative Commons Attribution 4.0 International License.

This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.