Construction of green fluorescent protein based bacterial biosensor for heavy metal remediation

  • C. Edward Raja
  • G. S. SelvamEmail author


Environmental contamination by heavy metals is a worldwide problem. Therefore, it is necessary to develop sensitive, effective and inexpensive methods, which can efficiently monitor and determine the level of hazardous metals in the environment. Conventional techniques to analyze metals, suffer from the disadvantages of high cost. Alternatively, development of simple system for monitoring heavy metals pollution is therefore needed. The present approach is based on the use of bacteria that are genetically engineered so that a measurable signal is produced when the bacteria are in contact with the bioavailable metal ions. Reporter genes are widely used as genetic tools for quantification and detection of specific cell population, gene expression and constructing whole cell biosensors as specific and sensitive devices for measuring biologically relevant concentrations of pollutants. An attempt has been made to construct the reporter gene enhanced green fluorescent protein and was expressed under the control of cadR gene, responsible for cadmium resistance. Recombinant strain Escherichia coli cadR30 was used, that carried cadR gene in pET30b expression vector and cloned. Clones confirmed by the expression of enhanced green fluorescent protein was detected under ultraviolet illumination and separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The construction of green fluorescent protein based Escherichia coli bacterial biosensor was developed based on green fluorescent protein expression under the control cadR gene of Pseudomonas aeruginosa BC15. The constructed bacterial biosensor is useful and applicable in determining the availability of heavy metals in soil and wastewater.


Aquatic environment Contamination E.coli Pseudomonas aeruginosa Reporter gene 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Andreazza, R.; Pieniz, S.; Wolf, L.; Lee, M.; Camargo, F. A. O.; Okeke, B. C., (2010). Characterization of copper bioreduction and biosorption by a highly copper resistant bacterium isolated from copper-contaminated vineyard soil. Sci. Total Environ., 408(1), 1501–1507 (7 pages).CrossRefGoogle Scholar
  2. APHA; AWWA; WEF, (1992). Standard methods for the examination of water and wastewater. 18th edition. American Public Health Association, American Water Works Association and the Water Environment Federation. Washington DC., USA.Google Scholar
  3. Barker, L. P.; Brooks, D. M.; Small, P. L. C., (1998). The identification of Mycobacterium marinum genes differentially expressed in macrophage phagosomes using promoter fusions to green fluorescent protein. Molecular Microbiol., 29(5), 1167–1177 (11 pages).CrossRefGoogle Scholar
  4. Barros, F. C. F.; Sousa, F. W.; Cavalcante, R. M.; Carvalho, T. V.; Dias, F. S.; Queiroz, D. C.; Vasconcellos, L. C. G.; Nascimento, R. F., (2008). Removal of copper, nickel and zinc ions from aqueous solution by chitosan-8-hydroxyquinoline beads. Clean., 36(3), 292–298 (7 pages).Google Scholar
  5. Casper, S.; Holt, C., (1996). Expression of the green fluorescent protein-encoding gene from a tobacco mosaic virus-based vector. Gene., 173(1), 69–73 (5 pages).CrossRefGoogle Scholar
  6. Cassidy, M. B.; Leung, K. T.; Lee, H.; Trevors, J. T., (2000). A comparison of enumeration methods for culturable Pseudomonas fluorescens cells marked with green fluorescent protein. J. Microbiol. Mtds., 40(2), 135–145 (11 pages).CrossRefGoogle Scholar
  7. Chakraborty, P.; Babu, G.; Alam, A.; Chaudhari, A., (2008). GFP expressing bacterial biosensor to measure lead contamination in aquatic environment. Curr. Sci., 94(6), 800–805 (6 pages).Google Scholar
  8. Chalfie, M.; Tu, Y.; Euskirchen, G.; Ward, W. W.; Prasher, D. C., (1994). Green fluorescent protein as marker for gene expression. Sci., 263(5148), 802–805 (4 pages).CrossRefGoogle Scholar
  9. Chien, M. K.; Shih, L. H., (2007). An empirical study of the implementation of green supply chain management practices in the electrical and electronic industry and their relation to organizational performances. Int. J. Environ. Sci. Tech., 4(3), 383–394 (12 pages).Google Scholar
  10. D’Souza, S. F., (2001). Microbial biosensors. Biosen. Bioelec., 16(6), 337–353 (17 pages).CrossRefGoogle Scholar
  11. Edward Raja, C.; Selvam, G. S., (2009). Plasmid profile and curing analysis of Pseudomonas aeruginosa as metal resistant. Int. J. Environ. Sci. Tech., 6(2), 259–266 (8 pages).Google Scholar
  12. Giaginis, C.; Gatzidou, E.; Theocharis, S., (2006). DNA repair systems as targets of cadmium toxicity. Toxicol. Appl. Pharmacol., 213(3), 282–290 (9 pages).CrossRefGoogle Scholar
  13. Gronow, M., (1984). Biosensors. Trends Biochem. Sci., 9(8), 336–340 (5 pages).CrossRefGoogle Scholar
  14. Gu, M. B.; Mitchell, R. J.; Kim, B. C., (2004). Whole-cell-based biosensors for environmental biomonitoring and application. Adv. Biochem. Eng. Biotech., 87, 269–305 (37 pages).Google Scholar
  15. Gueu, S.; Yao, B.; Adouby, K.; Ado, G., (2007). Kinetics and thermodynamics study of lead adsorption on to activated carbons from coconut and seed hull of the palm tree. Int. J. Environ. Sci. Tech., 4(1), 11–17 (7 pages).CrossRefGoogle Scholar
  16. Il’yasova, D.; Schwartz, G. G., (2005). Cadmium and renal cancer. Toxicol. Appl. Pharmacol., 207(2), 179–186 (8 pages).CrossRefGoogle Scholar
  17. Ivask, A.; Hakkila, K.; Virta, M., (2001). Detection of organomercurials with sensor bacteria. Analys. Chem., 73(21), 5168–5171 (4 pages).CrossRefGoogle Scholar
  18. Karbassi, A. R.; Nouri, J.; Mehrdadi, N.; Ayaz, G. O., (2008). Flocculation of heavy metals during mixing of freshwater with Caspian Sea water. Environ. Geo., 53(8), 1811–1816 (6 pages).CrossRefGoogle Scholar
  19. Kopera, E.; Schwerdtle, T.; Hartwig, A.; Bal, W., (2004). Co (II) and Cd (II) substitute for Zn (II) in the zincfinger derived from the DNA repair protein XPA, demonstrating a variety of potential mechanisms of toxicity. Chem. Res. Toxicol., 17(11), 1452–1458 (7 pages).CrossRefGoogle Scholar
  20. Laemmli, U. K., (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685 (6 pages).CrossRefGoogle Scholar
  21. Liao, V. H. C.; Chien, M. T.; Tseng, Y. T.; Ou, K. L., (2006). Assessment of heavy metal bioavailability in contaminated sediments and soils using green fluorescent protein based bacterial biosensors. Environ. Poll., 142(1), 17–23 (7 pages).CrossRefGoogle Scholar
  22. Liu, J.; Olsson, G.; Mattiasson, B., (2004). Short-term BOD (BODst) as a parameter for on-line monitoring of biological treatment process Part I. A novel design of BOD biosensor for easy renewal of bio-receptor. Biosen. Bioelect., 20(3), 562–570 (9 pages).CrossRefGoogle Scholar
  23. Ludin, B.; Doll, T.; Meill, R.; Kaech, S.; Matus, A., (1996). Application of novel vectors for GFP-tagging of proteins to study microtubule-associated proteins. Gene., 173(1), 107–111 (5 pages).CrossRefGoogle Scholar
  24. Malakootian, M.; Nouri, J.; Hossaini, H., (2009). Removal of heavy metals from paint industries wastewater using Leca as an available adsorbent. Int. J. Environ. Sci. Tech., 6(2), 183–190 (8 pages).Google Scholar
  25. Moss, J. B.; Price, A. L.; Raz, E.; Driever, W.; Rosenthal, N., (1996). Green fluorescent protein marks skeletal muscle in murine cell lines and zebrafish. Gene., 173(1), 89–98 (10 pages).CrossRefGoogle Scholar
  26. Mulchandani, A.; Bassi, A. S., (1995). Principles and application of biosensors for bioprocess monitoring and control. Crit. Rev. Biotech., 15(2), 105–124 (20 pages).CrossRefGoogle Scholar
  27. Niedenthal, R. K.; Riles, L.; Johnston, M.; Hegemann, J. H., (1996). Green fluorescent protein as a marker for gene expression and subcellular localization in budding yeast. Yeast., 12(8), 773–786 (14 pages).CrossRefGoogle Scholar
  28. Nouri, J.; Khorasani, N.; Lorestani, B.; Karami; M. Hassani; Hassani, A. H.; Yousefi, N., (2009). Accumulation of heavy metals in soil and uptake by plant species with phytoremediation potential. Environ. Earth Sci., 59(2); 315–323 (9 pages).CrossRefGoogle Scholar
  29. Nouri, J.; Lorestani, B.; Yousefi, N.; Khorasani, N.; Hasani, A. H.; Seif, S.; Cheraghi, M., (2011). Phytoremediation potential of native plants grown in the vicinity of Ahangaran lead-zinc mine (Hamedan, Iran). Environ. Earth Sci., 62(3), 639–644 (6 pages).CrossRefGoogle Scholar
  30. Prachayasittikul, V.; Isarankura Na Ayudhaya, C.; Bulow, L., (2001). Lighting E. coli cells as biological sensors for Cd. Biotech. Let., 23(16), 1285–1291 (7 pages).CrossRefGoogle Scholar
  31. Roberto, F.; Barnes, J.; Bruhn, D., (2002). Evaluation of a GFP reporter gene construct for environmental arsenic detection. Talanta, 58(1), 181–188 (8 pages).CrossRefGoogle Scholar
  32. Samarghandi, M. R.; Nouri, J.; Mesdaghinia, A. R.; Mahvi, A. H.; Nasseri, S.; Vaezi, F., (2007). Efficiency removal of phenol, lead and cadmium by means of UV/TiO2/H2O2 processes. Int. J. Environ. Sci. Tech., 4(1), 19–25 (7 pages).CrossRefGoogle Scholar
  33. Sambrook, J.; Fritsch, E. F.; Maniatis, T., (). Molecular Cloning: A laboratory manual, 2nd Ed. Cold spring harbour, NY: Cold spring harbour laboratory press.Google Scholar
  34. Selifonova, O.; Burlage, R.; Barkay, T., (1993). Bioluminescent sensors for detection of bioavailable Hg (II) in the environment. Appl. Environ. Microbiol., 59(9), 3083–3090 (8 pages).Google Scholar
  35. Shetty, R.; Ramanathan, S.; Badr, I.; Wolford, J.; Daunert, S., (1999). Green fluorescent protein in the design of a living biosensing system for L-arabinose. Analys. Chem., 71(4), 763–768 (6 pages).CrossRefGoogle Scholar
  36. Shetty, R. S.; Deo, S. K.; Shah, P.; Sun, P.; Rosen, B. P.; Daunert, S., (2003). Luminescence based whole cell sensing systems for cadmium and lead using genetically engineered bacteria. Analys. Bioanalys. Chem., 376(1), 11–17 (7 pages).Google Scholar
  37. Stiner, L.; Halverson, L. J., (2002). Development and characterization of a green fluorescent protein based bacterial biosensor for bioavailable toluene and related compounds. Appl. Environ. Microbiol., 68(4), 1962–1971 (10 pages).CrossRefGoogle Scholar
  38. Tauriainen, S.; Karp, M.; Chang, W.; Virta, M., (1998). Luminescent bacterial sensor for cadmium and lead. Biosen. Bioelect., 13(9), 931–938 (8 pages).CrossRefGoogle Scholar
  39. Townsend, A. T.; Miller, K. A.; Mclean, S.; Aldous, S., (1998). The determination of copper, zinc, cadmium and lead in urine by high resolution inductively coupled plasma mass spectrometry. J. Analys. Atom. Spect., 13, 1213–1219 (7 pages).CrossRefGoogle Scholar
  40. Vandeermeer, J. R.; Tropel, D.; Jaspers, M., (2004). Illuminating the detection chain of bacterial bioreporters. Environ. Microbiol., 6(10), 1005–1020 (16 pages).CrossRefGoogle Scholar
  41. Wang, S.; Hazelrigg, T., (1994). Implications for bcd mRNA localization from spatial distribution of exu protein in Drosophila oogenesis. Nature, 69, 400–403 (4 pages).CrossRefGoogle Scholar
  42. Willardson, M. B. B.; Wilkins, F. J.; Rand, A. T.; Schupp, M. J.; Hill, K. K.; Keim, P.; Jackson, J. P., (1998). Development and testing of a bacterial biosensor for toluene based environmental contaminants. Appl. Environ. Microbiol., 64(3), 1006–1012 (7 pages).Google Scholar
  43. Yagi, K., (2007). Application of whole cell bacterial sensors in biotechnology and environmental science. Appl. Microbiol. Biotech., 73(6), 1251–1258 (8 pages).CrossRefGoogle Scholar

Copyright information

© Islamic Azad University 2011

Authors and Affiliations

  1. 1.Department of Biochemistry, School of Biological SciencesCenter for Excellence in Genomic Sciences, Madurai Kamaraj UniversityMaduraiIndia

Personalised recommendations