Validation and calibration of a novel GEM biosensor for specific detection of Cd2+, Zn2+, and Pb2+
Abstract Background In this study, we designed a novel genetic circuit sensitive to Cd2+, Zn2+ and Pb2+ by mimicking the CadA/CadR operon system mediated heavy metal homeostasis mechanism of Pseudomonas aeruginosa. The regular DNA motifs on natural operon were reconfigured and coupled with the enhan...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2023-12-01
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| Series: | BMC Biotechnology |
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| Online Access: | https://doi.org/10.1186/s12896-023-00820-7 |
| _version_ | 1797397910499885056 |
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| author | H. M. L. P. B. Herath W. R. M. de Silva R. S. Dassanayake Y. I. N. S. Gunawardene J. R. P. Jayasingha M. K. Gayashan L. O. B. Afonso K. M. N. de Silva |
| author_facet | H. M. L. P. B. Herath W. R. M. de Silva R. S. Dassanayake Y. I. N. S. Gunawardene J. R. P. Jayasingha M. K. Gayashan L. O. B. Afonso K. M. N. de Silva |
| author_sort | H. M. L. P. B. Herath |
| collection | DOAJ |
| description | Abstract Background In this study, we designed a novel genetic circuit sensitive to Cd2+, Zn2+ and Pb2+ by mimicking the CadA/CadR operon system mediated heavy metal homeostasis mechanism of Pseudomonas aeruginosa. The regular DNA motifs on natural operon were reconfigured and coupled with the enhanced Green Fluorescent Protein (eGFP) reporter to develop a novel basic NOT type logic gate CadA/CadR-eGFP to respond metal ions mentioned above. A Genetically Engineered Microbial (GEM)-based biosensor (E.coli-BL21:pJET1.2-CadA/CadR-eGFP) was developed by cloning the chemically synthesised CadA/CadR-eGFP gene circuit into pJET1.2-plasmid and transforming into Escherichia coli (E. coli)-BL21 bacterial cells. Results The GEM-based biosensor cells indicated the reporter gene expression in the presence of Cd2+, Zn2+ and Pb2+ either singly or in combination. Further, the same biosensor cells calibrated for fluorescent intensity against heavy metal concentration generated linear graphs for Cd2+, Zn2+ and Pb2+ with the R2 values of 0.9809, 0.9761 and 0.9758, respectively as compared to non-specific metals, Fe3+ (0.0373), AsO4 3− (0.3825) and Ni2+ (0.8498) making our biosensor suitable for the detection of low concentration of the former metal ions in the range of 1–6 ppb. Furthermore, the GEM based biosensor cells were growing naturally within the concentration range of heavy metals, at 37 °C and optimum pH = 7.0 in the medium, resembling the characteristics of wildtype E.coli. Conclusion Finally, the novel GEM based biosensor cells developed in this study can be applied for detection of targeted heavy metals in low concentration ranges (1–6 ppb) at normal bacterial physiological conditions. |
| first_indexed | 2024-03-09T01:18:02Z |
| format | Article |
| id | doaj.art-dd18f3529a0f429aae0659bbf3eaf325 |
| institution | Directory Open Access Journal |
| issn | 1472-6750 |
| language | English |
| last_indexed | 2024-03-09T01:18:02Z |
| publishDate | 2023-12-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Biotechnology |
| spelling | doaj.art-dd18f3529a0f429aae0659bbf3eaf3252023-12-10T12:23:08ZengBMCBMC Biotechnology1472-67502023-12-0123111910.1186/s12896-023-00820-7Validation and calibration of a novel GEM biosensor for specific detection of Cd2+, Zn2+, and Pb2+H. M. L. P. B. Herath0W. R. M. de Silva1R. S. Dassanayake2Y. I. N. S. Gunawardene3J. R. P. Jayasingha4M. K. Gayashan5L. O. B. Afonso6K. M. N. de Silva7Centre for Advanced Materials and Devices (CAMD), Department of Chemistry, Faculty of Science, University of ColomboCentre for Advanced Materials and Devices (CAMD), Department of Chemistry, Faculty of Science, University of ColomboCentre for Advanced Materials and Devices (CAMD), Department of Chemistry, Faculty of Science, University of ColomboMolecular Medicine Unit, Faculty of Medicine, University of KelaniyaCentre for Advanced Materials and Devices (CAMD), Department of Chemistry, Faculty of Science, University of ColomboCentre for Advanced Materials and Devices (CAMD), Department of Chemistry, Faculty of Science, University of ColomboSchool of Life and Environmental Sciences, Faculty of Science, Engineering and Built Environment, Deakin UniversityCentre for Advanced Materials and Devices (CAMD), Department of Chemistry, Faculty of Science, University of ColomboAbstract Background In this study, we designed a novel genetic circuit sensitive to Cd2+, Zn2+ and Pb2+ by mimicking the CadA/CadR operon system mediated heavy metal homeostasis mechanism of Pseudomonas aeruginosa. The regular DNA motifs on natural operon were reconfigured and coupled with the enhanced Green Fluorescent Protein (eGFP) reporter to develop a novel basic NOT type logic gate CadA/CadR-eGFP to respond metal ions mentioned above. A Genetically Engineered Microbial (GEM)-based biosensor (E.coli-BL21:pJET1.2-CadA/CadR-eGFP) was developed by cloning the chemically synthesised CadA/CadR-eGFP gene circuit into pJET1.2-plasmid and transforming into Escherichia coli (E. coli)-BL21 bacterial cells. Results The GEM-based biosensor cells indicated the reporter gene expression in the presence of Cd2+, Zn2+ and Pb2+ either singly or in combination. Further, the same biosensor cells calibrated for fluorescent intensity against heavy metal concentration generated linear graphs for Cd2+, Zn2+ and Pb2+ with the R2 values of 0.9809, 0.9761 and 0.9758, respectively as compared to non-specific metals, Fe3+ (0.0373), AsO4 3− (0.3825) and Ni2+ (0.8498) making our biosensor suitable for the detection of low concentration of the former metal ions in the range of 1–6 ppb. Furthermore, the GEM based biosensor cells were growing naturally within the concentration range of heavy metals, at 37 °C and optimum pH = 7.0 in the medium, resembling the characteristics of wildtype E.coli. Conclusion Finally, the novel GEM based biosensor cells developed in this study can be applied for detection of targeted heavy metals in low concentration ranges (1–6 ppb) at normal bacterial physiological conditions.https://doi.org/10.1186/s12896-023-00820-7Microbial biosensorsCadmiumLeadZincGenetic circuitsHeavy metal detection |
| spellingShingle | H. M. L. P. B. Herath W. R. M. de Silva R. S. Dassanayake Y. I. N. S. Gunawardene J. R. P. Jayasingha M. K. Gayashan L. O. B. Afonso K. M. N. de Silva Validation and calibration of a novel GEM biosensor for specific detection of Cd2+, Zn2+, and Pb2+ BMC Biotechnology Microbial biosensors Cadmium Lead Zinc Genetic circuits Heavy metal detection |
| title | Validation and calibration of a novel GEM biosensor for specific detection of Cd2+, Zn2+, and Pb2+ |
| title_full | Validation and calibration of a novel GEM biosensor for specific detection of Cd2+, Zn2+, and Pb2+ |
| title_fullStr | Validation and calibration of a novel GEM biosensor for specific detection of Cd2+, Zn2+, and Pb2+ |
| title_full_unstemmed | Validation and calibration of a novel GEM biosensor for specific detection of Cd2+, Zn2+, and Pb2+ |
| title_short | Validation and calibration of a novel GEM biosensor for specific detection of Cd2+, Zn2+, and Pb2+ |
| title_sort | validation and calibration of a novel gem biosensor for specific detection of cd2 zn2 and pb2 |
| topic | Microbial biosensors Cadmium Lead Zinc Genetic circuits Heavy metal detection |
| url | https://doi.org/10.1186/s12896-023-00820-7 |
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