Heavy metals from natural and man-made sources can be a great threat to human and animal life. As small inorganic ions they are challenging to detect, usually requiring expensive and complicated machinery. Several heavy metals can accumulate in the human body, leading to long term toxic effects on the nervous system. Many bacteria have developed strategies to survive in heavy metal rich environments. One of these strategies is a bacterial operon containing genes for detoxification mechanisms controlled by a promoter and a regulatory protein. In this work some of these promoter-protein pairs, Pars-ArsR, PcopA-CueR, PmerTPAD-MerR and PzntA-ZntR from Escherichia coli have been employed in the design and construction of a set of biosensors aimed at the detection of heavy metals in drinking water. Biosensors usually employ biological recognition elements, transducing the signal from these to produce an output that can be integrated into electronic circuitry. The sensors presented in this work focus on reducing complexity and on providing a controlled sensor reaction. The arsenic biosensor ‘AsGard’ is based on the Pars-ArsR pair and functions by making the dissociation of an ArsR-mCherry fusion protein from its binding site in the Pars promoter visible. In the cell, ArsR dissociates from Pars upon binding of trivalent arsenic ions. Immobilising the relevant part of the Pars sequence on a solid plastic support allows for the mobilisation of previously bound ArsR-mCherry proteins in the presence of arsenic to become the sensor output. The AsGard sensor detects arsenic within minutes in a concentration range overlapping with the arsenic thresholds for drinking water as set by the World Health Organisation. Additional prototype sensors are presented bringing a reporter gene under the control of the aforementioned promoters. These sensors have been tested in vivo and in vitro in a cell free transcription translation system and partially detect metal concentrations close to relevant ranges. The Pars based sensor is tuneable in vitro by modifying the ratio of the supplied regulatory protein ArsR and is able to detect arsenic well within the relevant range. Spinach2, a fluorescent RNA aptamer, may make future designs independent from translation, drastically reducing complexity of cell free biosensors based on cis-trans transcriptional regulation.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:721256 |
| Date | January 2017 |
| Creators | Oltmanns, Jan |
| Contributors | French, Chris ; Elfick, Alistair |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/22854 |
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