Metal Biosensors: Development and Environmental Testing
|Principal Investigator||Anne J. Anderson|
|Institution||Utah State University|
|Relevance to Implications||Some|
|Class of Nanomaterial||Incidental Nanomaterials|
|Broad Research Categories||
|Anticipated Total Funding||$336,000.00|
|Anticipated End Year||2006|
Metals, such as Cu and Cd, threaten the environment and human health because they impair cellular function when present above threshold levels. Currently, assessments of risk are based on chemical techniques that determine only total levels of the metal. These total levels may differ from those that are bioavailable and having an impact on living cells. Bioavailability is influenced by the association of the metal with solids, colloids or low molecular weight ligands. Ligands include inorganic cations (chloride, nitrate and phosphate) as well as the organic structures (di- and tri-carboxylic acids and humic acids). Our objectives are to develop and test biosensors and DNA arrays that will detect Cu and Cd specifically and indicate the bioavailability of these metals to a bacterium.
The biosensors will be constructed with a root-colonizing bacterium, Pseudomonas putida, KT2440. This bacterium is robust, offers no environmental threat and its genomic sequence, almost completed, will be released in 2003. Fusions will be generated between the promoters of P. putida genes responding with increased transcription when exposed to Cu or Cd and the open reading frame of a reporter gene. We will use a reporter encoding for light production to screen for constructs showing increased light emission when exposed to low non-lethal levels of the metals. These promoter-fusion biosensors will be used singly for quantitative assays of Cu/Cd concentration. The response to the metals associated with the different ligands will be assayed to determine how the response relates to total metal concentration and free metal availability. In the laboratory, we will use in situ detection for real-time imaging of the promoter fusion activities while these bacterial sensors are colonizing roots of plants growing in metal-contaminated soils. The fusions will be deployed in plate arrays to permit rapid assessment of Cu and Cd bioavailability. A second detector system will array the KT2440 genes that react to both metals. Differential hybridization of the gene arrays to RNAs extracted from control and sample-exposed P. putida cells will indicate the level of impact of any metals in the samples. We will compare the responses from the promoter fusion constructs to Cu and Cd complexed with different ligands to their responses at the transcriptional level.
We will test the sensitivity and specificity of the biosensor plate arrays and the gene arrays using metal-contaminated soils and water from mine and government sources that are undergoing a remediation process.
We predict that we will generate an array of promoter fusions that respond selectively to Cu and to Cd. We will use these constructs to probe how response to free metal and complexed metals differ. We will explore the use of arrays of these biosensor cells to assess metal bioavailability in environmentally-contaminated samples. Mine spoils and run-off waters will be surveyed. Other DNA arrays of metal responsive genes will permit RNA analysis in cells exposed to metals. Their use will reveal at a global level how the bacterium responds to the metals and to the different complexes. We will probe the usefulness of these tools in monitoring the progress of a bioremediation or treatment process. Understanding how complexed metals are perceived by living cells is essential to predict the risk associated with a toxic metal-contaminated source.