Grantee Research Project Results
2004 Progress Report: Metal Biosensors: Development and Environmental Testing
EPA Grant Number: R830907Title: Metal Biosensors: Development and Environmental Testing
Investigators: Anderson, Anne J. , Miller, Charles D. , McLean, Joan E
Institution: Utah State University
EPA Project Officer: Hahn, Intaek
Project Period: May 1, 2003 through April 30, 2006 (Extended to April 30, 2007)
Project Period Covered by this Report: May 1, 2004 through April 30, 2005
Project Amount: $336,000
RFA: Environmental Futures Research in Nanoscale Science Engineering and Technology (2002) RFA Text | Recipients Lists
Research Category: Nanotechnology , Safer Chemicals
Objective:
The objective of this research project is to develop biosensor systems to detect the presence of Cu and Cd in environmental samples.
Current aquatic limits set by the U.S. Environmental Protection Agency for Cu and Cd are based on the concentration of the dissolved ion, water hardness, and pH. The water hardness factor and the pH attributes are considered because of the likelihood of competition between these metal ions and others for binding at the organism’s cell surface. In current practice, many of the tests for metal toxicity have been based on determining the lethal concentrations of Cu or Cd for a specific aquatic organism. The goal of our study is to understand how the metals interact at the gene level with a soil-borne bacterium, Pseudomonas putida, so that we can use molecular techniques to develop biosensors for the rapid determination of the bioavailability of Cu or Cd in environmental samples.
Progress Summary:
Our findings with biosensors in the soil bacterium, Pseudomonas putida, generated with three redox-sensitive promoters : lux fusions, show that Cu and Cd exert different cellular effects. With KCl as a background solution, Cu levels of 0.1 mg/L and above, added as CuCl2, decreased Lux activity from the biosensors in a manner that corresponded with cell death. In contrast, with Cd at levels of 1 and 10 mg/L, added as Cd(NO3)2, a decrease in Lux activity occurred only with a temporary stasis in growth. Current efforts are directed towards generating more specific and sensitive Lux fusions by using promoters selected to be metal responsive.
2D-gel proteomics analysis revealed that discrete soluble- and membrane-associated proteins increased in accumulation when cells were exposed to 5 or 10 mg/L Cu in a minimal medium for 6 hours . Under these conditions, no cell death occurred, and modeling studies predict that Cu is present as the soluble citrate form. Exposure to 10 mg/L Cd caused temporary stasis of growth and the accumulation of different membrane-associated proteins. At 5 mg/L Cd, no differences in soluble proteins were detected from the control treatment. Identification of the proteins is currently underway. According to the biotic ligand model, less than 2 percent free Cd ion was present following the addition of 10 mg/L Cd. Data indicate that t he metal is bound to citrate or phosphate due to the medium composition. The a ddition of Cd phosphate as an authentic complex did not cause changes in cell growth, suggesting that it was not bioavailable.
Future Activities:
We will make presentations at American Society of Microbiology and Institute of Biological Engineering 2006 spring national meetings.
Preparation of DNA Probes for Metal Responsive Genes
Primers have been designed for genes with responses specific to either Cu or Cd. We will use the PCR products from these genes to generate a spotted gene array. Exposure of this array to RNA extracted from exposed and control bacterial cells will determine whether differential expression from these genes can be used as a biosensor. This work will be initiated in the fall of 2005.
Journal Articles:
No journal articles submitted with this report: View all 8 publications for this projectSupplemental Keywords:
metal chelation, growth stasis, proteomics, gene arrays, microbiology, bioavailability, Pseudomonas putida, bacterial sensors, bioengineering, biosensors, cadmium and cadmium compounds, Cd, copper and copper compounds, Cu,, RFA, Scientific Discipline, Toxics, Water, Ecosystem Protection/Environmental Exposure & Risk, POLLUTANTS/TOXICS, Sustainable Industry/Business, Environmental Chemistry, Chemicals, Monitoring/Modeling, New/Innovative technologies, 33/50, Engineering, Chemistry, & Physics, Environmental Engineering, environmental monitoring, nanosensors, cadmium & cadmium compounds, chromium & chromium compounds, bioengineering, environmental measurement, nanotechnology, field monitoring, bacterial sensors, field detection, biosensors, cadmium, heavy metalsProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.