To identify trace metal binding peptides that have high affinity for lead, cadmium, copper and mercury.
To introduce genes encoding trace metal binding peptides into transgenic Chlamydomonas to increase its trace metal binding capacity.
To develop production and delivery systems for the use of trace metal accumulating algae to remediate areas of concern in the Great Lakes Region.

Heavy metal contamination of water and sediments in the Great Lakes Basin has been an on-going problem since the industrialization of the Great Lakes region. Heavy metals are known to cause a number of human health disorders and result in environmental degradation. Since heavy metals can not be decomposed they must be sequestered from the environment. Current heavy metal clean-up technologies are largely non-renewable, expensive, or not feasible for many aqueous environments. There is a need for renewable, cost effective, and environmentally compatible technologies for the recovery of heavy metals from contaminated Areas of Concern in the Great Lakes. We propose to develop transgenic single-celled algae for the selective recovery of heavy metals from contaminated sites and effluents. Algae expressing heavy metal specific binding proteins will be used for high efficiency recovery of heavy metals from contaminated sites. The recovered metals then can be released from the algae and recycled or disposed in an environmentally benign manner.

We will screen a phage combinatorial DNA library encoding all possible heptapeptide amino acid combinations for cadmium, copper, mercury and lead binding peptides. The metal binding affinities of the peptides then will be determined by equilibrium dialysis under a variety of conditions. The genes encoding metal-binding peptides will be fused to cell wall and membrane proteins of Chlamydomonas and transformed into wild type and transgenic cells expressing genes that enhance trace metal accumulation. Both live and dead transgenic Chlamydomonas cells that express trace metal-binding peptides will be assayed for their metal binding properties under a variety of conditions. Finally, we will conduct field trials in the Great Lakes region to determine the efficiency of our process for trace metal recovery.

We have identified twelve novel nickel-binding hepta-peptides from ours screens of the combinatorial phage display library. Molecular modeling of these peptides suggests they form a pocket around the metal. We have generated a fusion between a plasma membrane protein and a nickel binding domain. This construct has been transformed into Chlamydomonas and the phenotype is being characterized.In addition, we have isolated a novel copper binding heptamer from the phage display library screen. This peptide will be fused to the plasma membrane protein and transformed into Chlamydomonas to decorate the cell surface with metal bindingdomains. In addition, we have identified novel cadmium-induced genes in alage which we believe will have capacity for enhanced heavy metal recovery. In addition, we have generated fusions between a plasmamembrane protein and eith intact metallothionein or its alpha or beta domains in units fro 1-5. These heavy metal binding proteins are expressed on the surface of the cell. The best transgenic lines bind up to five-fold more cadmium than wil-dtype cells and grow at normal rates in the presence of toxic cadmium concentrations that kill wild-type cells.We also have determined that expression of the P5CS gene in Chalmydomonas enhances cadmium binding by four-fold. This gene increases the proline concentration about two-fold over wild-type cells. Significantly, we have demonstrated that the benefit of expressing proline is to regulate the redox state of the cells so as to protect cells from free-radical damage. Finally, we have determined the molecular responses of Chalmydomonas to cadmium stress and have demonstrated that an iron uptake protein is induced in its expression enhancing iron uptake to counteract cadmium poisoning.