Ewalt J¹ and Sayre RT².

OSBP¹ and Department of Plant Biology²

Trace heavy metals pollution is a global concern in soils as well as waters. There is a need for a more cost effective, renewable, effective remediation technique, but most importantly, for a recovery method that is selective for specific metals of concern. Bioremediation is the use of living organisms to sequester or make a pollutant less toxic to serve in environmental clean-up. We have been studying the ability of the unicellular algae, Chlamydomonas reinhardtii, to acquire and sequester heavy metals, specifically cadmium (Cd). The alga contain the enzyme phytochelatin synthetase which catalyzes the non-translational synthesis of phytochelatin a multi-conjugate of glutathione; (g Glu-Cys)_n-Gly where n=2-11. We are interested in increasing the heavy metal sequestration by increasing the effective concentration on the selected metal around the surface of the algal cells. The fusion of a specific-metal-binding peptide to a cell membrane protein of Chlamydomonas presumably will chelate the metal of interest on the surface. It is predicted that the increased effective concentration will increase the uptake of the metal by the cell. To identify metal binding peptided metals first are chelated to a polysepharose resin via IDA (iminodiacetate) and a solution of a phage displayed heptamer peptide library is added in batch. A random library consists of 70 copies of each of 2.8x10 ⁸different sequences. After washing at a neutral pH the unspecific peptides are removed. To remove the peptides with affinity to the metal of interest, the pH is lowered. The phage from each screening step is amplified. The amplified phage is reapplied for further selection and eliminations of less specific peptides. Three rounds of batch selection screening is carried out before the heptamer fused to the bacteriophage coat protein is sequenced.