T11 AM Aquatic Plants: Methods, Mechanisms and Markers
Tuesday, 15 November 2005: 8:00 AM - 11:40 AM in 343-344

(TOR-1117-597531) Transgenic Microalgae as Heavy Metal Biosensors.

Torres, M.A.¹, Falcao, V.R¹, Colepicolo, P.¹, Rajamani, S.², Ewalt, J.², Sayre, R.T.², ¹ Chemistry Institute, Departament of Biochemistry, University of Sao Paulo, Sao Paulo, Sao Paulo, Brazil² Department of Plant Cellular and Molecular Biology, Ohio State University, Columbus, OH, USA

ABSTRACT- Here we describe the design and properties of a heavy metal biosensor for the measurement of bio-available heavy metals in aqueous environments. The biosensor protein includes an N-terminal, cyan variant of the green fluorescent protein (mCFP), an intervening metallothionein (MT), and a C-terminal yellow variant of the green fluorescent protein (mYFP) from jellyfish. The genes encoding these proteins were codon-optimized for expression in Chlamydomonas and the modified green-fluorescent variants were engineered to reduce pH effects on fluorescence emission quantum yield and to reduce end-to-end dimerization. In the absence of heavy metals MT assumes an unfolded, random-coil structure. Following metal binding MT folds into a compact dumbbell shaped protein. The metal metal-induced folding of MT brings the two fluorescent proteins mCFP (excitation = 440 nm: emission = 485 nm) and mYFP (excitation = 515 nm: emission = 527nm) within 70 of each other facilitating fluorescence resonance energy transfer (FRET) between the two fluorophores, resulting in a shift of the relative ratio of yellow and blue fluorescence. We have expressed this heavy metal biosensor in a cell wall-less and arg- strain of Chlamydomonas reinhardtii. Fluorescence analyses confirm that the protein is expressed in the algae. When grown in TAP media the ratio of yellow to blue fluorescence is 1.0. Experiments show the effects of Cd²⁺, Cu²⁺ and Pb²⁺ ions on the fluorescence emission spectra of the biosensor. These results point microalgal biosensors like important organisms and could be used in biomonitoring of heavy metals in various water bodies.

Key words: Biosensors, Heavy metal, Chlamydomonas reinhardtii, Biomonitoring