Bastian presented the progress of his experiment about developing Forster Resonance Energy Transfer (FRET) biosensors for measuring bioavailable heavy metals in soil remediation. The biosensors exploit the fusion of a metal binding protein, metallothionein (MT) to fluorescent proteins (FPs) – in this case, enhanced Cyan FP (eCFP) and the yellow Venus FP (Rajamani et al., 2014).
Binding of heavy metals by MT changes the molecular distance between eCFP and Venus, bringing them closer to each other. This proximity enables energy transfer from eCFP (donor) to Venus (acceptor) changing the fluorescent properties of the system (Tsien, 1998; Carter et al, 2014).
The occurrence of FRET, and hence binding of metals to MT, can be measured by changes in the ratio of fluorescence emission of eCFP and Venus – metal binding enhances Venus fluorescence at the expense of eCFP. The expression of a FRET sensor inside a host bacterial cell allows direct, rapid measurement of intracellular metal concentrations.
Bastian has constructed FRET biosensors inside two different types of bacteria; Escherichia coli and Pseudomonas putida. These modified bacteria can respond directly to the presence of heavy metals potentially to be used to assess the bioavailable fraction in a soil.
As the site of action of heavy metals is inside the bacterial cell, bacteria-based biosensors that report cytoplasmic metal concentration directly can be developed as a more appropriate indicator. This biosensor is an alternative approach to measure the toxic effect of metals on cell physiology.
Bastian is currently working with the application of this biosensor as monitoring tools to measure the change of bioavailable heavy metals due to biochar amendment in contaminated soil (in collaboration with Rosa Soria, ESR 8). This is being conducted by extracting the soil pore water from the contaminated soil samples and adding them to the biosensor assay and monitoring results using a spectrofluorometer. Alternatively, microbes can be mixed directly with the soil and metal concentrations monitored using fluorescence microscopy.
The measurement of metal concentrations from the biosensors will be integrated with an analysis of plant-bioavailable metal concentrations and changes in soil microbial community to assess the remediation performance. The outcomes of these analysis are expected to provide information about the conditions that can improve the soil quality to support plant growth and microbial activity for the restoration of contaminated land.
References
Carter, K. P., Young, A. M. and Palmer, A. E. (2014) ‘Fluorescent Sensors for Measuring Metal Ions in Living Systems’. doi: 10.1021/cr400546e.
Rajamani, S. et al. (2014) ‘Noninvasive Evaluation of Heavy Metal Uptake and Storage in Micoralgae Using a Fluorescence Resonance Energy Transfer-Based Heavy Metal Biosensor’, Plant Physiology, 164(2), pp. 1059–1067. doi: 10.1104/pp.113.229765.
Tsien, R. Y. (1998) ‘the Green Fluorescent’, Annual Review of Biochemistry, 199(2), pp. 293–306. doi: 10.1146/annurev.biochem.67.1.509.