Construct Design

The sequestration of our targeted heavy metals by metallothioneins has a noticeable rate change when approaching saturation.^[1] Therefore, a saturation detection system for each of the three metals, composed of a reporter downstream from a heavy metal inducible promoter, was proposed. The expression of the reporter, amilCP, would then negatively correlate to the amount of heavy metals present in the cells and thus the amount that the metallothioneins had failed to sequester. Two BioBricks for the metallothionein saturation detection system were designed: amilCP was inserted behind a nickel/cobalt activated promoter, Prcn (BBa_K1460009), and behind a mercury activated promoter, PmerT (BBa_K1460010).

These reporter systems would be used in tandem with sequestering strains. Cultures of these cells would be placed downstream of cultures of sequestering strains in a continuous system. Once the metallothioneins in the upstream culture became saturated with heavy metals (so they can no longer take any metal out of solution), metal concentrations entering the reporter cultures would be higher. We could, therefore, continuously monitor concentrations of heavy metals leaving our filtration system by detecting colorimetric changes in these reporter cultures.

Dry lab's filtration box is designed to incorporate the metallothionein saturation system. The reporter constructs would be placed into a second hollow fiber reactor after the first. If the water contains metal ions after passing through the fiber reactor with the metallothioneins, amilCP will be expressed and turn blue. It would then be possible to find the relationship between concentrations of heavy metal in the water and the cultures’ color gradient. This could be used to indicate that the metallothioneins are saturated and the sequestration cultures need to be replaced.

References

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1. Krishnaswamy, R., & Wilson, D. (2000). Construction and Characterization of an Escherichia coli Strain Genetically Engineered for Ni(II) Bioaccumulation. Applied and Environmental Microbiology, 5383-5386.