Construct Design

In order to introduce heavy metal ions into our bacteria and allow the metallothionein proteins to bind and sequester these contaminants, we created BioBricks for the expression of heavy metal membrane transporters. The gene CPB4 codes for a membrane transporter that has a high capacity for the uptake of lead as well as a reduced affinity for other heavy metals, notably cadmium and cobalt. This gene was originally isolated from a resistant strain of Bacillus spp. found in heavy metal contaminated soil in Korea, although our plasmids utilize the gene from the plant Nicotiana tabacum. It has been found in previous research that bacterial strains possessing this gene have the capacity to remove lead from water and soil and could be useful in bioremediation applications [1]. Our first lead transporter construct (BBa_K1460005) consists of the constitutive Anderson promoter and the CPB4 gene for constitutive expression of the heavy metal membrane transporter and uptake of lead. The primary construct for lead sequestration (BBa_K1460008) consists of this first lead transporter construct put upstream of our metallothionein construct with the T7 promoter and GST-YMT. This construct allows for the constitutive expression of the lead transporter as well as the inducible expression of the metallothionein in BL21 by arabinose activating the araBAD promoter and allowing expression of the highly active T7 polymerase. This allows for our bacterial strains to grow to stationary phase before being induced to produce metallothioneins and being used to sequester lead.

Results

References

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1. Ref 1
2. Ref 2
3. Ref 3