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 cbp4 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-crs5. 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

Cells successfully expressing cbp4 should be transporting more lead ions past the cell wall. This would lead to increased lead sensitivity. To test for lead sensitivity, E.coli BL21 and engineered BL21 with part BBa_K1460005 in the amp^r plasmid pUC57 were grown for a 24 hour period in LB with 1 mM Pb.

What we see after 24 hours of growth is no significant difference in growth between the two strains (figure 1). However, what we consistently observed is that there is no inhibition of growth of BL21 at high concentrations of lead (figure 2). Even if cpb4 is expressed and is actively transporting lead ions into cells, it is possible that the concentration of lead is still not high enough to be toxic to the organisms. We were, unfortunately, unable to test lead concentrations higher than those shown above because these working concentrations are approaching the maximum solubility of the lead nitrate that we were using for testing.

Part BBa_K1460005 in pUC57 was co-transformed with part BBa_K1460001 (GST-crs5) in pSB1C3 and selected for with both ampicillin and chloramphenicol to effectively create the lead sequestration part BBa_K1460008. To test for sequestration efficiency, both BL21 and BL21 engineered with BBa_K1460001 and BBa_K1460005 were grown with LB + 0.1% Arabinose for 8 hours and then diluted in half with LB + 2 mM Pb for a final lead concentration of 1 mM. These cultures were grown for 8 more hours. The cells were then removed and supernatant was tested for lead concentration using Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) with the help of Cornell's Nutrient Analysis Lab. Error bars in chart represent standard deviation of three biological replicates.

The chart on the left shows the average final concentration of lead in the cultures. There was no statistically significant difference between BL21 and BL21 engineered with BBa_K1460001 and BBa_K1460005 (figure 3). However, when we consider cell density and plot the amount of metal removed per OD (figure 4) there is a statistically significant difference between the two strains. This data suggests that cells engineered with cbp4 and GST-crs5 are in fact able to remove lead ions from water, and to the best of our knowledge this is the first successful bacterial lead sequestration system involving transport proteins and metallothioneins.

Ideally, this experiment would be run with the OD of both strains remaining the same to prevent changes in metabolite concentrations. This is difficult in this experiment as, as we have shown, cells expressing metallothionein have inhibited growth.

References

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1. Arazi, T., Sunkar, R., Kaplan, B., & Fromm, H. (1999). A tobacco plasma membrane calmodulin-binding transporter confers Ni2 tolerance and Pb2 hypersensitivity in transgenic plants. The Plant Journal, 171-182.