As detailed on our "Experiments" page, we measured chrR6 chromium reduction activity using DPC assay. The strong r² value of 0.9944 for the linear trend line of our standard DPC curve indicates that this is an accurate Cr(VI) detection method within this range of concentrations. Therefore, when we investigate the reduction efficiencies of our reductases, we will use initial Cr(VI) concentrations within this range.

Figure 1: DPC standard curve

Figure 2.A compares the Cr(VI) reduction rate standardized for OD₆₀₀ of E. coli MG1655 versus E. coli MG1655 cotransformed with the sulfate transporter system (BBa_K2194004) and constitutively expressed chrR6 reductase enzyme (BBa_K2194000). The data indicates that below an initial [Cr(VI)] of 80 uM the cotransformed bacteria are more efficient at reducing Cr(VI) than wild type bacteria. Above 80 uM initial [Cr(VI)], the trend reverses and wild type bacteria are more efficient. This supports the hypothesis that increasing cell permeability to chromate (in this case via sulfate transporters) increases the reduction efficiency of chrR6, but that after a threshold concentration the increased permeability becomes toxic for the cell.

Figure 2A: DPC Assay Reveals that MG1655 E. coli more efficient Cr(VI) reducers above threshold initial [Cr(VI)]

In the experiment, which results shown below in Figure 3, we compared the Cr(VI) reduction efficiency of chrR6 versus nemA at different initial concentration of Cr(VI). The two reductases were expressed under constitutive promoters in E. coli MG1655. Final concentrations of Cr(VI) for each sample were measured at 12 hours after adding chromate. This data shows that at every initial concentration of Cr(VI), the enzyme chrR6 is a more efficient reductase than nemA.

Figure 2A: DPC Assay Reveals that MG1655 E. coli more efficient Cr(VI) reducers above threshold initial [Cr(VI)]

Figure 2.B presents the same data as Figure 2.A but as an amount of total Cr(VI) reduced rather than the OD₆₀₀ standardized rate. (The x-axis and the light blue bars display the initial concentration of Cr(VI) and the y-axis displays the final concentration of Cr(VI). Comparison of the dark blue bars to the light blue bar at each different initial concentration of Cr(VI) reveals the total change in [Cr(VI)].

Figure 2B: DPC Assay Reveals that MG1655 E. coli more efficient Cr(VI) reducers above threshold initial [Cr(VI)]

cysPUWA Effect on Cell Membrane Permeability to Cr(VI)

Kill Switch Failsafe Mechanism

Figure 1: IPTG induces toxin expression leading to cell death

Figure 4: IPTG-induced mCherry fluorescence vs. time

Figure 4: IPTG-induced mCherry fluorescence/OD vs. time

PgntK Promoter Activity

Project Achievements