Team:Rice/Results

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Hexatri

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RESULTS

Overview

chrR6 Chromium Reduction Activity

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)]

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)]

In the experiment which results are 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 3: chrR6 reductase enzyme is more efficient than nemA reductase in reducing Cr(VI)

cysPUWA Effect on Cell Membrane Permeability to Cr(VI)

In this experiment, we added varying concentrations of Cr(VI) to 0.05 OD₆₀₀ cultures of: E. coli MG1655, E. coli MG1655 transformed with a plasmid constitutively expressing reductase chrR6, and E. coli MG1655 cotransformed with a plasmid constitutively expressing reductase chrR6 and the sulfate transporter system cysPUWA-sbp. We grew the cultures for 12 hours shaking at 37C and took their final OD₆₀₀ measurements (presented here). The data indicates that the sulfate transporter system introduces toxicity to the cell (because it has the lowest OD₆₀₀ values for every initial concentration of Cr(VI) except [Cr(VI)]_i=0). Because the OD₆₀₀ values of the reductase and co-transformed reductase/transporter cultures are comparable at time 12 hours for no chromate, this indicates that the additional toxicity from the sulfate transporters is due to increase permeability of the cell to Cr(VI) rather than increased metabolic load of expressing a sythetically engineered plasmid.

Figure 4: Effect of Chromate on E. coli Growth

PgntK Promoter Activity

In this experiment, we compared the reporter protein expression of E. coli MG1655 transformed with a PgntK promoter+mCherry construct versus a Pcon promoter+mCherry construct. The data indicates that PgntK promoter has a comparable transcription initiation rate to Pcon promoter when no membrane stress is present.

Figure 5: PgntK (BBa_K2194002) Promoter similar strength to Pcon (BBa_J23108)

Kill Switch Failsafe Mechanism

Figure 6: IPTG induces toxin expression leading to cell death

In this experiment, we transformed E. coli MG1655 with a plasmid containing promoter Ptrc-2 and a fluorescent reporter protein mCherry and added varying amounts of IPTG to culture with the same initial OD₆₀₀. Time course measurements of mCherry fluorescence (excitation/emission=587nm/610nm) over 14 hours are shown in Figure 7. This graph shows a standard dose-responsive curve, indicating that the Ptrc-2 promoter is lacI-repressed as predicted.

In this initial test of the Kill Switch we used mCherry instead of the “toxin” BamHI because including the toxin would require culturing our bacteria in chromate to keep them alive. Instead, we measure mCherry fluorescence as an approximate and proportional cell death response to IPTG induction. In a future test, we would substitute mCherry in our construct for BamHI and measure OD₆₀₀ over time in cultures with chromate and varying concentrations of IPTG.

Figure 7: IPTG-induced mCherry fluorescence/OD vs. time