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