Part:BBa_M50039:Experience

Escherichia coli (E. coli) naturally possesses a pathway to respond within a few minutes to sudden copper metal induction. The main purpose of this system is to protect the cell from copper shock. The membrane protein cusS and cytoplasmic protein cusR are always present at low levels within the cell. The presence of copper phosphorylates the membrane protein cusS, which then trans-phosphorylates cytoplasmic cusR. Once cusR is activated, it binds to the appropriate sequence within the cusR-inducible promoter region, initiating bidirectional transcription of cusS and cusR. This pathway increases cusS and cusR concentration in the presence of copper, acting as a positive feedback loop that allows the cell to display an amplified response to toxic copper ions. These proteins then go on to activate downstream proteins that bind the copper ions to inactivate them, thereby increasing the cell’s survivability in copper ion solutions.


DNAse Experiment:
We aimed to transform E. coli to 1) confer additional survivability in solutions with elevated copper concentrations by producing the cusR protein and 2) respond to the presence of copper by producing green fluorescent protein (GFP). To do so, we took advantage of the cusS/cusR pathway. We designed 2 separate plasmids that differed in the sequence of the cusR-inducible promoting region, BBa_M50039 and BBa_M50041. We found that neither plasmid imbibed increased survivability or fluorescence in copper ion.

Procedure:
We transformed E. coli with BBa_M50039, BBa_M50041, or no plasmid using the protocol outlined in Practical 4 of the BIOE44 course, calling the first two our “transformed” populations, and the latter our control or “untransformed” group. We then streaked the populations transformed with plasmids conferring ampicillin (amp) resistance, BBa_M50039 and BBa_M50041, onto plates of LB and 1% amp. The E. coli population that was mock transformed was streaked onto a plate with no antibiotic. To further confirm growth of the correct populations, we also streaked BBa_M50039 and mock transformed group onto agar plates with 1% kanamycin, where no colonies formed, as expected. From the resulting colonies, we grew populations in 20 mL LB for 24 hours at 37°C, with 1% amp for the populations transformed with BBa_M50039 and BBa_M50041 and no antibiotic for the mock transformed-group, resulting in tubes of bacteria with OD 600nm equal to approximately 0.6-0.8.

Copper Response
As our plasmids were designed to produce GFP in response to copper ion, we measured the effect of copper ions on fluorescence. We diluted each bacterial population to OD 600nm = 0.5; dissolved copper ions to form concentrations of 0, 0.2, 5, and 20 mM into the growth medium with copper sulfate; let the bacteria continue to grow for 24 hours at 37°C; and determined the resulting fluorescence and OD 600nm . We selected these ion concentrations based on the knowledge that the cusR response is saturated at approximately 5 mM. We expected the transformed bacteria to have higher growth rates, due to the presumed increase in cusR protein that recruits copper ion-inactivating proteins, and higher fluorescence. We conducted each experiment in triplicate, with the resulting averages and standard deviations plotted in the figures below. Increasing the concentration of copper ion generally caused lower growth rates among all bacterial populations, consistent with the known toxicity of copper ion. Comparing growth rates among BBa_M50039, BBa_M50041, and the mock transformed control group, we found no significant differences at copper ion concentrations of 0, 0.2, and 5 mM. At 20 mM, however, the control group has nearly twice the concentration of bacteria, a result that is contrary to our prediction that the groups transformed to produce additional cusR in the presence of copper ion would exhibit increased growth rates. The plasmids appear to diminish survivability in copper. Comparing fluorescence between the groups at different concentrations, we found that increasing copper ion concentration did not significantly affect the fluorescence of BBa_M50039 and BBa_M50041 groups. This result suggests that the plasmids we designed do not function as intended. Furthermore, the control group exhibited significantly higher fluorescence at copper ion concentrations of 5 and 20 mM. The control group was intended to serve as the baseline fluorescence for bacteria without GFP, indicating that the autofluorescence of our transformed groups is suppressed by the copper ion more so than the autofluorescence of the untransformed group. The first figure (a) below shows bacterial growth after incubation with the copper ion after 24 hours, and figure (b) shows fluorescence after incubation with copper ion after 24 hours. Growth was normalized to the maximum OD 600nm among all bacterial populations and growth conditions, including incubation in copper, nickel, and iron ions.

Nickel and Iron ion Reponse
Given the chemical similarity between transition metal ions in biological systems, we studied the effect of two other transition metal ions, nickel and iron, on bacterial growth rate and fluorescence to determine the selectivity of our plasmid. We conducted the experiments the same as we did for copper ion, except copper was replaced with either nickel or iron ions from the sulfate salt. These averages and standard deviations can be seen below in the figures.

Nearly all growth rates between groups at all concentrations of nickel and iron are not significantly different from each other, which suggests that both transformed and mock transformed groups demonstrate similar growth responses to these ions. The only exception is at 20 mM iron ion, as can be seen in the figure below: The control group maintains growth, while the transformed populations are unable to survive in this environment, indicating that the transformed populations may have increased sensitivity to iron concentration. Regarding fluorescence, there are no significant differences between the transformed and control population for both ions at all ion concentrations. Interestingly and unrelated to the main goal of the study, increasing nickel ion depressed autofluorescence across all bacterial groups, while increasing iron ion augmented it, suggesting that these ions differentially affect production of autofluorescent components.

Upon testing the growth and fluorescence upon incubation with copper ion (to test functionality) and nickel and iron ions (to test specificity), we found that fluorescence and growth were not significantly different from the control group for all ions. Thus, we were unable to create a biological fluorescent copper sensor.

Stanford Location

Plasmid Name: DNAse_BBa_M50039
DNA 2.0 Gene #: 273824
Organism: E. coli
Device Type: Sensor
Glycerol Stock Barcode: 0133027170
Box Label: BIOE44 F16

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