Part:BBa_M50039:Experience

Escherichia coli (E. coli) naturally possesses a pathway to respond within a few minutes to sudden copper metal induction. 4 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.

Stanford Location

Plasmid Name: DNAse pColi P1
DNA 2.0 Gene #: 273824
Organism: E. coli
Device Type: Sensor
Glycerol Stock Barcode: 0133027170
Box Label: BIOE44 F16

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