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.
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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. Nonetheless, the creation of one would still have a positive impact on our ability to monitor water supplies for copper contamination.

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.

Discussion:
We can troubleshoot our device to pinpoint why the device does not function appropriately. So far, we have investigated the expression of cusR and concluded that cusR was not being produced in significantly higher quantities in the transformed groups than in the control group, as seen in SI 1. To ensure that the plasmids in their current forms do not function as designed, we could measure the growth, fluorescence, and presence of cusR at different time points. This is because it is possible that the fluorescent response or increased cusR production occurs at shorter or longer timescales than the initially investigated 24 hours. To better increase the precision of measuring cusR, we could do a Western blot with cusR antibodies.

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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