Part:BBa_K135000:Experience

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Applications of BBa_K135000

We have used this part, fused upstream of BBa_E0840 (BBa_K135001), and have shown GFP activation in reponse to adhesion on glass slides. The conclusion that this is activated in response to adhesion follows from Ottos and Silhavys work (see references), in which they demonstrated activation of a pCpxR-LacZ fusion in response to adhesion to glass microspheres. Filamentous bacteria fluoresced significantly, while it was hard to ascertain whether smaller bacteria were fluorescing. Due to time constraints, a better characterisation of the efficiency of the promoter could not be performed.

Characterizing the cpxR promoter's response to varying temperatures over different time periods

Protocol:

The cpxR promoter was constructed upstream of an RFP construct (BBa_I13507). Top 10 competent cells were transformed with k135000-I13507 plasmids and plated. 5 ml overnight cultures were made from 5 different colonies using LB broth with appropriate antibiotics. Each of these cultures were aliquoted into six different tubes containing 600 µL of culture. These tubes were then placed in hot water baths at 30 C, 37C, 42C, 47C. Measurements were taken every hour for 5 hours after placing the tubes in different temperatures at 555 nm excitation and 632 nm emission.

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Figure 3: RFP output produced by the CpxR-I13507 system when the system is heat shocked at different temperature for different lengths of time. The RFP output was measured at 555 nm excitation and 632 nm emission frequencies

Discussion of Results and Conclusion

This graph shows that the CpxR system does respond to temperature activated stress. When the system is placed at 42 C the RFP output is much higher at t=0 compared to the system placed at 37 C or 30 C. This indicates that the system does get activated due to heat shock which matches the literature parameters. At 47 C, the system gets activated faster because the linear regression has a steeper slope. This indicates that the system is being stressed and it produces its downstream product which is RFP in this case and DegP and other chaperones in the genomic DNA much faster in order to cope with periplasmic protein denaturation. Also, it seems that the system gets activated dramatically after 3 hours regardless of the temperature, this could indicate that the system peaks after 3 hours and the genomic CpxR produces enough downstream chaperones and proteases in order for the system to be able to cope with stress which allows the RFP reading to decrease at 4 hours time because the cell reaches homeostasis. This allows the cell to get rid of misfolded protein and other factors that might be contributing to stressing it out and causing the Cpx regulon to be activated. The cell then shows a rapid rise again because it is still under heat shock stress. But, if the cell was placed at 37 degrees, the cell would show a flatline pattern rather than an oscillating pattern.

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