Part:BBa_K339007
CpxR Reporter
cpxR promoter upstream of an RFP generator.
Usage and Biology
This part was used in order to characterixe the cpxR promoter, specifically its repsonse to folding and misfolding proteins in the cytoplasm.
E coli hav several native sigma factors in the genome that are upregulated during envelope stress. Some of the sigma factors include sigma E and sigma 70. These two factors act upon and induce different proteases and chaperones which act to correct the misfolding agent. There is also a new regulon that is currently studied widely, called the Cpx regulon. A well characterized periplasmic regulon found in E. coli is the Cpx regulon. Cpx pathway is involved in maintaining cellular adhesion and keeping periplasmic environment in check. It regulates proteins which are responsible for cellular adhesion and pili formation (Diguiseppe and Silhavy, 2003). Cpx also acts as a periplasmic heat shock pathway. The Cpx pathway is activated by factors which cause protein aggregation in the periplasmic space of gram negative bacteria. Extracellular stresses such as exposure to ethanol, change in pH and temperature change induce the activation of the Cpx heat shock regulon. This increases the transcription of proteins such as CpxP, DegP and CpxR. The Cpx pathway is activated by misfolded proteins or excessive protein concentration in the periplasmic space as well.
"http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/Cpxmechanism.png"
Figure illustrates Cpx regulon activation pathway. In the lack of stress, the CpxP protein is docked on CpxA transmembrane protein. In the presence of misfolded protein in the envelope, CpxP binds to the misfolded protein which allows CpxA to phosphorylate CpxR which acts as a TF for downstream proteases such as DegP.(Silhavy and Raivio,2001)
CpxR is a transcription factor that is generally dephoshorylated (inactive form). However, in occasion of misfolded protein in the periplasmic space, CpxR is phosphorylated by CpxA which autophosphorylates itself and also has kinase activity towards CpxR. The phosphorylated CpxR then binds to promoter regions that code for proteases such as DegP, ppiD etc.
Sequence and Features
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 636
Illegal AgeI site found at 748 - 1000COMPATIBLE WITH RFC[1000]
Functional Parameters
Characterization of the cpxR promoter's response to folding and misfolding proteins through co-transformation of MalE and MalE31 coupled to arabinose promoter in cpxR reporter competent cells- Unversity of Calgary 2010
Protocol:
Arabinose inducible promoter (I0500) coupled with standard ribosome binding site (B0034) and the respective maltose binding protein were transformed into competent cells containing pCpxR coupled with RFP generator (I13507). These cells were plated and incubated overnight. Colonies from each of the plates were selected and overnight cultures were prepared at 37 C. These 5 ml overnight cultures were then sub-cultured in 20 ml broth. These were shaken for 6-8 hours and aliquoted into 5 ml cultures and induced with varying levels of arabinose(percent). This was incubated in the shaker for 12-14 hours and RFP output was measured using 555 excitation and 632 nm emission frequency.
Results
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Figure 2: RFP output produced by the CpxR-I13507 system when co-transfected with I0500-B0034-MalE (red) and I0500-B0034-MalE31 (blue) at different arabinose concentrations. RFP levels were measured at 555 nm excitation and 632 nm emission frequencies
"http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/lineofbestfitCpxR.png"
Figure 3: RFP output produced by the CpxR-I13507 system when co-transfected with I0500-B0034-MalE (red) and I0500-B0034-MalE31 (blue) at different arabinose concentrations. RFP levels were measured at 555 nm excitation and 632 nm emission frequencies.
Discussion of Results and Conclusion
Figure 2 and 3 indicate the RFP output normalized with growth ratio (OD) at different levels of arabinose. Figure 1 shows that CpxR-I13507 is activated at the highest level when MalE31, the periplasmic misfolder, is expressed. This occurs around 0.2% arabinose concentration. Similar trends are observed in the case of MalE which is a periplasmic folder. MalE and MalE31 activate the system at different levels. MalE31 has similar trends to MalE but has a higher level of RFP expression. These results prove that MalE and MalE31 can both activate the CpxR system however, MalE31, which misfolds, activates it more rapidly and at a lower level of arabinose concentration compared to MalE. If the line of best fit is studied, it is seen that MalE has very minimal level of Cpx activation. Whereas, malE31 has a linear regression which flattens out as the system reaches its upper threshold of detection. Biologically, this could mean that the MalE31 is activated at levels that saturate the cellular chaperones and cause the system to reach its threshold level of proteolytic and chaperone activities. Another interesting pattern observed is the fact that when MalE is constructed with CpxR-I13507 on the same plasmid (Green), the cell RFP output is much lower compared to cells co-transfected with CpxR-I13507 and I0500-B0034 –MalE. This indicates that insertion of high copy plasmid also induces stress in the periplasmic region of the cell consequently inducing the activation of CpxR system.
Characterization of the cpxR promoter's response to varying temperatures over different time periods - University of Calgary 2010
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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