Characterization

Experiment 1: Testing of MalE and MalE31 with literature established reporter constructs

Protocol:

We obtained degP and cpxR reporter constructs from Dr. Tracy Raivio's lab. These constructs contain the promoters upstream of lacZ. They were characterized with NlpE, an outermembrane lipoprotein that activates the Cpx pathway. We made TOP10 E. Coli competent cells with plasmids of these constructs and then transformed in MalE and MalE31 circuits with arabinose inducible promoters. We also transformed in the NlpE expression construct which we received fro the Raivio lab as well. The purpose of this assay was to confirm that promoters coupled reporters could indeed be induced by misfolding protein and to show that the various maltose binding proteins that we received were functional. From plates, we made 5 mL LB cultures and induced with 1uL IPTG for cultures containing the NLPE constructs. 75uL of X-Gal was also added to each culture. The cultures were then grown up overnight in a 30°C shaking incubator and observed for color.

Results

Figure 1: Image of overnight cultures. From left to right: NLPE in cells with the degP reporter construct, NLPE in cells with the cpxR reporter, malE31 in cells with the degP reporter, malE31 in cells with the cpxR reporter, malE in cells with the degP reporter contruct and malE in cells containing the cpxR reporter.

Discussion of Results and Conclusion

Figure 1 indicates that malE31 is able to activae the cpxR and and degP promoers while malE is not. This allowed us to conlucde that these parts are working as expected. Although these parts are both entered in the registry, the sequences are not complete, so we are submitting new versions of them, constructed ourselves. Once we knew that these parts were functional, we went o to characterize them with our reporter constructs.

Experiment 2: 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

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

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

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.

Experiment 3: Characterizing the cpxR promoter's response to varying temperatures over different time periods

Protocol:

Top 10 competent cells were transformed with CpxR-I13507 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.

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.

Experiment 4: Characterization of the degP promoter's response to folding and misfolding proteins

Protocol

Arabinose inducible promoter (I0500) coupled with standard ribosome binding site (B0034) and the respective maltose binding protein were transformed into competent cells containing pDegP 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 subcultured in 20 ml LB broth. These were shaken for 6-8 hours and aliquoted into 5 ml cultures and induced with varying levels of arabinose. This was incubated in the shaker for 12-14 hours and RFP output was measured using 555/632 nm.

This figure demonstrates that the DegP promoter activated with 15 different concentrations of arabinose. This diagram shows that the DegP promoter (K239000) is not particularly sensitive to misfolding proteins.

Discussion and conclusions

The figure shows that MalE and MalE31 express the DegP promoter in a similar fashion. This is slightly contradictory compared to the literature. The literature claims that the DegP promoter is upregulated in the case of protein misfolding, graph shown . Since MalE and MalE31 have been tested using other experiments described in this page, it is reasonable to conclude that K230009 is not very responsive to protein folding stress, that is , the DNA might not be consistent.

Experiment 5: Characterization of the ibpAB-fsxA fusion promoter's response to properly folding and misfolding proteins

Purpose/ Protocol

The purpose of this assay is to characterize the ibpAB fusion promoter (ibpAB-FxSA) with a protein that is known to fold correctly (MalEΔSS) and with a protein that are known to misfold (MalE31ΔSS) while remaining in the cytoplasm. The promoter was coupled with green fluorescent protein so when activated, the reporter would be produced. A construct received from Jean-Michel Betton's lab containing MalEΔSS and MalE31ΔSS downstream from a maltose-induced promoter were transformed into Top10 competent cells containing the plasmid with the ibpAB-fsxA GFP reporter plasmid (ibpAB-I13504). Overnight cultures were made from these transformations in 5 mL of LB Lennox Broth and left to grow for sixteen hours. Induction was done with multiple concentrations of maltose to produce different quantities of protein and the cells were shaken at 30°C. Four hours after induction, GFP fluorescence was measured and can be seen below.

Results

Figure 4: This graph shows the GFP fluorescence produced when MalEΔSS and MalE31ΔSS downstream of maltose-inducible promoters were transformed into Top10 competent cells containing the ibpAB-fsxA promoter coupled with a GFP reporter.

Discussion and Conclusions

The graph trend lines show a sharp decrease from the opening GFP output before rising again to level off. The induction using varying concentrations of maltose to produce known misfolding protein shows that the properly folding maltose binding protein (MalEΔSS) causes more fluorescence output than the misfolding maltose binding protein (MalE31ΔSS). This is in contradiction with the literature data. However, the last data value at maltose concentration of 0.5% maltose added to the solution is what creates this. In the future, more characterization assays should be run with inductions using more variants of maltose concentrations. These would allow for further verification and confirmation of either literature data or our data. There have been very few studies using this fusion promoter so little is known about it. In addition, more known folding and misfolding proteins can be used to enhance the data gained with maltose binding protein.

Experiment 6: Preliminary characterization of Nms6 for the University of Lethbridge Team

Purpose/ Protocol

The University of Lethbridge is working with a protein meant to degrade catechol in tailing ponds. They have had trouble expressing it and they believe it was misfolding in the cytoplasm. It was constructed downstream of an arabinose-inducible promoter (I0500) and ribosomal binding site (B0034). Then, it was transformed into Top10 competent cells containing a plasmid with the ibpAB-fsxA promoter coupled with a GFP reporter. 5 mL overnight cultures were made of these and induced with varying concentrations of arabinose after 16 hours of growth. The cultures were then grown in 30°C conditions and green fluorescence output was measured four hours after induction.

Results

This figure shows the GFP fluorescence output when Nms-6 was ligated with an arabinose-inducible promoter and transformed into cells containing the ibpAB-fsxA fusion promoter coupled with GFP.

Discussion and Conclusions

These preliminary results are not conclusive, however they seem to indicate that there is a much higher GFP output when the expression of Nms6 protein was being induced at higher levels through arabinose induction. The graph shows an upward line of best fit, indicating the rise in quantities of GFP present. This suggests that at higher levels of expression, this protein may be misfolding in the cytoplasm. These results are not conclusive however as we do not have enough characetrization data for the ibpAB fusion promoter. With more characterization on the activation of the ibpAB-fsxA fusion promoter and multiple trials to assay GFP production, more conclusive results could be obtained. Regardless, the Univeristy of Lethbridge may want to try expressing thier protein at lower levels, either through an inducible promoter, or through the use of a lower copy plasmid.