Difference between revisions of "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.

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.

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

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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.

Leeds 2013 Team Experience

Membrane Stress

Investigation of literature regarding the action and regulation of the Cpx pathway lead to the discovery that the system as a whole responds to a large variety of membrane stresses, something that could be true of the specific promoter we chose to use (pCpxR). As a result of this we deemed it necessary to investigate the response of our promoter to each individual membrane stress.
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Individually many of the experiments produced erratic and seemingly random data which is listed independently below. However plotting all average readings together shows that there is a dramatic increase in response to the stress induced by the silica beads comparative to all the chemically induced membrane stresses we used.

This indicates that it would be possible to detect the presence of silica beads in solution, in principle, using our binding and detection system, certainly when silica beads are present at relatively high concentrations. However further work needs to be done to assess whether this high response remains across a range of silica bead concentrations in solution.

In light of these results, some of these experiments were repeated for more accurate results. How the Cpx pathway reacts to temperature is already well characterised in the literature and by the [http://2010.igem.org/Team:Calgary/Parts/Characterization Calgary 2010 iGem team.]

The Ethanol gradient, Triton X-100 gradient and pH gradient were all repeated. In the second experiment, a robotic pipette was used to directly fill the 96 well plate to reduce human error. Excluding wells with imposed pH gradient, all wells were kept at pH 7 using Tris.HCl buffer, the pH gradient was also achieved with a Tris.HCl buffer.

The 96 well plates were then incubated while shaking while shaking for 2 hours at 37 degrees to allow time for GFP to mature. A longer incubation time was introduced to give more conclusive results.

We also performed preliminary investigation on the effects of ethanol and temperature extremes on the CpxR promoter but the results were inconclusive warranting further studies, possibly by the next years Leeds iGEM team. Listed below is the data for detergent and pH extremes, this type of data was typical for all the chemical stresses we employed with detergent and pH producing the most coherent results.

Triton X-100

Between the concentration values of 0.1-9% triton X-100 the data shows roughly the relationship that was expected, a rise in GFP production as the triton concentration increases. This is likely due to triton acting as a detergent resulting in damage to the plasma membrane of cells, activating the Cpx pathway in general, including the cpxR promoter. Past 10% Triton X-100 concentration it is assumed that cells are dying in large quantities, being unable to produce GFP at all or in very limited amounts before they are killed. This explains the corrected fluorescence readings reducing to below zero. The reason for the large spike in fluorescence at Triton concentrations >20% is unclear, a possibly explanation for this is the total lysis of the cell, expelling all cellular components resulting in background GFP levels having a larger fluorescence reading due to reduced scattering from within the cell.</p>

pH Effects

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The slight fluctuations across the pH scale are in line with the background fluorescence readings from our cell cultures, representing the standard levels of cpx activation as the cells collide, disperse in solution and divide during replication.

The higher levels of expression at both low and high pH are likely to be due to the pH extremes denaturing proteins and disrupting interactions within the plasma membrane. It could be assumed that pH’s further into the extremes of acid and base would completely lyse and kill the cells resulting in a possibly lower fluorescence.

Atomic Force Microscopy

Fig.1: An image gained from Atomic Force Microscopy Experiments showing expression cells containing the pCpxR promoter coupled with GFP coding region.

The Atomic Force Microscopy experiments were designed to quantify the force necessary to trigger the CpxR promoter, generating GFP. This was to be done by observing the culture under the fluoroscope portion of the F-AFM, and then "prod" a bacterium at a low force, slowly increasing the stress until a fluorescence response was seen. This would then give a direct measure of the stress threshold for pCpxR, a value currently unknown in the literature. However, as with all new experiments, there were some teething issues. Firstly, it can be seen from the image that the culture fluoresced at a low level prior to exposure to the AFM tip. This was likely due to smaller stresses in solution and some can clearly be seen in the middle of binomial fission, which is known to also activate the Cpx pathway in general. There were also issues with trying to keep the bacterial cells stable enough to continually poke them with the AFM tip, initially a high glycerol concentration was employed in an attempt to achieve this however the cells remained very mobile.

We are Team Tianjin of iGEM 2016, we aim at biodegrading PET by enzymes and we want to introduce the inclusion body regulation system in our project. This part can be used to report the generation of inclusion body in E.coli, and what we did is that we change the mRFP gene to the ddpX gene, which can cause cell lysis when overexpress. Then the E.coli will break down after too many inclusion bodies are in the periplasm so that we can directly purify the protein without the complicated step of breaking cells. Application improvement of the CpxR promoter by using it to regulate the ddpX gene- Team Tianjin, 2016

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