Part:BBa_K1486008

CxpR & Split IFP1.4 [Cterm + Cterm][1]

<P> This construct aimed to evaluate the activation and presumed dimerization of CpxR in E.Coli by fusing the novel split IFP1.4 (Infrared Fluorescent Protein) fragments - IFP[1] and IFP[2] - developed by the Michnick Lab (see reference). We chose the split IFP1.4 as it is a reversible split fluorescent protein emitting little noise. Upon dimerization, CpxR allows the two parts of the split IFP1.4 to re-fold and acquire their ability to emit fluorescence. Not knowing how CpxR might dimerize, we built 4 different biobricks with the various possible orientations that the dimerization of CpxR might acquire. </P> <P> An experiment on all possible CpxR - Split IFP fragments was launched to determine whether CpxR dimerized in E.Coli, as well as how this dimerization occured. The experiment conditions can be found here. The basics were the following: induction of stress by 50 mM KCl and reading on a plate reader with excitation and emission wavelengths of 640nm and 708 nm respectively. </P> <P> The results of this experiment show that CpxR dimerizes, and that it does so via its C-terminal. Hence, the functional biobrick is CxpR & Split IFP1.4 [Cterm + Cterm] (BBa_K1486008). </P>

Experiment 1: Demonstration of CpxR's dimerization & Elucidation of its dimerization orientation [BBa_K1486008]

               <p>
               Introduction 

CpxR is the relay protein in the stress resonsive CpxAR two component regulatory system. It has been shown by split beta galactosidase assay that CpxR dimerizes when phosphorylated (activated) in yersinia pseudotuberculosis. Moreover, following other in vitro FRET studies, it was shown that E. coli CpxR interacted with itself. We therefore hypothesised that dimerization would also be true in vivo in E. coli.

Aim
This experiment aimed to determine if and how CpxR dimerised in vivo in E. coli. This experiment intended to get a first idea of the real-time temporal dynamics of the activation of CpxR (the cytoplasmic relay protein of the CpxA-R pathway) by KCl stress via CpxA (the periplasmic sensor protein of the CpxA-R pathway). This experiment is a first of its kind.

Methods
To evaluate if and how CpxR dimerized under KCl stress, we built by gibson assembly four constructs with the various possible orientations that the split IFP1.4 fragments could have with CpxR: IFP[1] and IFP[2] on the N-terminus of CpxR, IFP[1] on the N-terminus of CpxR and IFP[2] on the C-terminus of CpxR, and finally IFP[1] and IFP[2] on the N-terminus of CpxR. The split IFP fragments were provided by the Michnick Lab, and the CpxR coding region was amplified by PCR from extracted E. coli genome (Bacterial Genomic Miniprep Kit from Sigma Aldrich). The protocol for stressing the cells and reading the fluorescence can be downloaded <a href="https://static.igem.org/mediawiki/2014/a/a5/EPFL_Protocol_IFP_stress_1.pdf">here</a>.

Results
As seen in the graph bellow, induction of the signal was done at minute 24 (marked via a vertically spoted line). The construct with IFP fragments on the C-termina responded immediately to stress. In a fact we observed a 3 fold signal increase in 2 minutes. All other constructs we observed a low baseline signal non responsive to KCl stress. It is to be noted that the C-termina constructs always had higher signal levels than the other constructs. This leads us to believe that the PBS used to resuspend our cultures led to small levels of stress (the PBS we use does not contain KCl but traces of NaCl). The 30-fold signal increase from the baseline allows us to assert that our constructs responds to KCl stress.

Discussion
We successfully proved that CpxR dimerized in vivo and that dimerization led to close interaction of its C-terminus. This finding suggests that CpxR binds via its C-termina. This leads us to hypothesise that the CpxR dimerisation mechanisms is the same for other members of the highly conserved OmpR/PhoB subfamily. This hypothesis could allow the development of similar system that could the study other components of the OmpR/PhoB subfamily and thus lead to a new generation of highly senstitive and reactive biosensors.

Experiment 2: Signal induction by various concentrations of KCl & signal shutdown by centrifugation

Aim
Having found that KCl was a good signal inducer for our signal, we decided to characterise our biobrick by testing if the signal could be modulated by various concentrations of KCl and if we were able to remove the signal by centrifugation and medium change. To do so, we read our signal for 20 minutes without stress and then added KCl. At minute 144 we centrifuged our cells and replaced the medium with PBS to be able to get a shutdown of the signal.

Methods
To evaluate if a modulation in KCl concentrations affected the intensity of the intensity of the fluorescent signal, and if a change in medium by centrifugation shutdown the signal; we read our signal on a plate reader for 20 minutes without stress and then added KCl. At minute 144 we centrifuged our cells and replaced the medium with PBS to be able to get a shutdown of the signal. The protocol for this experiment can be downloaded <a href="https://static.igem.org/mediawiki/2014/a/a5/EPFL_Protocol_IFP_stress_1.pdf">here</a>.

Results
We successfully showed that increasing concentrations of KCl led to stronger signals up to a saturation concentration of about 80 mM KCl. Moreover we were able to shut the signal down, thus proving the reversibility of our system. These results prove the reversibility of the split IFP1.4 and suggest that real-time temporal dynamics analysis are possible for our system.

Experiment 3: Visualization of the the CpxR split IFP1.4 activation by KCl stress

Aim
Having shown that we were able to monitor the temporal dynamics of our construct, we wanted to see if we were able to analyze the spatial dynamics by microscopy.

Methods
To visualize the activation of our construct, we prepared cells as above for the previous plate-reader experiments, spread 10 µl on a glass slide added a coverslip and imaged them on a Zeiss Axioplan with a x100 objective and a APC (Cy5.5) filter. The pictures shown bellow were taken with a 5.1(s) integration time.

Results
As seen in the pictures bellow, we were able to distinguish specific patterns within bacteria. We observed two phenotypes within our population: elongated and normal cells. The difference in these phenotypes was noticed in previous experiments and is most certainly due to the CpxR overexpression as we observed this also in non-stressed conditions. In the first phenotype (elongated) we were able to distinguish several bands that seem fairly uniformly distributed. In the second phenotype (normal) we observed a single band in the center of the bacteria. These observations led us to believe that CpxR might be involved in the division process of E. coli as it seems coherent for cells to slow down division upon stress. After looking into the literature, similar bands were visualizable in E. coli for factors related to septum formation such as ftsZ or pbpB. Nevertheless when comparing our patterns to the ftsZ and pbpB patterns, we noticed that CpxR might be localized in opposition to these factors. Further experiments comparing the sub-localization of CpxR and ftsZ could help the scientific community better understand how E. coli monitor division under various environments.

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References

Michnick, S., Tchekanda, E., & Sivanesan, D. (2014, April 20). An infrared reporter to detect spatiotemporal dynamics of protein-protein interactions. Nature Methods, 6-6.

WARNING: This Biobrick was built by Gibson Assembly and contains illegal restriction restriction enzyme sites !

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 2213
  Illegal PstI site found at 3446
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1205
  Illegal PstI site found at 2213
  Illegal PstI site found at 3446
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 3657
  Illegal BamHI site found at 1144
  Illegal XhoI site found at 1283
  Illegal XhoI site found at 2470
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 2213
  Illegal PstI site found at 3446
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 2213
  Illegal PstI site found at 3446
  Illegal AgeI site found at 979
  Illegal AgeI site found at 1694
  Illegal AgeI site found at 2881
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961