RESULTS

Characterisation of the spatiotemporal dynamics of the CpxR - split IFP 1.4 stress sensor

Experiment 1: Promoter characterisation and folding ability of fused GFP to CpxR via 10 amino acid 2 x (GGGGS) flexible linker

This construct aimed to evaluate the expression and correct folding of our CpxR construct, and the function of the arabinose promoter in E. coli by fusing a superfolder GFP protein to the N terminus of CpxR. The sfGFP was chosen because of its higher intensity compared to GFP.

Not knowing if CpxR would react the same way if sfGFP were attached to the N or C terminus, 2 biobricks were built, one with each of the orientations: BBa_K1486002 (N terminus) and BBa_K1486005 (C terminus).

An experiment on both possible CpxR - sfGFP orientations was launched to determine whether the proteins were well expressed and folded, and if the arabinose promoter worked well. It was also done on a microfluidic chip. The N terminus GFP biobrick results can be seen below; fluorescence intensity plotted against time.

The increasing standard deviation for the cells with arabinose can be explained as some chambers did not have a lot of cells and so there was a low intensity. As it can be seen in the following picture :

These are chambers with arabinose in the medium, you can see that there are different cell density and thus different intensity in the chambers. Inducing a high standard deviation

Experiment 2: CpxR dimerization & Dimerization Orientation

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 here.

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 3: 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 here.

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 4: 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.

Characterisation of the split luciferase

Experiment 1: CheY/CheZ fused to split Firefly/Renilla luciferase, and full Firefly/Renilla luciferase characterisation

Introduction
CheY and CheZ are two proteins involved in the bacterial chemotaxis pathway. It has been shown by split luciferase complementation assay that these two proteins are not interacting in presence of chemoattractant, but start to interact (CheZ being the phosphatase of CheY) in absence of chemoattractant or presence of chemorepellent. Based on the work of Waldor¹ Laboratory, we wanted to redo and adapt the experiment to test our own splits.

Aim
This experiment aimed to test the efficiency of split Renilla luciferase and split Firefly luciferase. We wanted to study the speed of the signal and the amount of substrate needed to have a performant response.

Method
To proceed to this complementation assay, we built two constructs, one to test split Renilla Luciferase and the other for split Firefly Luciferase The CheY was fused to the N-terminal part of each split, while the CheZ was fused to the C-terminal part. We used the full luciferases (Renilla : BBa_K1486022 and Firefly : BBa_K325108 from Cambridge 2010 team) as positive controls and the non-fused splits (Renilla : BBa_K1486021 and Firefly : BBa_K1486018 ) as negative controls.

The bioluminescence assay was performed as described here.
The constructs were designed and assembled as described here.
.

Results
As shown in the graphs below (fig.1A and 1B), we couldn't really observe a high signal for our complementation assay. However, the signal being higher than the blanks, it is an encouraging sign that the splits luciferase can be used for other experiments of this kind. A possible explanation for these results is that arabinose being a chemoattractant, we might need to do more wash steps with PBS to get rid of the arabinose before taking the measurements. Moreover, CheY and CheZ being endogenously expressed in bacteria, the edogenous proteins could interfere with our fusion proteins and weaken our signal. This complementation assay should be tested with CheY/CheZ knock out strains, as it was done in Waldor Laboratory.

We also could determine which of the luciferases would best suit our following experiments. As shown in fig. 2, for the same concentration of substrate, we see that firefly luciferase has a more stable and higher signal. Moreover, the difference between the background noise (negative control, non fused split luciferase) and the full luciferase is bigger for Firefly luciferase, which is also preferable.

Microfluidic Achievements

Experiment 1:

Microfluidic Accomplishments

Empty	MITOMI	MITOMI modified	SmashColi	BioPad	FilterColi	CleanColi
Full chip
Unit Cell						N/A
Designed	N/A	CHECK	CHECK	CHECK	CHECK	CHECK
Mold fabrication	CHECK	CHECK	CHECK	CHECK	N/A	N/A
Fabrication of the chip	CHECK	CHECK	CHECK	CHECK	N/A	N/A
Application	CHECK	CHECK	CHECK	N/A	N/A	N/A
Reference	MITOMI paper	N/A	N/A	N/A	N/A	N/A

Experiment 2: Culturing E. coli with constitutive GFP on chip

We loaded E. coli, which contained constitutive GFP, in the chip. By using LabVIEW, a protocol was launched overnight to ensure the growth of the cells (the protocol can be found here).

The next morning, a scan of the chip was done to see the intensity of the GFP in the chip.

Experiment 3: Inducing the pBAD promoter of our E. coli that has CpxR linked with GFP

The experiment that was done on wetbench to show that CpxR linked with GFP was expressed with an arabinose promoter was replicated on a MITOMI chip. LB medium with arabinose was flowed in the upper half whereas LB medium without arabinose was flowed in the lower half. We scanned every hour for 5h (to know how it was done click here).

Figure 1. Scan of the microfluidic chip at t = 0min. No signal is detected

 

Figure 2. Scan of the microfluidic chip at t = 300min.

We analysed the scans and obtained the following results.

Figure 3. Evolution of CpxR-GFP fluorescence over time

Yeast stuff ?

Experiment 1:

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References

1: S.K. Hatzios, S. Ringgaard, B. M. Davis, M. K. Waldor (2012, August 15). Studies of Dynamic Protein-Protein Interactions in Bacteria Using Renilla Luciferase Complementation Are Undermined by Nonspecific Enzyme Inhibition. Plos One.