RESULTS

Experiment 1: Characterisation of the AraC/PBad Promoter and folding ability of GFP fused to CpxR

This construct aimed to evaluate the expression of our construct and the characteristics of the arabinose promoter in E. coli by fusing a superfolder GFP protein to 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 if the arabinose promoter worked well. It was also done on a microfluidic chip and on the wet bench. The N results can be seen below; fluorescence intensity plotted as a function of arabinose concentration.

Experiment 2: Demonstration of CpxR's dimerization & Elucidation of its dimerization orientation

Previous in vitro experiments (FRET) have shown that CpxR dimerizes. Currently little in vivo information about CpxR’s dimerization is available. To characterize our stress responsive bacteria, we had to confirm that CpxR dimerized in vivo as well as elucidate CpxR’s dimerization orientation.

We synthesized four constructs with combinations of the split IFP1.4 fragments fused to the C or N terminal of CpxR. As seen in the graph bellow, induction of IFP1.4 signal by 50 mM KCl was done at t=24min. We clearly see that the construct with IFP fragments on the C-terminal immediately responded to stress by emitting fluorescence. In fact we observe a 3 fold signal increase in 2 minutes. On the other hand, the three other orientations were non-responsive to KCl stress.

This amazing 30-fold signal increase in little time from the baseline allows us to assert that only the strain synthesizing the IFP fragments on the C-terminals of CpxR responded to KCl stress. For a further analysis of this experiment check out this link!

Experiment 3: Signal induction by various concentrations of KCl & signal shutdown by centrifugation

Having confirmed that KCl was a good signal inducer for our signal, we tested different concentration of KCL to modulate the signal and better characterize our biobrick. We also aimed to shutdown the signal by centrifugation and medium change. The signal was measured on a plate reader 20 minutes, before the addition of KCL. After 2 hours of measurement we centrifuged the cells for ten minutes and replaced the medium with PBS to be able to get a shutdown of the signal.

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 drastically shut down the signal, 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 is possible for our system. For a thorough analysis of this experiment check out this link!

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

Having shown that we were able to monitor the temporal dynamics of CpxR activation, we wanted to see if we could analyze CpxR’s spatial dynamics by microscopy. 10μl of our cells, previously stressed with 80 mM KCL were spread on a glass slide and imaged with a x100 objective and a APC (Cy5.5) filter.

We noticed various characteristics of the cells from the picture bellow. First, IFP signal was much more present in stressed bacteria rather than in non-stressed bacteria. Secondly, we distinguished two specific phenotypes within bacteria: elongated and normal cells. We noticed that this difference was due to CpxR overexpression as we saw this phenomenon also in non-stressed conditions.

In elongated cells, we were able to distinguish several bright bands of IFP signal that seem fairly uniformly distributed. In the normal phenotype we distinguished a single band in the centre 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. For a thorough analysis of this experiment check out this link!

---

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

	MITOMI	MITOMI modified	SmashColi	BioPad	FilterColi	CleanColi
Full chip
Unit Cell
Designed
Mold fabrication
Fabrication of the chip
Application
Reference	MITOMI paper

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: CpxR linked with GFP on the N terminal, induced by arabinose in E. coli

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).

We analysed the scans and obtained the following results.

Figure 3. Evolution of CpxR-GFP fluorescence over time

Characterization of the PBS2-HOG1& split Renilla Luciferase stress-sensitive response

Experiment 1: Confirmation of successful transformation via whole Renilla Luciferase tag

Aim
Transforming S.Cerevisiae cells for the first time, our first experiment consisted of the confirmation of whole protein tag transformations as a positive control for subsequent experiments and the determination of which concentration of substrate for the luciferase we should use.

Methods
Linear fragments containing homologous regions to the target gene surrounding our Luciferase tag and selective markers were created by overlap PCR from GFP tagging plasmids from AddGene. S.Cerevisiae cells were transformed to obtain two different strains : HOG1-rLuc and PBS2-rLuc.The strains were then tested in a 96 well plate using a plate reader.

Results
The test was performed using two different concentrations of the substrate, coelenterazine-h(benzyl coelenterazine) : 1μM and 5μM. The plate started to be read directly after addition of the substrate in the dark and measures were taken every 56 seconds thereafter. Three wells for each sample were measured and below are shown the graphs of their average plotted over time. The results are shown in the figure below. We observed a signal-to-noise ratio of 4 at 1μM of substrate and 10 at 5μM for the PBS2-rLuc strain. The HOG1-rLuc strain died quickly in the plate, and we had to reproduce the strain, of which we have not been able to confirm the success yet.

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.