Envelope Stress Responsive Bacteria

The EPF Lausanne iGEM team has been working on engineering E. coli responding to stress - in particular menchanical stress – quickly and efficiently. Taking in consideration that bacteria naturally respond to various envelope stresses through the Cpx pathway, we combined Protein Complementation techniques with biosensors to achieve fast spatiotemporal analysis of bacteria response to stimuli.

To help you understand how our engineered bacteria work, we would like to start out by introducing two major components of our project: the Cpx pathway and the split reporter proteins.

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The Cpx pathway

How can Bacteria sense and adapt themselves to environmental stress? The key lies in a fairly unknown two component regulatory system: the Cpx pathway. Its natural function is to control the expression of ‘survival’ genes whose products act in the periplasm to maintain membrane integrity. The first member of the pathway, CpxA, "senses" the environmental stress and activates its corresponding relay protein, CpxR (acts like a transcription factor). This ensures continued bacterial growth even in environments with harmful stresses.
Sensors, responder, inhibitor...The Cpx two component regulatory system belongs to the class I histidine kinases and includes three main proteins:

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Split reporter proteins: Infrared Fluorescent Protein

Protein complementation techniques consist in splitting reporter proteins with fluorescent, bioluminescent, or colorimetric properties at specific locations and fusing them to proteins of interest. The fused split fragments remain inactive until physical interaction of the proteins of interest leads to the reconstitution of the chemical properties and the proper folding of the split reporter proteins. This technique is widely used to characterise the interactions between proteins of interest in a given pathway. The EPFL iGEM team however chose to implement protein complementation techniques in a new way. Our team "hijacked" the Cpx pathway in E. coli by fusing split protein fragments to CpxR to develop fast and precise stress biosensors. On top of being used in basic science as a stress sensor and a proof of concept of our novel way of viewing biosensors, we aimed to integrate our engineered bacteria in our BioPad to allow fast and precise signal emission upon touch.

The split protein we chose to fuse to CpxR was the Infrared Fluorescent Protein (IFP 1.4). The IFP1.4 is an engineered chromophore-binding domain of a bacteriophytochrome from Deinococcus radiodurans. Its split was an important landmark in the field of protein complementation techniques, as the split IFP1.4 is the first fluorescent split protein to be reversible. Moreover, due to its emission in the infrared wavelengths, the IFP1.4 benefits of high signal-to-noise ratios allowing precise analysis of spatial dynamics. The split IFP was developped in 2014 by the Michnick lab in the following extraordinary paper.

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Experiment planification

Having chosen a protein complementation candidate- IFP1.4 - and a stimuli responsive dimerizing protein (CpxR), we planned out a set of experiments that would allow us to prove that fast and specific spatiotemporal analysis of stimuli is possible.
We therefore set ourselves three intermediate objectives:

• Prove that CpxR dimerizes and find out its dimerization orientation.

• Prove that the system can be activated and shut down quickly.

• Prove that the system can provide spatial information and that sub-localisation of the protein within bacteria is detectable.

Moreover, to assert that our system could be used to build a BioPad, we also had two extra objectives:

• Show that our system can be activated by envelope deformation - particularily by mechanically stress in a microfluidic chip.

• Prove that after envelope deformation the system can shut down

Experiment 1: CpxR dimerization

In order to prove that CpxR dimerizes and to find out its dimerization orientation, synthesis of 4 constructs will be necessary: each part of the split being at the C or N terminal of the CpxR. After genome extraction for CpxR amplification, amplification of IFP1 and IFP2 from Michnick's lab, many PCR's and Gibson Assemblies, we finally obtained our 4 constructs:

The 4 different strains (resulting from the transformation) put under stress will be analysed on a plate reader to measure the IFP signal. KCL is known to trigger the activation of CpxA and should lead to a signal. In function of its intensity in stressed cells, non-stressed cells and DH5α, we will be able to deduce on which orientation the CpxR does dimerize. -> check out our result! Only the orientation where both IFP1 and IFP2 are fused at the C terminal of the CpxR allows the emission of a signal.

Experiment 2: Activation and deactivation of the signal

After activation achievement, the signal also has to be deactivated once the source of stress is removed. This switch is of great importance, as we want to characterize the response in a fast and precise way. Activation and deactivation need to be directly related to the source of stress.
Bacteria IFP signal will be measured on the plate reader again. In a first step, signal emission will be measured after addition of the source of stress (50 mM KCL), and after the removal of this source in a second step. 96 wells plate centrifugation will allow us to remove the KCL-PBS solution and resuspend cells in PBS with no KCL, which should shut down the signal.
Look at our results! The signal drop in a very impressive way…

Experiment 3: Localisation of the signal within Bacteria

After a temporal analysis of the CpxR dimerization, spatial analysis is also of interest to characterize the CpxR in more details. Samples of stressed cells (80 mM of KCL) on a glass light can be observed under a microscope with appropriate filter. As well as precise information on the localization of the signal, this experiment will allow us to see a clear difference between stressed and non-stressed cells.

Experiment 4: Split Luciferase Complementation Assay

Luciferase complementation assay is a powerful tool to study interaction between proteins in vivo. Thus, it is important to choose carefully which signal is best suited for these kind of experiments.
Advantages of luciferase for our project :

• Fast and reversible signal (quasi-immediate)
• Light excitation not needed (minimal autofluorescence) => simplification of the signal detection and processing mechanisms
• Signal highly correlated with concentration of substrate => very modulable
• Small concentrations of substrate are needed

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 Waldor Laboratory, we wanted to redo and adapt the experiment to test our own splits.

We created two different constructs, one with split Firefly luciferase and one with split Renilla luciferase, to test different possibilities with different substrates (Firefly luciferase oxidizes d-luciferin and Renilla luciferase oxidizes coelenterazine). Check out out results to see how went our experiments!