Team:EPF Lausanne/Envelope stress responsive bacteria

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Envelope Stress Responsive Bacteria





The EPF Lausanne iGEM team has been working on engineering Bacteria 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.


The Cpx pathway


Cpx_pathway_description_diagram

The natural function of the Cpx two component regulatory system in bacteria is to control the expression of ‘survival’ genes whose products act in the periplasm to maintain membrane integrity. This ensures continued bacterial growth even in environments with harmful extracytoplasmic stresses. The Cpx two component regulatory system belongs to the class I histidine kinases and includes three main proteins:


Cpx_pathway_description


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 very represented a step forward in the field of protein complementation techniques as it 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. The IFP1.4 and the split IFP1.4 both have excitation and emission maxima of 640 and 708 nm respectively. The chromophore Biliverdin is easily incorporated in the cells.
Both have the advantages to be reversible. Split IFP allows the characterization of the homodimerization of the CpxR in a very specific spatiotemporal manner, as the emission of light is highly localized. Moreover, IFP creates a lot less background noise than other proteins used in protein complementation assay. Luciferase light emission can be monitored by the concentration of substrates, the Luciferin, which can be on our advantage in order to increase the signal for our BioPad.




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

  • Prove that after envelope deformation the system can shut down


In order to characterize the homodimerization of the CpxR, the various orientations of split IFP-CPXR (fusion at the C or N terminal) need to be synthesized and experimented on a plate reader. Different kind of stresses should be tested on our cell to trigger a signal, with final purpose to obtain a signal under pressure. Temporal dynamics of the functional CpxR-IFP signal emission will also be interesting, to see the signal shut on and off by changing the medium. We will finally analyze the spatial dynamics of the CpxR-IFP fusion by microscopy.

Exp_IFP
fig1. This is the figure's legend

DRAW

Various orientations of CpxR-Split IFP synthesis


We aim to synthesize four different plasmids containing:

1. Both IFP[1] and IFP[2] at the C terminal of CPXR
2. IFP[1] at the C terminal and IFP[2] at the N terminal
3. IFP[1] at the N terminal and IFP[2] at the C terminal
4. Both IFP[1] and IFP[2] at the N terminal of the CPXR

(DRAW) To do so, we first extracted the genome of E.Coli strain K-12 MG1655 and amplified by PCR the CpxR sequence. In order to insert CpxR sequence in iGEM backbone PSB1C3, addition of overlaps on the CpxR sequence was achieved by PCR. Gibson assembly allowed us to insert CpxR inside the backbone PSB1C3. We obtained IFP[1] and IFP[2] from Michnick lab. IFP[1] and IFP[2] were fused with the same technique (addition of overlap and Gibson assembly) at the N or C terminal of CpxR in the newly synthesized plasmid. In order to avoid co-transformation, IFP[1]- CpxR and IFP[2]-CpxR were fused in the same plasmid, resulting on the four plasmid containing the orientations cited above.


First experiment: Testing our four strains under stresses on a plate reader


The first experiment was achieved on a plate reader in order to measure the signal of the four different strains under different stresses: KCL, cupper, KOH or silica beads, which are thought to activate the pathway (link). Pressure will be more difficult to quantitate, so We also measured as negative control the signal of strains expressing one part of the split only (IFP[1]-CpxR or IFP[2]-CpxR). Three measurements were necessary to finally conclude that only the first configuration works, when both split part of IFP are at the C terminal of the CpxR.

GRAPH


Second experiment: Testing different stresses


Characterize the response that we could obtain with different kind of stresses, the final goal being to obtain a response under pressure. Salts and different of PH are easier to monitor than the pressure, because of its quantification and their response are better known.

Third experiment: shutting on and down the signal

  1. Antibiotics hypothesis

  2. AFM pictures



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