• First subsection
• First Text
  • Part I
  • Part II

Envelope Stress Responsive Bacteria

We have been working on showing that biologically engineered Bacteria can detect and process signals quickly and efficiently. With this in mind, our team brought forward a novel idea: combining Protein Complementation techniques with biosensors to achieve fast spatiotemporal analysis of bacteria response to stimuli.

Cpx pathway

The pathway we engineered in Bacteria is the Cpx two component regulatory system. It's natural function 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 extractoplasmic stresses. The Cpx two component regulatory system belongs to the class I histidine kinases and includes three main protein (3 blocks with the descriptions of the proteins) (pathway draw)

Split reporter proteins: Infrared Fluorescent Protein

Among the different possibilities we could choose, we decided to use the Infrared Fluorescent Protein (IFP). Infrared-fluorescent proteins (IFPs) are engineered chromophore-binding domain of a bacteriophytochrome from Deinococcus radiodurans, with 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.

Solving the orientation of CpxR homodimerization: Split IFP

As the orientation of CpxR homodimerization is not very well studied, we had to resolve which end (C or N terminal) of the CpxR would be the most suitable for the fusion of the IFP fragments. We designed the four following constructs:

• Both IFP[1] and IFP[2] at the C terminal of CpxR

• IFP[1] at the C terminal and IFP[2] at the N terminal

• IFP[1] at the N terminal and IFP[2] at the C terminal

• Both IFP[1] and IFP[2] at the N terminal of the CpxR

Procedure

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). 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

What could be the other sources of stress activating the pathway