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

Envelope Stress Responsive Bacteria

We have been working on engineering Bacteria so that they can trigger quickly and efficiently a signal upon stress. Knowing Bacteria naturally have a pathway to respond to stress, we combined 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. Its 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

Protein complementation assay aims to characterize interaction between proteins of interest. Based on this technique, we could engineer Bacteria emitting light upon stress. Split proteins are two inactive fragments of a reporter protein genetically fused to interacting proteins (CpxR). When two CpxR interact, the two reporter fragments can reversibly associate and reconstitute enzyme activity, triggering a very fast response to stimuli, which was highly required for the idea to make a Touch Pad.

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:

• GA1: Both IFP[1] and IFP[2] at the C terminal of CpxR

• GA2: IFP[1] at the C terminal and IFP[2] at the N terminal

• GA3: IFP[1] at the N terminal and IFP[2] at the C terminal

• GA4: Both IFP[1] and IFP[2] at the N terminal of the CpxR

Procedure

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 (ara promoter, chloramphenicol resistance, prefix and suffix containing respectively EcorI, XbaI and PstI, SpeI), 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 combinations 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: shutting on and down the signal

Stressing the cell with different concentration of KCL will allow us to quantify more precisely the amount of stress necessary to trigger a signal.

1. Antibiotics hypothesis

2. AFM pictures