Revision as of 16:17, 13 October 2014

Envelope Stress Responsive Bacteria

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

DRAW

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

Antibiotics hypothesis

AFM pictures

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 <p class="lead>
-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.
+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. </p>
 <br/>
+<p class="lead>
 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.  <br/>
 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.
+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.</p>
   <br/> <br/>
 <div class="cntr>
 <img src="https://static.igem.org/mediawiki/2014/1/14/IFP_CpxR_Scheme_-_copie.png" alt="" />
 </div>
-</p>
 <h2>Solving the orientation of CpxR homodimerization: Split IFP</h2>

Team:EPF Lausanne/Envelope stress responsive bacteria

From 2014.igem.org