Team:EPF Lausanne/Envelope stress responsive bacteria

From 2014.igem.org

(Difference between revisions)
Line 102: Line 102:
<br/>
<br/>
 +
2) Final constructs: Split-IFP
 +
 +
The split IFP was used for the very fast response triggered upon stimuli. Find more about its construction and the experiments achieved to characterize the CPXR homodimerization and to test the signal resulting from different stimuli.
 +
 +
The reason why we decided to use split protein is because of the very fast response to stimuli that triggers protein complementation techniques, and that was highly required for the idea to make a Touch Pad. Moreover, we were also able to characterize more in details the CPX pathway, particularly how the CPXR homodimerize and the spatiotemporal dynamics of homodimerization upon different kinds of stresses.
 +
 +
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:
 +
 +
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)
 +
 +
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.
 +
 +
(draw)
 +
 +
First experiment
 +
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
 +
 +
As KCL worked better than the PH stress, mechanical stress or cupper stress, we tried with different types of salts:
 +
 +
Antibiotics hypothesis
 +
 +
AFM pictures

Revision as of 14:21, 13 October 2014

Envelope Stress Respsonsive Bacteria


2) Final constructs: Split-IFP The split IFP was used for the very fast response triggered upon stimuli. Find more about its construction and the experiments achieved to characterize the CPXR homodimerization and to test the signal resulting from different stimuli. The reason why we decided to use split protein is because of the very fast response to stimuli that triggers protein complementation techniques, and that was highly required for the idea to make a Touch Pad. Moreover, we were also able to characterize more in details the CPX pathway, particularly how the CPXR homodimerize and the spatiotemporal dynamics of homodimerization upon different kinds of stresses. 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: 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) 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. (draw) First experiment 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 As KCL worked better than the PH stress, mechanical stress or cupper stress, we tried with different types of salts: Antibiotics hypothesis AFM pictures

Sponsors