Part:BBa_K1486008

CxpR & Split IFP1.4 [Cterm + Cterm][1]

Purpose of the Biobrick

This construct aimed to evaluate the activation and presumed dimerization of CpxR in E.Coli by fusing the novel split IFP1.4 (Infrared Fluorescent Protein) fragments - IFP[1] and IFP[2] - developed by the Michnick Lab (see reference). We chose the split IFP1.4 as it is a reversible split fluorescent protein emitting little noise. Upon dimerization, CpxR allows the two parts of the split IFP1.4 to re-fold and acquire their ability to emit fluorescence. Not knowing how CpxR might dimerize, we built 4 different biobricks with the various possible orientations that the dimerization of CpxR might acquire.

An experiment on all possible CpxR - Split IFP fragments was launched to determine whether CpxR dimerized in E.Coli, as well as how this dimerization occured. The experiment conditions can be found here. The basics were the following: induction of stress by 50 mM KCl and reading on a plate reader with excitation and emission wavelengths of 640nm and 708 nm respectively.

The results of this experiment show that CpxR dimerizes, and that it does so via its C-terminal. Hence, the functional biobrick is CxpR & Split IFP1.4 [Cterm + Cterm] (BBa_K1486008).

Experiment 1: CpxR dimerization & Dimerization Orientation

CpxR is the relay protein in the CpxAR two component regulatory system. It has been shown through Protein Complementation techniques that CpxR dimerizes when phosphorylated (activated) in yersinia pseudotuberculosis. Moreover, following other in vitro FRET studies, it was shown that CpxR interacted with itself. We therefore hypothesised that dimerization would also be true in vivo in E.Coli. To determine this, we built four constructs with the various possible orientations as seen under the associated Biobricks section bellow. As seen in the graph bellow, we successfully proved that CpxR dimerized in vivo and that dimerization led to close interaction of its C-terminus. Thanks to the fusion of the split IFP1.4, our biobrick allows fine tuned spatiotemporal analysis of the activation of CpxR in vivo. This finding is important as CpxR is part of the highly conserved OmpR/PhoB subfamily - especially for their C-terminus. This system could be used to study various other components of the OmpR/PhoB subfamily and thus lead to a new generation of highly senstitive and reactive biosensors.

As seen in the graph, induction of the signal was done at minute 24 (marked via a vertically spoted line). It is to be noted that the signal is immediate (3 fold increase in 2 minutes) and that the signal overall increased 30-fold.

Experiment 2: Signal induction by various concentrations of KCl & signal shutdown by centrifugation

Having found that KCl was a good signal inducer for our signal, we decided to characterise our biobrick by testing if the signal could be modulated by various concentrations of KCl and if we were able to remove the signal by centrifugation and medium change. To do so, we read our signal for 20 minutes without stress and then added KCl. At minute 144 we then centrifuged our cells and replaced the medium with PBS.

As seen in the figure above, we successfully showed that increasing concentrations of KCl led to stronger signals up to a saturation concentration of about 80 mM KCl. Moreover we were able to shut the signal down, thus proving the reversibility of our system.

Associated Biobricks

In the context of the same experiment, we designed three more constructs with different CpxR - IFP fragment orientations. This construct was made in all orientations:

• CxpR & Split IFP1.4 [Nterm + Nterm] ( https://parts.igem.org/Part:BBa_K1486009 )
• CxpR & Split IFP1.4 [Nterm + Cterm] ( https://parts.igem.org/Part:BBa_K1486010 )
• CxpR & Split IFP1.4 [Cterm + Nterm] ( https://parts.igem.org/Part:BBa_K1486011 )

WARNING: This Biobrick was built by Gibson Assembly and contains illegal restriction restriction enzyme sites !

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 2213
  Illegal PstI site found at 3446
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1205
  Illegal PstI site found at 2213
  Illegal PstI site found at 3446
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 3657
  Illegal BamHI site found at 1144
  Illegal XhoI site found at 1283
  Illegal XhoI site found at 2470
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 2213
  Illegal PstI site found at 3446
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 2213
  Illegal PstI site found at 3446
  Illegal AgeI site found at 979
  Illegal AgeI site found at 1694
  Illegal AgeI site found at 2881
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961