CxpR & Split IFP1.4 [Cterm + Nterm]
Purpose of the Biobrick
This construct aimed to evaluate the activation and presumed dimerization of CpxR in E.Coli by fusing split IFP1.4 (Infrared Fluorescent Protein) - IFP and IFP. Upon dimerization, CpxR allows the two parts of the split protein 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_K1486056).
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 [Cterm + Cterm] BioBrick compatible ( http://parts.igem.org/Part:BBa_K1486056 )
- CxpR & Split IFP1.4 [Cterm + Cterm] ( http://parts.igem.org/Part:BBa_K1486008 )
- CxpR & Split IFP1.4 [Nterm + Nterm] ( http://parts.igem.org/Part:BBa_K1486009 )
- CxpR & Split IFP1.4 [Nterm + Cterm] ( http://parts.igem.org/Part:BBa_K1486010 )
|negative_regulators||CpxA (phosphatase activity)|
|positive_regulators||CpxA (Histidine kinase activity when activated)|