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| − | ===Purpose of the Biobrick===
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| − | This construct aimed to evaluate the activation and presumed dimerization of CpxR in E.Coli by fusing split IFP1.4 (Infrared Fluorescent Protein) - IFP[1] and IFP[2]. 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.
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| − | 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.
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| − | 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).
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| − | ===Experiment 1: CpxR dimerization & Dimerization Orientation===
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| − | CpxR is the relay protein in the CpxAR two component regulatory system. It has been shown that CpxR dimerizes when phosphorylated (activated) in yersinia pseudotuberculosis. We therefore hypothesised that this would also be true 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 association of its C-terminus. This finding is important as the C-terminus of
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| − | https://static.igem.org/mediawiki/2014/c/c2/KCL_Construct_Comparison_small.jpg
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| − | 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.
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| − | ===Associated Biobricks===
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| − | <FONT SIZE="2"><P>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:
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| − | <LI> CxpR & Split IFP1.4 [Nterm + Nterm] ( https://parts.igem.org/Part:BBa_K1486009 )
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| − | <LI> CxpR & Split IFP1.4 [Nterm + Cterm] ( https://parts.igem.org/Part:BBa_K1486010 )
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| − | <LI> CxpR & Split IFP1.4 [Cterm + Nterm] ( https://parts.igem.org/Part:BBa_K1486011 )
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| − | </UL></P></FONT>
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| | + | ===Usage and Biology=== |
| | + | This biobricked was an intermediate for [https://parts.igem.org/Part:BBa_K1486056 BBa_K1486056]. |
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| − | <!-- Add more about the biology of this part here | + | <!-- Add more about the biology of this part here |
| | ===Usage and Biology=== | | ===Usage and Biology=== |
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