Difference between revisions of "Part:BBa K1486009"

 
 
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<partinfo>BBa_K1486009 short</partinfo>
 
<partinfo>BBa_K1486009 short</partinfo>
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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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This part has two fused proteins downstream of an arabinose-inducible promoter: CpxR-IFP[1] and CpxR-IFP[2].  
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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_K1486056).
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This construct was made in all orientations:
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===Associated Biobricks===
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- CpxR-IFP[1] + CpxR-IFP[2] (BBa_K1286008)  
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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 [Cterm + Cterm] BioBrick compatible ( https://parts.igem.org/Part:BBa_K1486056 )
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<LI> CxpR & Split IFP1.4 [Cterm + Cterm] ( https://parts.igem.org/Part:BBa_K1486008 )
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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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- IFP[1]-CpxR + CpxR-IFP[2] (BBa_K1486010) 
 
  
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- CpxR-IFP[1] + IFP[2]-CpxR (BBa_K1486011)
 
  
 
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<span class='h3bb'>Sequence and Features</span>
 
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<partinfo>BBa_K1486008 SequenceAndFeatures</partinfo>
  
  
 
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===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K1486008 parameters</partinfo>
 
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Latest revision as of 21:14, 17 October 2014

CxpR & Split IFP1.4 [Nterm + 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[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.

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).

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:


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