Difference between revisions of "Part:BBa K2097000"
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<partinfo>BBa_K2097000 short</partinfo>
 
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<p>CpxA-CpxR is a two-component mechanism that is activated at pH 7.4 and repressed at pH 6.0. CpxA is an intermembrane protein that autophosphorylates at a certain external pH, CpxR (a kinase) then gets phosphorylated by CpxA and acts as a transcription factor for the downstream gene, YGCP in this case. This system originally is a transcription factor for the virF gene, but virF was replaced with a reporter. The original sequence was found in <i>Shigella sonnei</i>, but <i>E. coli</i> has a homolog of these proteins so only the appropriate BioBrick prefix/suffix and CpxR binding site were required.  Used <a href="https://parts.igem.org/Part:BBa_K2097002">BBa_K2097002</a> as the control series.</p></html>
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<partinfo>BBa_K2097000 SequenceAndFeatures</partinfo>
  
 
<h3>Usage and Biology</h3><p></p>
 
<h3>Usage and Biology</h3><p></p>
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<p>CpxA-CpxR is a two-component mechanism that is activated at pH 7.4 and repressed at pH 6.0. CpxA is an intermembrane protein that autophosphorylates at a certain external pH, CpxR (a kinase) then gets phosphorylated by CpxA and acts as a transcription factor for the downstream gene, YGCP in this case. This system originally is a transcription factor for the virF gene, but virF was replaced with a reporter. The original sequence was found in <i>Shigella sonnei</i>, but <i>E. coli</i> has a homolog of these proteins<sup>[1] [2]</sup>. Only the appropriate BioBrick prefix/suffix and CpxR binding site were required to design the part.  Used <a href="https://parts.igem.org/Part:BBa_K2097002">BBa_K2097002</a> as the control series.</p></html>
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[1] Nakayama, S.-I., and Watanabe, H. (1998) Identification of cpxR as a Positive Regulator Essential for Expression of the Shigella sonnei virF Gene. Journal of Bacteriology 180, 3522–3528
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[2]Nakayama, S.-I., and Watanabe, H. (1995) Involvement of cpxA, a Sensor of a Two-Component Regulatory System, in the pH-Dependent Regulation of Expression of Shigella sonnei virF Gene. Journal of Bacteriology 177, 5062–5069
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<h3>Experimental Design</h3>
 
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https://static.igem.org/mediawiki/2016/a/ad/T--Austin_UTexas--pH_Dependent_Promoter.jpeg
 
https://static.igem.org/mediawiki/2016/a/ad/T--Austin_UTexas--pH_Dependent_Promoter.jpeg
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<p><b>Figure 1.</b>The blue data points represent fluorescent readings with the control series (<a href="https://parts.igem.org/Part:BBa_K2097002">BBa_K2097002</a>), while the orange data points are the Cpx construct. As one can see, the Cpx construct shows increased fluorescence as the pH increases from 6 to 9.</p>
 
<p><b>Figure 1.</b>The blue data points represent fluorescent readings with the control series (<a href="https://parts.igem.org/Part:BBa_K2097002">BBa_K2097002</a>), while the orange data points are the Cpx construct. As one can see, the Cpx construct shows increased fluorescence as the pH increases from 6 to 9.</p>
 
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<partinfo>BBa_K2097000 SequenceAndFeatures</partinfo>
 

Revision as of 19:00, 25 October 2016


CpxR binding site attached to a yellow-green color protein (YGCP) acts as a neutral pH indicator.




Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Usage and Biology

CpxA-CpxR is a two-component mechanism that is activated at pH 7.4 and repressed at pH 6.0. CpxA is an intermembrane protein that autophosphorylates at a certain external pH, CpxR (a kinase) then gets phosphorylated by CpxA and acts as a transcription factor for the downstream gene, YGCP in this case. This system originally is a transcription factor for the virF gene, but virF was replaced with a reporter. The original sequence was found in Shigella sonnei, but E. coli has a homolog of these proteins[1] [2]. Only the appropriate BioBrick prefix/suffix and CpxR binding site were required to design the part. Used BBa_K2097002 as the control series.

[1] Nakayama, S.-I., and Watanabe, H. (1998) Identification of cpxR as a Positive Regulator Essential for Expression of the Shigella sonnei virF Gene. Journal of Bacteriology 180, 3522–3528

[2]Nakayama, S.-I., and Watanabe, H. (1995) Involvement of cpxA, a Sensor of a Two-Component Regulatory System, in the pH-Dependent Regulation of Expression of Shigella sonnei virF Gene. Journal of Bacteriology 177, 5062–5069

Experimental Design

T--Austin_UTexas--pH_Dependent_Promoter.jpeg

Figure 1.The blue data points represent fluorescent readings with the control series (BBa_K2097002), while the orange data points are the Cpx construct. As one can see, the Cpx construct shows increased fluorescence as the pH increases from 6 to 9.