Difference between revisions of "Part:BBa K2097000"
 
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<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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<p>CpxA-CpxR is a two-component mechanism that is activated at pH 7.4 and remains activated at higher pH. The mechanism is repressed at pH 6.0 and remain repressed at lower pH. 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 the virF coding sequence 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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<h3>Experimental Design</h3>
 
<h3>Experimental Design</h3>
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Top10 E. coli are grown in unaltered M9 minimal media with chloremphenicol (CAM) resistance to ensure healthy growth until the OD600 was around 0.6, which correlates to log phase growth. Next, aliquots of these cells were "washed" in either pH 6, 7, 8, or 9 M9 media to create a 1:3 dilution. This washing step ensured no pH alteration occurred due to previous unaltered media. The culture is grown overnight in the pH media with CAM resistance. The next day, aliquots of those cultures were placed in a 96 well plate reader to calculate relative fluorescence values using the 455 nm excitation of the YGCP. Fluorescence data was normalized using OD600 values. These results are in figure 1.
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Top10 E. coli are grown in unaltered neutral M9 minimal media with chloremphenicol (CAM) resistance to ensure healthy growth until their OD600 was around 0.6, which correlates to log phase growth. Next, aliquots of these cells were "washed" in either pH 6, 7, 8, or 9 M9 media to create a 1:3 dilution. This washing step ensured no pH alteration occurred due to the previous unaltered neutral media. The culture is grown overnight in the pH media with CAM resistance. The next day, aliquots of those cultures were placed in a 96 well plate reader to calculate relative fluorescence values using the 455 nm excitation of the YGCP. Fluorescence data were normalized using OD600 values. These results are in figure 1.
 
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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>
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<p><b>Figure 1.</b>The fluorescent data were normalized by dividing it by the OD600 readings, and therefore only show relative, normalized fluorescence. 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 8.</p>
  
 
<h3>References</h3>
 
<h3>References</h3>

Latest revision as of 02:15, 25 October 2017


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 remains activated at higher pH. The mechanism is repressed at pH 6.0 and remain repressed at lower pH. 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 the virF coding sequence 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.

Experimental Design

Top10 E. coli are grown in unaltered neutral M9 minimal media with chloremphenicol (CAM) resistance to ensure healthy growth until their OD600 was around 0.6, which correlates to log phase growth. Next, aliquots of these cells were "washed" in either pH 6, 7, 8, or 9 M9 media to create a 1:3 dilution. This washing step ensured no pH alteration occurred due to the previous unaltered neutral media. The culture is grown overnight in the pH media with CAM resistance. The next day, aliquots of those cultures were placed in a 96 well plate reader to calculate relative fluorescence values using the 455 nm excitation of the YGCP. Fluorescence data were normalized using OD600 values. These results are in figure 1.

T--Austin_UTexas--pH_Dependent_Promoter.jpeg

Figure 1.The fluorescent data were normalized by dividing it by the OD600 readings, and therefore only show relative, normalized fluorescence. 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 8.

References

[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