Difference between revisions of "Team:Austin UTexas/Results"

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<h4>CpxA-CpxR</h4>
 
<h4>CpxA-CpxR</h4>
  
<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. 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 all that is required on the construct is the appropriate prefix/suffix and CpxR binding site.<sup>5,6</sup></p>
 
 
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[[File:T--Austin_UTexas--Cpx_pH_Culture_Tubes_2.png|thumb|right|500px| Figure 1. Testing the CpxR Construct in pH 6-9. From left to right is control pH 6-9 and then experimental pH 6-9. These are showing the gradient change in expression accordingly with the change of pH due to a pH-dependent promotor compared to consistent expression accordingly with a promoter that is always "on". Credit: Sofia Chinea]]
 
[[File:T--Austin_UTexas--Cpx_pH_Culture_Tubes_2.png|thumb|right|500px| Figure 1. Testing the CpxR Construct in pH 6-9. From left to right is control pH 6-9 and then experimental pH 6-9. These are showing the gradient change in expression accordingly with the change of pH due to a pH-dependent promotor compared to consistent expression accordingly with a promoter that is always "on". Credit: Sofia Chinea]]
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[[File:T--Austin_UTexas--pH_Dependent_Promoter.jpeg|thumb|right|500px| Figure 2. Normalized fluorescent values from CpxR construct vs control (YGCP). The fluorescence per cell count stayed generally the same throughout the range of pH while the CpxR has a clear increase in fluorescence per cell. Credit: Sofia Chinea]]
 
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<p>The order from left to right in figure 1 is control pH 6-9 and then Experimental pH 6-9. These are showing the gradient change in expression accordingly with the change of pH due to a pH-dependent promotor compared to consistent expression accordingly with a promoter that is always "on". The main point is that the control at pH 6 has more expression of the yellow-green chromoprotein than the Experimental at pH 6. The pH-dependent promoter of the experimental group is down-regulated at pH 6 whereas the control is not. Also, there is an increase in YGCP expression between the experiment pH 7 and pH 8 that is not seen in the control between pH 7 and pH 8. The normalized data in figure 2 shows the relative expression of YGCP. The CpxA-CpxR construct can be found on the iGEM registry as: <a href=“http://parts.igem.org/Part:Bba_K2097000”>BBa_K2097000</a>, while the construct utilized as a control can be found on the iGEM registry as <a href="http://parts.igem.org/Part:BBa_2097002">BBa_K2097002</a> as well as in figure 3.</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. 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 all that is required on the construct is the appropriate prefix/suffix and CpxR binding site.<sup>5,6</sup></p>
 
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[[File:T--Austin_UTexas--pH_Dependent_Promoter.jpeg|thumb|right|500px| Figure 2. Normalized fluorescent values from CpxR construct vs control (YGCP). The fluorescence per cell count stayed generally the same throughout the range of pH while the CpxR has a clear increase in fluorescence per cell. Credit: Sofia Chinea]]
 
 
[[File:T--Austin_UTexas--YGCPtube.png|thumb|left|275px|Figure 3. amajLime expressed in <i>E. coli</i> in liquid LB. Credit: Sofia Chinea]]
 
[[File:T--Austin_UTexas--YGCPtube.png|thumb|left|275px|Figure 3. amajLime expressed in <i>E. coli</i> in liquid LB. Credit: Sofia Chinea]]
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<p>The order from left to right in figure 1 is control pH 6-9 and then Experimental pH 6-9. These are showing the gradient change in expression accordingly with the change of pH due to a pH-dependent promotor compared to consistent expression accordingly with a promoter that is always "on". The main point is that the control at pH 6 has more expression of the yellow-green chromoprotein than the Experimental at pH 6. The pH-dependent promoter of the experimental group is down-regulated at pH 6 whereas the control is not. Also, there is an increase in YGCP expression between the experiment pH 7 and pH 8 that is not seen in the control between pH 7 and pH 8. The normalized data in figure 2 shows the relative expression of YGCP. The CpxA-CpxR construct can be found on the iGEM registry as: <a href=“http://parts.igem.org/Part:Bba_K2097000”>BBa_K2097000</a>, while the construct utilized as a control can be found on the iGEM registry as <a href="http://parts.igem.org/Part:BBa_2097002">BBa_K2097002</a> as well as in figure 3.</p>
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<h4>P-atp2</h4>
 
<h4>P-atp2</h4>
 
<p>The P-atp2 promoter, native to the bacterium <i>Corynebacterium glutamicum</i> is reportedly induced at pH 7, to pH 9 (<a href="https://2015.igem.org/Team:BIT-China/Parts">BIT-China-2015</a> and <a href="http://parts.igem.org/Part:BBa_K1675021">BBa_K1675021</a>).<sup>1</sup> Utilizing the blue chromoprotein (<a href="http://partsregistry.org/Part:BBa_K592009">BBa_K592009</a>), a test was designed in which a plasmid containing the P-atp2 promoter with the blue chromoprotein was grown alongside an <i>E. coli</i> line that contained a plasmid with just the blue chromoprotein. We expected to see constant blue chromoprotein production in the control series (those that lacked P-atp2) and a visual increase in blue chromoprotein as the pH was raised from 6 to 9 in the cells that contained the P-atp2 construct. The construct utilized as a control can be found on the iGEM registry <a href="http://parts.igem.org/Part:BBa_2097001">BBa_K2097001</a> as as in figure 5.</p>
 
<p>The P-atp2 promoter, native to the bacterium <i>Corynebacterium glutamicum</i> is reportedly induced at pH 7, to pH 9 (<a href="https://2015.igem.org/Team:BIT-China/Parts">BIT-China-2015</a> and <a href="http://parts.igem.org/Part:BBa_K1675021">BBa_K1675021</a>).<sup>1</sup> Utilizing the blue chromoprotein (<a href="http://partsregistry.org/Part:BBa_K592009">BBa_K592009</a>), a test was designed in which a plasmid containing the P-atp2 promoter with the blue chromoprotein was grown alongside an <i>E. coli</i> line that contained a plasmid with just the blue chromoprotein. We expected to see constant blue chromoprotein production in the control series (those that lacked P-atp2) and a visual increase in blue chromoprotein as the pH was raised from 6 to 9 in the cells that contained the P-atp2 construct. The construct utilized as a control can be found on the iGEM registry <a href="http://parts.igem.org/Part:BBa_2097001">BBa_K2097001</a> as as in figure 5.</p>

Revision as of 22:42, 18 October 2016

Austin_UTexas

Results


Click on one of the images below to learn more about our results!




Kombucha Strains

Conjugation

Recapitulation

Ethanol

pH Sensors