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

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<h2>Registry Parts</h2>
 
<h2>Registry Parts</h2>
 
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<h3>pH Sensors</h3>
 
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[[File:T--Austin_UTexas--Cpx_pH_Culture_Tubes_2.png|thumb|left|300px| 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".]]
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[[File:T--Austin_UTexas--pH_Dependent_Promoter.jpeg|thumb|left|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.]]
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<h4>CpxA-CpxR</h4>
 
<h4>CpxA-CpxR</h4>
 
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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 we replaced virF with the Reporter. The original sequence was found in <i>Shigella sonnei</i>, but E. coli has a homolog of these proteins so all that is required on the construct is the appropriate prefix/suffix and CpxR binding site.
 
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 we replaced virF with the Reporter. The original sequence was found in <i>Shigella sonnei</i>, but E. coli has a homolog of these proteins so all that is required on the construct is the appropriate prefix/suffix and CpxR binding site.
 
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[[File:T--Austin_UTexas--Cpx_pH_Culture_Tubes_2.png|thumb|left|600px| 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".]]
 
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Figure 1 qualitatively depicts the Control at pH 6 with 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 since the qualitative data is ambiguous. The construct can be found on the iGEM registry as: <a href="http://parts.igem.org/Part:BBa_K2097000">K2097001</a>.
 
Figure 1 qualitatively depicts the Control at pH 6 with 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 since the qualitative data is ambiguous. The construct can be found on the iGEM registry as: <a href="http://parts.igem.org/Part:BBa_K2097000">K2097001</a>.
 
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[[File:T--Austin_UTexas--pH_Dependent_Promoter.jpeg|thumb|left|600px| 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.]]
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<h3>Composite Parts</h3>
 
<h3>Composite Parts</h3>
 
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[[File:T--Austin_UTexas--BCPplate.png|thumb|left|600px| Figure 3. Control amilCP.  This control was used to compare the color intensity to the pH sensitive P-atp2 while testing with a range of pH values. This part was documented to not be affected by changes in pH.]]
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[[File:T--Austin_UTexas--BCPplate.png|thumb|left|500px| Figure 3. Control amilCP.  This control was used to compare the color intensity to the pH sensitive P-atp2 while testing with a range of pH values. This part was documented to not be affected by changes in pH.]]
  
 
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<a href="http://parts.igem.org/Part:BBa_K2097001">K2097001</a> is a composite part made up of BBa_K592009 (blue chromoprotein) and BBa_K608002 (a strong RBS and promoter). The protein exhibits a strong blue/purple color when expressed. BBa_K608002 is made up of a promoter, BBa_J23104, and an RBS, BBa_B003. <b>LINKS</b>
 
<a href="http://parts.igem.org/Part:BBa_K2097001">K2097001</a> is a composite part made up of BBa_K592009 (blue chromoprotein) and BBa_K608002 (a strong RBS and promoter). The protein exhibits a strong blue/purple color when expressed. BBa_K608002 is made up of a promoter, BBa_J23104, and an RBS, BBa_B003. <b>LINKS</b>
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<h4>Yellow-Green Chromoprotein</h4>
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<u>Yellow-Green Chromoprotein</u>
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<a href="http://parts.igem.org/Part:BBa_K2097002">K2097002</a> is a composite part made up of BBa_K1033916 (amajLime, a yellow-green chromoprotein)and BBa_K608006 (a medium promoter and RBS). <b>LINKS</b>
 
<a href="http://parts.igem.org/Part:BBa_K2097002">K2097002</a> is a composite part made up of BBa_K1033916 (amajLime, a yellow-green chromoprotein)and BBa_K608006 (a medium promoter and RBS). <b>LINKS</b>

Revision as of 21:36, 17 October 2016

Austin_UTexas

Registry Parts


pH Sensors

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".
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.

CpxA-CpxR

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 we replaced virF with the Reporter. The original sequence was found in Shigella sonnei, but E. coli has a homolog of these proteins so all that is required on the construct is the appropriate prefix/suffix and CpxR binding site.

Figure 1 qualitatively depicts the Control at pH 6 with 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 since the qualitative data is ambiguous. The construct can be found on the iGEM registry as: K2097001.

Composite Parts

Figure 3. Control amilCP. This control was used to compare the color intensity to the pH sensitive P-atp2 while testing with a range of pH values. This part was documented to not be affected by changes in pH.

Blue Chromoprotein

K2097001 is a composite part made up of BBa_K592009 (blue chromoprotein) and BBa_K608002 (a strong RBS and promoter). The protein exhibits a strong blue/purple color when expressed. BBa_K608002 is made up of a promoter, BBa_J23104, and an RBS, BBa_B003. LINKS

Yellow-Green Chromoprotein

K2097002 is a composite part made up of BBa_K1033916 (amajLime, a yellow-green chromoprotein)and BBa_K608006 (a medium promoter and RBS). LINKS