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

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<u>P-atp2</u>
 
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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>). 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.
 
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Revision as of 05:38, 17 October 2016

Registry Parts

pH Sensors

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. 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 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: Bba_K2097000.

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Next Steps and the GOX Sequences as Putative Promoters

Blue chromoprotein with strong promoter and RBS

Yellow-green chromoprotein with medium promoter and RBS