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

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<p>During the kombucha brewing process, the beverage becomes more acidic.  Additionally, it is unclear if or how the microbial community changes within the beverage over time.  Thus, our team decided to find pH sensitive promoters  that could be used to track not only the pH of the maturing beverage, but also the presence of the various microbes within the kombucha over time.  We successfully created a neutral range reporter, attempted to create acidic and basic range reporters, and found three putative acidic range reporters that are endogenous to one of our kombucha bacteria, <i>Gluconobacter oxydans</i></p>
 
<p>During the kombucha brewing process, the beverage becomes more acidic.  Additionally, it is unclear if or how the microbial community changes within the beverage over time.  Thus, our team decided to find pH sensitive promoters  that could be used to track not only the pH of the maturing beverage, but also the presence of the various microbes within the kombucha over time.  We successfully created a neutral range reporter, attempted to create acidic and basic range reporters, and found three putative acidic range reporters that are endogenous to one of our kombucha bacteria, <i>Gluconobacter oxydans</i></p>
 
<p>Though an acidic sensor was what was required for our kombucha analysis, the identification of sensors in other areas of the pH spectrum were explored as well. Three sequences were identified, the CadC operon for the acidic range, CpxA-CpxR complex for the neutral range, and the P-atp2 promoter from the BioBrick Registry (<a href="http://parts.igem.org/Part:BBa_K1675021">BBa_K1675021</a>) for the basic range. Each sequence was paired with a unique corresponding reporter sequence so that if each pH sensitive plasmid were in the same environment, the specific pH of the system could be seen. The reporters used were, <a href="http://parts.igem.org/Part:BBa_E1010">BBa_E1010</a> for the CadC construct, <a href="http://parts.igem.org/Part:BBa_K1033916">BBa_K1033916</a> for the CpxA-CpxR complex, and <a href="http://partsregistry.org/Part:BBa_K592009">BBa_K592009</a> for the P-atp2 promoter.</p>
 
<p>Though an acidic sensor was what was required for our kombucha analysis, the identification of sensors in other areas of the pH spectrum were explored as well. Three sequences were identified, the CadC operon for the acidic range, CpxA-CpxR complex for the neutral range, and the P-atp2 promoter from the BioBrick Registry (<a href="http://parts.igem.org/Part:BBa_K1675021">BBa_K1675021</a>) for the basic range. Each sequence was paired with a unique corresponding reporter sequence so that if each pH sensitive plasmid were in the same environment, the specific pH of the system could be seen. The reporters used were, <a href="http://parts.igem.org/Part:BBa_E1010">BBa_E1010</a> for the CadC construct, <a href="http://parts.igem.org/Part:BBa_K1033916">BBa_K1033916</a> for the CpxA-CpxR complex, and <a href="http://partsregistry.org/Part:BBa_K592009">BBa_K592009</a> for the P-atp2 promoter.</p>
 
<h4>CadC</h4>
 
 
<p>The CadC operon is a native pathway in <i>E. coli</i>, involved in the cadaverine synthesis pathway. The protein CadC protein on the operon is produced and activates segments downstream of the operon on the CadBA receptors. The CadC protein is pH sensitive to an external pH 5.5 and below, as well as lysine dependent. A point mutation on codon 265, in which argenine is converted to cystine, causes the CadC protein to become lysine independent.<sup>3</sup></p>
 
<p>Unfortunately, we have been unable to grow the modified CadC operon in <i>E. coli</i> suggesting some form of cell toxicity. Due to this apparent toxicity, no data regarding this mutant CadC could be collected. Alternative candidates are being explored for other pH sensors that sense in the acidic range.</p>
 
  
 
<h4>CpxA-CpxR</h4>
 
<h4>CpxA-CpxR</h4>
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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>
 
<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>CadC</h4>
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<p>The CadC operon is a native pathway in <i>E. coli</i>, involved in the cadaverine synthesis pathway. The protein CadC protein on the operon is produced and activates segments downstream of the operon on the CadBA receptors. The CadC protein is pH sensitive to an external pH 5.5 and below, as well as lysine dependent. A point mutation on codon 265, in which argenine is converted to cystine, causes the CadC protein to become lysine independent.<sup>3</sup></p>
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<p>Unfortunately, we have been unable to grow the modified CadC operon in <i>E. coli</i> suggesting some form of cell toxicity. Due to this apparent toxicity, no data regarding this mutant CadC could be collected. Alternative candidates are being explored for other pH sensors that sense in the acidic range.</p>
  
 
<h4>P-atp2</h4>
 
<h4>P-atp2</h4>

Revision as of 00:06, 19 October 2016

Austin_UTexas

Results


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