Team/CINVESTAV-IPN-UNAM MX/oxigenresponse.htm

From 2012.igem.org

(Difference between revisions)
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             <img src="http://2012.igem.org/wiki/images/c/c5/Osy01.jpg">
             <img src="http://2012.igem.org/wiki/images/c/c5/Osy01.jpg">
             <p>Figure 1. This BioBrick will show if our dependent promoter is functional, using the
             <p>Figure 1. This BioBrick will show if our dependent promoter is functional, using the
-
  constitutive (or natural) system from R. sphaeroides or the orthologue system from R.
+
  constitutive (or natural) system from <em>R. sphaeroides</em> or the orthologue system from <em>R.
-
  palustris.</p>
+
  palustris.</em></p>
     </div>
     </div>
           <div align="center"><img src="http://2012.igem.org/wiki/images/0/06/Osy02.jpg" width="562" height="192">
           <div align="center"><img src="http://2012.igem.org/wiki/images/0/06/Osy02.jpg" width="562" height="192">
  <p>Figure 2. This BioBrick will show if our complete system is functional because probably
  <p>Figure 2. This BioBrick will show if our complete system is functional because probably
  we need a synthetic system to promote GFP expression by binding its target sequence
  we need a synthetic system to promote GFP expression by binding its target sequence
-
  (dependent promoter) in R. palustris.</p>
+
  (dependent promoter) in <em>R. palustris.</em></p>
  </div>
  </div>
-
  <p>Both systems were cloned in pRK415 because it is a vector for Purple Non-Sulfur
+
  <p>Both systems were cloned in pRK415 because this is a vector for Purple Non-Sulfur
-
  Photosynthetic Bacteria, the plasmids were introduced in R. sphaeroides and R. palustris,
+
  Photosynthetic Bacteria, the plasmids were introduced in <em>R. sphaeroides</em> and <em>R. palustris</em>,
  by biparental and triparental conjugation.</p>
  by biparental and triparental conjugation.</p>
  <p>The measurement approach we used was:
  <p>The measurement approach we used was:
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<li>Anaerobic/Light</li>
<li>Anaerobic/Light</li>
  <li>Anaeroibic/darkness</li></p>
  <li>Anaeroibic/darkness</li></p>
-
<p>For all data results, we considered a negative control: Rhodobacter sphaeroides or Rhodopseudomonas palustris, conjugated bacteria with pRK415 vector without BioBrick.</p>
+
<p>For all data results, we considered a negative control: <em>Rhodobacter sphaeroides</em> or <em>Rhodopseudomonas palustris</em>, conjugated bacteria with pRK415 vector without BioBrick.</p>
<div align="center"><img src="http://2012.igem.org/wiki/images/7/73/Osy03.jpg" width="563" height="452">
<div align="center"><img src="http://2012.igem.org/wiki/images/7/73/Osy03.jpg" width="563" height="452">
<img src="http://2012.igem.org/wiki/images/e/ec/Osy04.jpg" width="563" height="452">
<img src="http://2012.igem.org/wiki/images/e/ec/Osy04.jpg" width="563" height="452">
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</div>
</div>
<p id="text2">Discussion</p>
<p id="text2">Discussion</p>
-
  <p>In R. sphaeroides, as we can see in figure 3 and 4, there was low GFP expression, probably
+
  <p>In <em>R. sphaeroides</em>, as we can see in figure 3 and 4, there was low GFP expression, probably
  because growing conditions were microaerophilic instead of extrictly anaerobic. In
  because growing conditions were microaerophilic instead of extrictly anaerobic. In
  anaerobic conditions PrrB autophosphorylates and passes a phosphate group to PrrA, this
  anaerobic conditions PrrB autophosphorylates and passes a phosphate group to PrrA, this
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<br>
<br>
-
  In R. palustris, we had GFP expression in dependent promoter (BBa_K776019), maybe
+
  In <em>R. palustris</em>, we had GFP expression in dependent promoter (BBa_K776019), maybe
  because orthologous proteins activated it, and the complete system (BBa_K776021) also
  because orthologous proteins activated it, and the complete system (BBa_K776021) also
  was functional in the expected condition.<br>
  was functional in the expected condition.<br>
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<p id="text2">Conclusion</p>
<p id="text2">Conclusion</p>
<p> Our two BioBricks (K776019 and BBa_K776021) are functional in two photosynthetic
<p> Our two BioBricks (K776019 and BBa_K776021) are functional in two photosynthetic
-
  bacteria R. palustris and R. sphaeroides. This is a functional system for controlling genetic
+
  bacteria <em>R. palustris</em> and <em>R. sphaeroides</em>. This is a functional system for controlling genetic
  expression with Oxygen tension.</p>
  expression with Oxygen tension.</p>
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<!-- end #page -->
<!-- end #page -->
<div id="piep">
<div id="piep">
-
<p align="center"> Rhodofactory 2012 </p>
+
<p align="center"> <strong>Rhodofactory</strong> 2012 </p>
<div id="sponsors">
<div id="sponsors">
  <div align="center"><img src="http://2012.igem.org/wiki/images/8/8a/Icytdf.png" alt="icytdf" width="90" height="82" /></div>
  <div align="center"><img src="http://2012.igem.org/wiki/images/8/8a/Icytdf.png" alt="icytdf" width="90" height="82" /></div>

Revision as of 03:05, 26 October 2012

Rho

Oxygen Control System!

PrrA/PrrB two component system

This system remains inactive under high oxygen tension, when oxygen concentration decreases, it is possible the GFP transcription. (See Rhodofactory section for a complete explanation).
We made two BioBricks (BBa_K776019 y BBa_K776021) to test the Oxygen Control System, each one has GFP as a reporter gene and the functionality was related to the fluorescence detection.

Figure 1. This BioBrick will show if our dependent promoter is functional, using the constitutive (or natural) system from R. sphaeroides or the orthologue system from R. palustris.

Figure 2. This BioBrick will show if our complete system is functional because probably we need a synthetic system to promote GFP expression by binding its target sequence (dependent promoter) in R. palustris.

Both systems were cloned in pRK415 because this is a vector for Purple Non-Sulfur Photosynthetic Bacteria, the plasmids were introduced in R. sphaeroides and R. palustris, by biparental and triparental conjugation.

The measurement approach we used was:

  • Fluorescence Microscopy: To have a qualitative detection of GFP in these bacteria.
  • Flow Cytometry: To have a quantitative detection of GFP expression, we calculated the percentage of bacterial population expressing GFP (GFP+) in 1000 bacteria.

  • We used 3 environmental growing conditions:

  • Aerobic/Darkness
  • Anaerobic/Light
  • Anaeroibic/darkness
  • For all data results, we considered a negative control: Rhodobacter sphaeroides or Rhodopseudomonas palustris, conjugated bacteria with pRK415 vector without BioBrick.

    Figure 3. Percentage of bacterial population expressing GFP.

    Figure 4. Representative images obtained by fluorescence microscopy, where our systems were functional in the expected conditions.

    Discussion

    In R. sphaeroides, as we can see in figure 3 and 4, there was low GFP expression, probably because growing conditions were microaerophilic instead of extrictly anaerobic. In anaerobic conditions PrrB autophosphorylates and passes a phosphate group to PrrA, this activated PrrA binds its promoter sequence to start GFP expression. Furthermore, when we introduced the complete system (BBa_K776021), actually we are overexpressing the regulatory proteins and the signaling could not be fully controlled.

    In R. palustris, we had GFP expression in dependent promoter (BBa_K776019), maybe because orthologous proteins activated it, and the complete system (BBa_K776021) also was functional in the expected condition.

    The GFP expression that we did not expected under environmental conditions, probably it is due to the complexity in the regulatory network where this system is involved, and there are interfering proteins.

    Conclusion

    Our two BioBricks (K776019 and BBa_K776021) are functional in two photosynthetic bacteria R. palustris and R. sphaeroides. This is a functional system for controlling genetic expression with Oxygen tension.

     

    Rhodofactory 2012

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