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      <p>In Rhodopseudomonas palustris, we can see in Figure 1A a perfect behavior of AppA/PpsR, the PpsR promoter do not show GFP expression, it indicates that other regulation systems do not bind this promoter, but when we introduce the complete system, we see a GFP expression of 31.89%, because the non natural system we designed is functional.  In the case of PrrA/PrrB, the PrrA  promoter do not show GFP expression, probably because the RegA/B system (the homologs system in this bacteria) do not have affinity for this sequence, but the complete construction is functional, we have to consider that it is an artificial system and the behavior can be different than expected, the functionality at these conditions is interesting because PrrB is supposed to be inactivated at aerobic conditions, or if the PrrA protein  need to be phosphorylated, how it is being inactivated in this chassis. R. palustris has a different PrrA/PrrB system, and its mechanism is quite different, in fact, the main regulator to start photosynthetic metabolism is not PrrA/PrrB system, actually it is FixK enzyme (Rey, 2010).
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The Anaerobic/Light conditions in R. Palustris (figure 2A) made possible the functionality of our Synthetic AppA/PpsR system, the PpsR promoter show a low (0.169%) GFP expression, but the complete construction also show a little GFP expression.
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In figure 3, the AppA/PpsR system is not functional, because in this condition, reduced PpsR has not affinity by its target sequence and the transcription is possible, AppA is is forming the complex with PpsR, but we can not see GFP expression. PrrA/PrrB show a low response, probably due to the independence of R. palustris for PrrA/PrrB mechanism, it functions with FixJ-FixK to regulate the change of metabolism aerobic to anaerobic (Metz, 2012).
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<h1><em>Oxygen Control System!</em></h1>
 +
            <p id="text2"> PrrA/PrrB two component system</p>
 +
          <p>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).<br><br>
 +
 
 +
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.</p>
 +
          <div align="center">
 +
            <img src="https://static.igem.org/mediawiki/2012/c/c5/Osy01.jpg">
 +
            <p>Figure 1. This BioBrick will show if our dependent promoter is functional, using the
 +
constitutive (or natural) system from <em>R. sphaeroides</em> or the orthologue system from <em>R.
 +
palustris.</em></p>
 +
    </div>
 +
          <div align="center"><img src="https://static.igem.org/mediawiki/2012/0/06/Osy02.jpg" width="562" height="192">
 +
  <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
 +
(dependent promoter) in <em>R. palustris.</em></p>
 +
  </div>
 +
  <p>Both systems were cloned in pRK415 because this is a vector for Purple Non-Sulfur
 +
Photosynthetic Bacteria, the plasmids were introduced in <em>R. sphaeroides</em> and <em>R. palustris</em>,
 +
by biparental and triparental conjugation.</p>
 +
<p>The measurement approach we used was:
 +
<li>Fluorescence Microscopy: To have a qualitative detection of GFP in these bacteria.</li>
 +
<li>Flow Cytometry: To have a quantitative detection of GFP expression, we calculated the
 +
percentage of bacterial population expressing GFP (GFP+) in 1000 bacteria.</li><br>
</p>
</p>
-
In R. palustris, the Median Fluorescence intensity (MIF) between complete systems and promoter is quite different. Figure 8 shows that PrrA promoter works better in Anaerobic/light conditions, than in others, it is the expected result considering the participation of homolog proteins, but the complete system is different because we saw a big change of fluorescence in aerobic/light conditions, introducing a synthetic system could affect the functionality of our constructions. AppA/PpsR system is functional because our promoter is being activated by other proteins, but the signal increase with our Synthetic Construction in the complete system, and also, as it is expected, the complete system works in aerobic/light conditions.
+
  <p>We used 3 environmental growing conditions:
 +
<li>Aerobic/Darkness</li>
 +
<li>Anaerobic/Light</li>
 +
<li>Anaeroibic/darkness</li></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="https://static.igem.org/mediawiki/2012/7/73/Osy03.jpg" width="563" height="452">
 +
<img src="https://static.igem.org/mediawiki/2012/e/ec/Osy04.jpg" width="563" height="452">
 +
<p>Figure 3. Percentage of bacterial population expressing GFP.<br>
</p>
</p>
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    </div>
+
</div>
 +
<div align="center">
 +
  <img src="https://static.igem.org/mediawiki/2012/6/6b/Osy05.jpg" width="556" height="384">
 +
  <p>Figure 4. Representative images obtained by fluorescence microscopy, where our systems
 +
were functional in the expected conditions.</p>
 +
</div>
 +
<p id="text2">Discussion</p>
 +
<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
 +
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.<br>
 +
<br>
 +
In <em>R. palustris</em>, we had GFP expression in PrrA dependent promoter (BBa_K776019), maybe
 +
because orthologous proteins activated it. The complete system (BBa_K776021) also
 +
was functional but in a lower level, assumably due to the interference of other proteins that regulate photosynthetic genes.<br>
 +
<br>
 +
The GFP expression that we did not expected was in aerobic condition in the complete system, probably
 +
is due to the complexity of the regulatory network where this system is involved.
</p>
</p>
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<p id="text2">Conclusion</p>
 +
<p> Our two BioBricks (K776019 and BBa_K776021) are functional in two photosynthetic
 +
bacteria <em>R. palustris</em> and <em>R. sphaeroides</em>, both in anaerobic/light expected condition. This is a functional system for controlling genetic
 +
expression with Oxygen tension.</p>
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<li><a href="Biobricks.htm" target="_parent">Biobricks</a></li>
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<li><a href="Lightandoxre.htm">Light and oxygen response</a></li>
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<li><a href="Notebook.htm">Notebook</a></li>
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<li><a href="oxigenresponse.htm">Oxygen response</a></li>
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<li><a href="Protocol.htm">Protocols</a></li>
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Latest revision as of 03:36, 27 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 PrrA dependent promoter (BBa_K776019), maybe because orthologous proteins activated it. The complete system (BBa_K776021) also was functional but in a lower level, assumably due to the interference of other proteins that regulate photosynthetic genes.

    The GFP expression that we did not expected was in aerobic condition in the complete system, probably is due to the complexity of the regulatory network where this system is involved.

    Conclusion

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

     

    Rhodofactory 2012

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