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      <p><strong></strong><strong> </strong></p>
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      <p><br />
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        As we  can see, in figure 1A the PpsR promoter system, in R.  sphaeroides,  shows a high signal (17.66%) of GFP expression, it implies that constitutive  proteins from this bacteria were able to activate our system. The growth  conditions were aerobic/darkness, although oxidized PpsR  binds its target promoter, it is known that AppA can avoid the binding affinity of PpsR in  the dark probably by the interference of an AppA-(PpsR)2  complex (Kim, 2006). In the case of AppA/PpsR complete system, we have a high GFP  expression due to activity of the extra AppA and PpsR enzymes that were introduced.</p>
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      <p>The  blue columns show the low GFP expression with both PrrA  promoter and PrrA/PrrB  complete system; in aerobic conditions PrrB autophosphorylates and passes a  phosphate group to PrrA,  this activated PrrA  binds to its promoter region as a transcriptional repressor (Bauer, 2003).  All the results show an equivalent result  with the images that were obtained by fluorescence microscope.</p>
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      <p>Figure  2A shows that in R. sphaeroides, the  AppA/PpsR  system promoted the GFP expression, it is possible because reduced PpsR is  unable to bind its promoter and AppA is a flavin with a photoreceptor, thus under light, AppA is  unable to forma a complex with PpsR and we can see GFP expression in the  bacterial population. </p>
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<p>In  opposite way, PrrA/PrrB  system shows less GFP expression, it is probably due to the low growth rate in R. sphaeroides,  under photosynthetic conditions (anaerobic/light), both systems were tested in  one and a half days of growing, and maybe it was not enough time to have a culture in exponential phase of growing. PrrA/PrrB is supposed to be functional because in  anaerobic conditions PrrB autophospholylate and  can transfer the phosphate group to PrrA, when PrrA receive the phosphate group, it can bind  its target promoter and the transcription is possible (Bauer, 2008).</p>
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      <p>AppA/PpsR system can be sense to signals: oxygen and light, in figure 3A, the GFP expression is high with PpsR  promoter because we have the natural R. sphaeroides AppA/PpsR  system working on our promoter. In anaerobic conditions the reduced PpsR has  low affinity to his target sequence and AppA is forming the complex ApA-(PpsR)2, (Bauer, 2001).  We can see it   with  the  level of GFP expression 21.21%, however, when  we introduce the complete system the GFP expression is much less, probably  because we are introducing extra AppA and PpsR enzymes that can interfere with the  constitutive system. PrrA/PrrB show  GFP expression higher in PrrA  promoter than in complete system, these results probably have the same problem,  that bacterial cultures were not in exponential phase of growing or that the  complete system can be interfering with the constitutive. </p>  
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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
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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
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constitutive (or natural) system from <em>R. sphaeroides</em> or the orthologue system from <em>R.
 +
palustris.</em></p>
     </div>
     </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>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">
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<img src="https://static.igem.org/mediawiki/2012/e/ec/Osy04.jpg" width="563" height="452">
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<p>Figure 3. Percentage of bacterial population expressing GFP.<br>
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</p>
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</div>
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<div align="center">
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  <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
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were functional in the expected conditions.</p>
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</div>
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<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>
 +
<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>
 +
 +
</div>
 +
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 +
  <div id="updates" class="orangebox">
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    <h2>Results</h2>
 +
<ul>
 +
<li><a href="Biobricks.htm" target="_parent">Biobricks</a></li>
 +
<li><a href="Lightandoxre.htm">Light and oxygen response</a></li>
 +
<li><a href="Notebook.htm">Notebook</a></li>
 +
<li><a href="oxigenresponse.htm">Oxygen response</a></li>
 +
<li><a href="Protocol.htm">Protocols</a></li>
 +
</ul>
 +
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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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    Retrieved from "http://2012.igem.org/Team/CINVESTAV-IPN-UNAM_MX/oxigenresponse.htm"