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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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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.
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<h1>Light and oxygen response!</h1>
 +
<p id="text2">AppA/PpsR Regulation System</p>
 +
<p>In this repressor/antirepressor system, under low oxygen tension, the GFP
 +
expression is possible because the repressor PpsR is forming a complex with the
 +
antirepresssor protein AppA. Moreover, when blue light fall upon the complex, a
 +
conformational change in AppA breaks the complex, and AppA avoid GFP expression. (See
 +
Rhodofactory section for a complete explanation).<br><br>
 +
 
 +
 
 +
We made two BioBricks (BBa_K776018 y BBa_K776020) to test the Light &
 +
Oxygen Control System, each one has GFP as a reporter gene and the functionality was
 +
related to the fluorescence detection.</p>
 +
<img src="https://static.igem.org/mediawiki/2012/4/40/Oxy01.jpg" alt="oxy01" width="561" height="241">
 +
<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>
 +
<img src="https://static.igem.org/mediawiki/2012/1/1c/Oxy02.jpg" width="563" height="183">
 +
<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>
 +
<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 was:
 +
<li>Fluorescence Microscopy: To have a qualitative GFP detection in these bacteria.</li>
 +
<li>Flow Cytometry: To have a quantitative GFP detection, we calculated the percentage of
 +
bacterial population expressing GFP (GFP+) in 1000 bacteria.</li></p><br>
 +
 
 +
<p>We used 3 environmental growing conditions:
 +
<li>Aerobic/Darkness</li>
 +
<li>Anaerobic/Light</li>
 +
<li>Anaeroibic/darkness</li>
 +
</p><br>
 +
 
 +
<p>For all data results, we considered a negative control: <em>R. sphaeroides</em> and
 +
<em>R.palustris</em>, conjugated bacteria with pRK415 vector without BioBrick.</p>
 +
<div align="center"><img src="https://static.igem.org/mediawiki/2012/f/f0/Oxygenres01.jpg" width="563" height="452">
 +
<img src="https://static.igem.org/mediawiki/2012/c/cc/Oxygenres02.jpg" width="563" height="452">
 +
<p>Figure 3. Percentage of bacterial population expressing GFP..<br>
 +
</p>
 +
  <img src="https://static.igem.org/mediawiki/2012/9/9b/Oxy03.jpg" width="561" height="465"></div>
 +
<div align="center">
 +
</div>
 +
<p>Figure 4. Images obtained by fluorescence microscopy, where our systems
 +
were functional in the expected conditions.</p>
 +
<p id="text2">Discussion</p>
 +
<p>In <em>R. sphaeroides</em>, the best functionality of our system was in aerobic and darknesss
 +
condition, it was not the expected result, but probably the activation is because to the
 +
incomplete repression of the system because we were overexpressing the constitutive
 +
proteins. Altought, we obtained GFP expression with a lower level, in the expected
 +
condition, both BioBricks (BBa_K776018 y BBa_K776020) were functional.<br>
 +
<br>
 +
 
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In <em>R.palustris</em>, our best condition was in anaerobic and light condition, expected result. The low level of GFP in BioBrick BBa_K77608 it could be due to low affinity of orthologous proteins to our promoter sequence, but when we introduced the complete system BBa_K776020 GFP expression increases showing the funcionality of our system. </p>
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<p id="text2">Conclusion</p>
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<p>Both BioBricks (K776018 and BBa_K776020) are functional in two photosynthetic
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bacteria <em>R.palustris</em> and <em>R. sphaeroides.</em> The best condicion was aerobic/darknes and anaerobic light respectively.
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This is a functional system for controlling genetic expression with Light and Oxygen signals.</p>
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<li><a href="Lightandoxre.htm">Light and oxygen response</a></li>
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Latest revision as of 22:59, 26 October 2012

Rho

Light and oxygen response!

AppA/PpsR Regulation System

In this repressor/antirepressor system, under low oxygen tension, the GFP expression is possible because the repressor PpsR is forming a complex with the antirepresssor protein AppA. Moreover, when blue light fall upon the complex, a conformational change in AppA breaks the complex, and AppA avoid GFP expression. (See Rhodofactory section for a complete explanation).

We made two BioBricks (BBa_K776018 y BBa_K776020) to test the Light & Oxygen Control System, each one has GFP as a reporter gene and the functionality was related to the fluorescence detection.

oxy01

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 was:

  • Fluorescence Microscopy: To have a qualitative GFP detection in these bacteria.
  • Flow Cytometry: To have a quantitative GFP detection, 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: R. sphaeroides and R.palustris, conjugated bacteria with pRK415 vector without BioBrick.

    Figure 3. Percentage of bacterial population expressing GFP..

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

    Discussion

    In R. sphaeroides, the best functionality of our system was in aerobic and darknesss condition, it was not the expected result, but probably the activation is because to the incomplete repression of the system because we were overexpressing the constitutive proteins. Altought, we obtained GFP expression with a lower level, in the expected condition, both BioBricks (BBa_K776018 y BBa_K776020) were functional.

    In R.palustris, our best condition was in anaerobic and light condition, expected result. The low level of GFP in BioBrick BBa_K77608 it could be due to low affinity of orthologous proteins to our promoter sequence, but when we introduced the complete system BBa_K776020 GFP expression increases showing the funcionality of our system.

    Conclusion

    Both BioBricks (K776018 and BBa_K776020) are functional in two photosynthetic bacteria R.palustris and R. sphaeroides. The best condicion was aerobic/darknes and anaerobic light respectively. This is a functional system for controlling genetic expression with Light and Oxygen signals.

     

    Rhodofactory 2012

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