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

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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 R. sphaeroides or the orthologue system from R.palustris.</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 R. palustris.</p>
 +
<p>Both systems were cloned in pRK415 because it is a vector for Purple Non-Sulfur
 +
Photosynthetic Bacteria, the plasmids were introduced in R. sphaeroides and R. palustris,
 +
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: Rhodobacter sphaeroides and
 +
Rhodopseudomonas palustris, conjugated bacteria with pRK415 vector without BioBrick.</p>
 +
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<div align="center"><br>
 +
    <br>
 +
</div>
 +
<p>Figure 4. Images obtained by fluorescence microscopy (dark field), where our systems
 +
were functional in the expected conditions.</p>
 +
<p id="text2">Discussion</p>
 +
<p>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 due 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>
 +
 +
In R. palustris, we had a good response in the expected condition, our Light and Oxygen
 +
Control System is functional in anaerobic and light condition because we had a lower GFP level in BioBrick BBa_K77608 because orthologous proteins probably have a low affinity by this sequence, but when we introduced the complete system BBa_K776020, the
 +
synthetic system promoted the GFP expression.</p>
 +
 +
<p id="text2">Conclusion</p>
 +
<p>Our two BioBricks (K776018 and BBa_K776020) are functional in two photosynthetic
 +
bacteria R. palustris and R. sphaeroides. This is a functional system for controlling genetic
 +
expression with Light and Oxygen signals.</p>
 +
 +
</div>
 +
 +
<div id="sidebar">
 +
  <div id="updates" class="orangebox">
 +
 +
    <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">Oxigen response</a></li>
 +
<li><a href="Protocol.htm">Protocols</a></li>
 +
</ul>
 +
</div>
 +
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 +
  <div style="clear: both;">&nbsp;</div>
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<p align="center"> Rhodofactory 2012 </p>
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Revision as of 21:49, 25 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 it 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: Rhodobacter sphaeroides and Rhodopseudomonas palustris, conjugated bacteria with pRK415 vector without BioBrick.



    Figure 4. Images obtained by fluorescence microscopy (dark field), 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 due 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, we had a good response in the expected condition, our Light and Oxygen Control System is functional in anaerobic and light condition because we had a lower GFP level in BioBrick BBa_K77608 because orthologous proteins probably have a low affinity by this sequence, but when we introduced the complete system BBa_K776020, the synthetic system promoted the GFP expression.

    Conclusion

    Our two BioBricks (K776018 and BBa_K776020) are functional in two photosynthetic bacteria R. palustris and R. sphaeroides. This is a functional system for controlling genetic expression with Light and Oxygen signals.

     

    Rhodofactory 2012

    icytdf
    osli
    bio
    fermentAS
    cinestav
    genscript
    unam
    gto
    quimica
    valaner
    ipn

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