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<h1><em>Light & Oxygen Response: AppA/PpsR <br /><br />Regulation System! </em></h1>
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<p>This is a repressor/antirepressor system, which under high oxygen tension; PpsR represses GFP expression by avoiding RNA polymerase binding
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the promoter sequence.
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When oxygen concentration decreases and in presence of light AppA has a conformational change and can bind with PpsR, this complex prevents the union of
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PpsR to its target sequence, thus GFP expression can begin.<br>
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<a href="">Results<span class="flecha">&#9660;</span></a>
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(See the next video for a visual explanation).</p>
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<p>The first biobrick consists in the complete light-oxygen dependent system, AppA and PpsR, each one with Ribosome Binding Site, under a
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Medium strength promoter (J23104), this first biobrick also it has the PpsR dependent promoter and GFP as a reporter gene.</p>
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<img src="https://static.igem.org/mediawiki/2012/b/b6/Rodo02.jpg" alt="rodo02" width="568" height="107"><br>
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  The second circuit is just the PpsR dependent promoter and GFP as a reporter gene.</p>
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<img src="https://static.igem.org/mediawiki/2012/a/a1/Rodo03.jpg" alt="rodo03" width="562" height="252">
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<p id="text2">We were inspired in:</p>
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<p>
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This system is inspired in AppA/PpsR repressor/antirepressor system from Rhodobacter sphaeroides. The PpsR protein is a master repressor of
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Photosynthesis (PS) genes (Moskvin and Gomelsky 2005). Inactivation of the ppsR gene is enough to turn on PS gene expression and formation
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of the photosynthetic apparatus even at a high oxygen concentration, whereas ppsR overexpression is sufficient to block PS development even
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in the absence of oxygen. PpsR directly represses transcription of most carotenoid and pigment synthesis genes, photosystems operons, and
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genes involved in tetrapyrrole biosynthesis (Gomelsky and Kaplan 1995). The upstream regions of these genes contain two PpsR binding sites,
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TGTcN10gACA.<br><br>
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A second protein called AppA, which has no known homologues, plays a role in controlling gene expression in <em>R. sphaeroides</em> in response to both
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light and O2 by acting as an antirepressor of PpsR. Our parts (appa, ppsr and ppsr-promoter) were synthesized by Genescript, and are codon
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optimized for <em>R. sphaeroides.</em></p>
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<hr>
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<p id="refe">References<br>
 +
 
 +
1. Gomelsky L., Moskvin L., Stenzel A., Jones D., Donohue T. and Gomelsky M.(2008) <strong>Hierarchical Regulation of Photosynthesis
 +
Gene Expression by the Oxygen-Responsive PrrBA and AppA-PpsR Systems of <em>Rhodobacter sphaeroides.</em></strong> J. Bacteriol.
 +
Dec. 2008, p. 8106–8114 Vol. 190, No. 24<br>
 +
 
 +
2. Moskvin, O. V., L. Gomelsky, and M. Gomelsky. (2005). <strong>Transcriptome analysis of the <em>Rhodobacter sphaeroides</em> PpsR
 +
regulon: PpsR as a master regulator of photosystem development.</strong> J. Bacteriol. 187:2148–2156.<br>
 +
 
 +
3. Gomelsky, M., and S. Kaplan. (1995). <strong>Genetic evidence that PpsR from <em>Rhodobacter sphaeroides.</em> 2.4.1 functions as a
 +
repressor of puc and bchF expression.</strong> J. Bacteriol. 177:1634–1637. <br>
 +
4. Gomelsky, M., and S. Kaplan. (1995). <strong>AppA, a novel gene encoding a transacting factor involved in the regulation of
 +
photosynthesis gene expression in <em>Rhodobacter sphaeroides.</em> 2.4.1.</strong> J. Bacteriol. 177:4609–4618.
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      <h2><strong>Light + Redox Response: AppA/PpsR Control System</strong></h2>
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      <p>This is a  repressor/antirepressor system, under high oxygen tension <strong>PpsR</strong> strongly repress GFP expression by binding a conserved  regulatory sequence that avoid RNA polymerase for promoter binding. When oxygen  concentration decreases AppA is activated, and bind PpsR avoiding this DNA  binding activity, thus, GFP expression can begin.</p>
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      <p><strong>How does it works?</strong></p>
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      <p>Our genetic system  consists in 2 modules, the first one is formed by <em>appa</em> and <em>ppsr </em>coupled to  B0030 RBS units, J23104 Strong constitutive promoter and B0014 Double  terminator to form the transcription unit. We will evaluate the functioning of  J23104, B0030 and B0014 in a new chassis. The second module consists in <strong>J54103</strong> GFP generator (B0030 RBS + E1010  GFP + B0014 Double terminator) coupled to PpsR repressible promoter.</p>
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<p><strong>Inspired in…</strong></p>
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  This  system is inspired in AppA/PpsR repressor/antirepressor system from <em>Rhodobacter sphaeroides</em>, PpsR protein is  a master repressor of Photosynthesis (PS) genes (Moskvin and Gomelsky 2005).  Inactivation of the <em>ppsR </em>gene is enough to turn on PS gene expression  and formation of the photosynthetic apparatus even at a high oxygen  concentration, whereas <em>ppsR </em>overexpression is sufficient to block PS  development even in the absence of oxygen. PpsR directly represses  transcription of most carotenoid and pigment synthesis genes, photosystems  operons, and genes involved in tetrapyrrole biosynthesis (Gomelsky and Kaplan  1995). <strong>The upstream regions of these  genes contain two PpsR binding sites, TGTcN10gACA.</strong></p>
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<p>A  second protein called AppA, which has no known homologues, plays a role in  controlling gene expression in <em>R. sphaeroides </em>in response to both light  and O2 by acting as an antirepressor of PpsR. Our parts (appa, ppsr and  ppsr-promoter) were synthesized with Genescript, and are codon optimized for <em>R. palustris.</em></p>
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  <li>Gomelsky L., Moskvin L.,  Stenzel A., Jones D., Donohue T. and Gomelsky  M.(2008) <strong>Hierarchical Regulation of  Photosynthesis Gene Expression by the Oxygen-Responsive PrrBA and AppA-PpsR  Systems of <em>Rhodobacter sphaeroides.</em></strong> J. Bacteriol. Dec. 2008, p.  8106–8114 Vol. 190, No. 24 </li>
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  <li>Moskvin, O. V., L. Gomelsky, and M. Gomelsky<strong>. (</strong>2005<strong>). Transcriptome  analysis of the </strong><strong><em>Rhodobacter sphaeroides </em></strong><strong>PpsR regulon:  PpsR as a master regulator of photosystem development</strong>. J. Bacteriol. <strong>187:</strong>2148–2156. </li>
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  <li>Gomelsky, M., and S. Kaplan.<strong> (</strong>1995). <strong>Genetic evidence that PpsR from </strong><strong><em>Rhodobacter  sphaeroides </em></strong><strong>2.4.1 functions as a repressor of </strong><strong><em>puc </em></strong><strong>and </strong><strong><em>bchF </em></strong><strong>expression</strong>. J. Bacteriol. <strong>177:</strong>1634–1637.</li>
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  <li>Gomelsky, M., and S. Kaplan. <strong>(</strong>1995). <strong><em>appA</em></strong><strong>, a novel gene encoding a </strong><strong><em>trans</em></strong><strong>acting factor involved in the regulation of photosynthesis gene  expression in </strong><strong><em>Rhodobacter sphaeroides </em></strong><strong>2.4.1</strong>. J.  Bacteriol. <strong>177:</strong>4609–4618.</li>
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Latest revision as of 03:31, 27 October 2012

Rho

Light & Oxygen Response: AppA/PpsR

Regulation System!

This is a repressor/antirepressor system, which under high oxygen tension; PpsR represses GFP expression by avoiding RNA polymerase binding the promoter sequence. When oxygen concentration decreases and in presence of light AppA has a conformational change and can bind with PpsR, this complex prevents the union of PpsR to its target sequence, thus GFP expression can begin.
(See the next video for a visual explanation).


Our biobricks

The first biobrick consists in the complete light-oxygen dependent system, AppA and PpsR, each one with Ribosome Binding Site, under a Medium strength promoter (J23104), this first biobrick also it has the PpsR dependent promoter and GFP as a reporter gene.

rodo02


The second circuit is just the PpsR dependent promoter and GFP as a reporter gene.

rodo03

We were inspired in:

This system is inspired in AppA/PpsR repressor/antirepressor system from Rhodobacter sphaeroides. The PpsR protein is a master repressor of Photosynthesis (PS) genes (Moskvin and Gomelsky 2005). Inactivation of the ppsR gene is enough to turn on PS gene expression and formation of the photosynthetic apparatus even at a high oxygen concentration, whereas ppsR overexpression is sufficient to block PS development even in the absence of oxygen. PpsR directly represses transcription of most carotenoid and pigment synthesis genes, photosystems operons, and genes involved in tetrapyrrole biosynthesis (Gomelsky and Kaplan 1995). The upstream regions of these genes contain two PpsR binding sites, TGTcN10gACA.

A second protein called AppA, which has no known homologues, plays a role in controlling gene expression in R. sphaeroides in response to both light and O2 by acting as an antirepressor of PpsR. Our parts (appa, ppsr and ppsr-promoter) were synthesized by Genescript, and are codon optimized for R. sphaeroides.

References
1. Gomelsky L., Moskvin L., Stenzel A., Jones D., Donohue T. and Gomelsky M.(2008) Hierarchical Regulation of Photosynthesis Gene Expression by the Oxygen-Responsive PrrBA and AppA-PpsR Systems of Rhodobacter sphaeroides. J. Bacteriol. Dec. 2008, p. 8106–8114 Vol. 190, No. 24
2. Moskvin, O. V., L. Gomelsky, and M. Gomelsky. (2005). Transcriptome analysis of the Rhodobacter sphaeroides PpsR regulon: PpsR as a master regulator of photosystem development. J. Bacteriol. 187:2148–2156.
3. Gomelsky, M., and S. Kaplan. (1995). Genetic evidence that PpsR from Rhodobacter sphaeroides. 2.4.1 functions as a repressor of puc and bchF expression. J. Bacteriol. 177:1634–1637.
4. Gomelsky, M., and S. Kaplan. (1995). AppA, a novel gene encoding a transacting factor involved in the regulation of photosynthesis gene expression in Rhodobacter sphaeroides. 2.4.1. J. Bacteriol. 177:4609–4618.

 

Rhodofactory 2012

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osli
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