Difference between revisions of "Team:Toulouse/Description/Regulation"

 
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  <ul>
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        <li><a href="#part1">- Regulate</i></a></li>
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        <li><a href="#part2">- Light and Dark conditions</a></li>
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<h3>Regulation</h3>
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<h3>Regulate</h3>
 
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         <p align="justify" style="font-size:15px;">
 
         <p align="justify" style="font-size:15px;">
 
In order to respect the bee life cycle  
 
In order to respect the bee life cycle  
−
and to optimize our solution fighting
+
and to optimize our solution to limit
−
Varroa destructor infestation of domestic
+
Varroa destructor infestation, we have integrated a regulation  
−
bees, we have integrated a regulation  
+
system to our genetic construction, in the form of <b>a NOT logic gate controled by a day/night (or circadian) switch</b>.  
−
system to our genetic construction: <b>a day/night (or circadian) switch</b>.  
+
In daytime, the bees go back and forth at the beehive entrance, bringing varroas inside or outside the hive.
−
During the day, the bees are working outside doing pollination,
+
In nighttime, bees stay in the hive. Hence,  
−
so they make back and forth at the beehive entrance.
+
we want our <b>ApiColi</b> <i>E. coli</i> strain to produce either butyric acid in daytime to  
−
This is when they can bring varroas into the hive.  
+
attract varroa into the physical trap, or formic acid to kill it by night.
−
During the night they are less active. Hence,  
+
−
we want our <b>ApiColi</b> to produce butyric acid to  
+
−
attract varroa into the physical trap by day, and formic acid to kill it by night.
+
  
 
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  <img src="https://static.igem.org/mediawiki/2015/f/f2/TLSE_regulation_schema.png" style="width:80%">
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<div class="group center" id="lightsensor">
 
<p align="justify" style="font-size:15px;">
 
<p align="justify" style="font-size:15px;">
  
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We based our reflexion on a light response system built in E. coli [1]
+
We based our reflexion on a light response system built in <i>E. coli</i> [1]
−
This system has then been used to design a genetic response
+
The genetic system has been designed to be switched on and off in response to light [2].
−
that can be switched on and off by light [2].
+
In our project, we have further improved the  
−
In our project, we have further enhanced the  
+
process in order to control two  
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process in order to be able to produce two  
+
alternative genetic programs depending on the light presence.
−
different genetic expressions depending on light.
+
 
<br>
 
<br>
 
<br>
 
<br>
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originating from cyanobacteria  
 
originating from cyanobacteria  
 
<i>Synechocystis sp.</i> PCC 6803. Cph8  
 
<i>Synechocystis sp.</i> PCC 6803. Cph8  
−
is the result of the fusion between  
+
is a hybrid protein between  
 
the <b>red light response domain of Cph1</b>  
 
the <b>red light response domain of Cph1</b>  
−
(a phytochrome-like protein that comes
+
(a phytochrome-like protein  
−
from Synechocystis sp PCC 6803) and the  
+
from <i>Synechocystis</i> sp PCC 6803) and the  
−
<b>intracellular histidin kinase EnvZ</b>  
+
<b>intracellular domain of the histidin kinase EnvZ</b>  
 
(an osmolarity sensor protein) from <i>E. coli.</i></b>
 
(an osmolarity sensor protein) from <i>E. coli.</i></b>
−
Synthesis of PCB requires the expression of Ho1  
+
The synthesis of PCB requires the expression of both Ho1  
 
(heme oxygenase gene) and PcyA (biliverdin reductase gene). [3]
 
(heme oxygenase gene) and PcyA (biliverdin reductase gene). [3]
 
</p>
 
</p>
 
</div>
 
</div>
  
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<div class="subtitle">
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<div class="subtitle" id="part2">
 
<h3>Light and Dark conditions</h3>
 
<h3>Light and Dark conditions</h3>
 
</div>
 
</div>
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<div class="group center">
 
<div class="group center">
 
<p align="justify" style="font-size:15px;">
 
<p align="justify" style="font-size:15px;">
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When there is no light, Cph8 autophosphorylates its intracellular EnvZ domain while consuming one molecule of ATP. This activates the transcription factor OmpR by transferring its phosphate to it. OmpR-P therefore upregulates genes under control of the promoter POmpC.
+
Whitout light, Cph8 <b>autophosphorylates</b> its EnvZ intracellular domain while consuming one molecule of ATP. The phosphoryl group will be subsequently transfered to the <b>transcription factor OmpR</b>, which then, will upregulate genes expressed from the <b>P<sub>OmpC</sub> promoter</b> .
−
<br>
+
−
On the contrary, when there is light, PCB prevents the Cph8 autophosphorylation. Hence OmpR is not activated and there is no expression of genes under PompC control.
+
 
</p>
 
</p>
 
</div>
 
</div>
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<center><img src="https://static.igem.org/mediawiki/2015/f/f1/TLSE_night_regu.png" style="width:55%"></center>
  
 
<div class="group center">
 
<div class="group center">
 
<p align="justify" style="font-size:15px;">
 
<p align="justify" style="font-size:15px;">
−
This circadian switch is further enhanced with a combination of cI repressor and lacI repressor which enables ApiColi to produce butyric acid (polycistron B) when there is light, while formic acid is not produced, and conversely to produce formic acid (polycistron A) during the night and no butyric acid. Without light, polycistronic A genes, corresponding to the formate pathway, are transcribed The first protein, encoded by the cI gene downregulates the pLac promotor, hence, the polycistronic gene B is not transcribed.
+
In contrast, with light, <b>PCB prevents</b> the Cph8 autophosphorylation. OmpR will not be activated and the genes under P<sub>OmpC</sub> will not be expressed.
−
When there is light, there is no more expression of the polycistronic gene A, thus there is no more repression of the polycistronic gene B. Thus, there is production of butyrate via the expression of polycistronic gene B. In this same genetic element, there is a protein that represses the polycistronic gene A expression.
+
 
</p>
 
</p>
 
</div>
 
</div>
 +
<center><img src="https://static.igem.org/mediawiki/2015/e/e5/TLSE_day_reg.png" style="width:55%"></center>
  
 +
 +
<div class="group center">
 +
<p align="justify" style="font-size:15px;">
 +
This circadian switch is further improved by integrating a NOT logic gate into the regulation circuitry. This is achieved using the <b>phage lambda cI repressor</b> (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1587006">BBa_K1587006</a>) as well as the bacterial <b>LacI repressor</b>. With such a design, in daytime, ApiColi will synthesize <b>butyric acid</b> (polycistron B) while repressing the synthesis of <b>formic acid</b> (polycistron A) and <I>vice versa</I> in nighttime. Without light, cI produced from the first gene of the polycistronic A, will repress the P<sub>Lac</sub> promoter, preventing the expression of the polycistronic B genes whose first gene codes for the LacI repressor.
 +
In daytime, polycistronic A genes will not be expressed, there will be no CI repressor to prevent the transcription of the polycistronic B genes. Thus, butyrate will be produced and the LacI repressor will repress the polycistronic A genes.</p>
 +
</div>
 +
 +
 +
 +
<center><img src="https://static.igem.org/mediawiki/2015/f/f3/Regulation2.gif" style="width:60%;"></center>
 +
 +
<center>
 +
<div class="title">
 +
<h3>READ MORE</h3>
 +
</div> </center>
 +
 +
<div class="group center">
 +
<div style="width:20%;">
 +
<a href="https://2015.igem.org/Team:Toulouse/Description/Attract">
 +
<div class="title">
 +
<h3>Attract</h3>
 +
</div>
 +
</a>
 +
</div>
 +
<div style="width:20%;">
 +
<a href="https://2015.igem.org/Team:Toulouse/Description/Eradicate">
 +
<div class="title">
 +
<h3>Eradicate</h3>
 +
</div>
 +
</a>
 +
</div>
 +
<div style="width:20%;">
 +
<a href="https://2015.igem.org/Team:Toulouse/Description/Regulation">
 +
<div class="title">
 +
<h3>Regulation</h3>
 +
</div>
 +
</a>
 +
</div>
 +
<div style="width:20%;">
 +
<a href="https://2015.igem.org/Team:Toulouse/Design">
 +
<div class="title">
 +
<h3>Device: TrApiColi</h3>
 +
</div>
 +
</a>
 +
</div>
 +
<div style="width:20%;">
 +
<a href="https://2015.igem.org/Team:Toulouse/Results">
 +
<div class="title">
 +
<h3>Results</h3>
 +
</div>
 +
</a>
 +
</div>
 +
</div>
 
   </main>
 
   </main>
 
</div>
 
</div>
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<ul>
 
<ul>
 
<li>
 
<li>
−
[1] 2005 Nature, levskaya, synthetic biology: engineering <i>E. coli</i> to see light
+
[1] Levskaya A, Chevalier AA, Tabor JJ, Simpson ZB, Lavery LA, Levy M, Davidson EA, Scouras A, Ellington AD, Marcotte EM & Voigt CA (2005) Synthetic biology: Engineering <i>Escherichia coli</i> to see light. Nature 438: 441–442
 
</li>
 
</li>
  
 
<li>
 
<li>
−
[2] 2013 PLOS, lee switchable gene expression in <i>E. coli</i> using a miniaturized photobioreactor</li>
+
[2] Lee JM, Lee J, Kim T & Lee SK (2013) Switchable Gene Expression in <i>Escherichia coli</i> Using a Miniaturized Photobioreactor. PLoS ONE 8: e52382
−
 
+
 
<li>
 
<li>
−
[3] PNAS, 2001, gabetta, Genetic engineering of phytochrome biosynthesis in bacteria
+
[3] Gambetta GA & Lagarias JC (2001) Genetic engineering of phytochrome biosynthesis in bacteria. PNAS 98: 10566–10571
 
</li>
 
</li>
 
</ul>
 
</ul>

Latest revision as of 19:23, 18 September 2015

iGEM Toulouse 2015

Regulate


Content

Regulate

In order to respect the bee life cycle and to optimize our solution to limit Varroa destructor infestation, we have integrated a regulation system to our genetic construction, in the form of a NOT logic gate controled by a day/night (or circadian) switch. In daytime, the bees go back and forth at the beehive entrance, bringing varroas inside or outside the hive. In nighttime, bees stay in the hive. Hence, we want our ApiColi E. coli strain to produce either butyric acid in daytime to attract varroa into the physical trap, or formic acid to kill it by night.

We based our reflexion on a light response system built in E. coli [1] The genetic system has been designed to be switched on and off in response to light [2]. In our project, we have further improved the process in order to control two alternative genetic programs depending on the light presence.

The core of the light sensor is composed of the membrane proteins PCB and Cph8. PCB is a chromophore (phycocyabilin) originating from cyanobacteria Synechocystis sp. PCC 6803. Cph8 is a hybrid protein between the red light response domain of Cph1 (a phytochrome-like protein from Synechocystis sp PCC 6803) and the intracellular domain of the histidin kinase EnvZ (an osmolarity sensor protein) from E. coli. The synthesis of PCB requires the expression of both Ho1 (heme oxygenase gene) and PcyA (biliverdin reductase gene). [3]

Light and Dark conditions

Whitout light, Cph8 autophosphorylates its EnvZ intracellular domain while consuming one molecule of ATP. The phosphoryl group will be subsequently transfered to the transcription factor OmpR, which then, will upregulate genes expressed from the POmpC promoter .

In contrast, with light, PCB prevents the Cph8 autophosphorylation. OmpR will not be activated and the genes under POmpC will not be expressed.

This circadian switch is further improved by integrating a NOT logic gate into the regulation circuitry. This is achieved using the phage lambda cI repressor (BBa_K1587006) as well as the bacterial LacI repressor. With such a design, in daytime, ApiColi will synthesize butyric acid (polycistron B) while repressing the synthesis of formic acid (polycistron A) and vice versa in nighttime. Without light, cI produced from the first gene of the polycistronic A, will repress the PLac promoter, preventing the expression of the polycistronic B genes whose first gene codes for the LacI repressor. In daytime, polycistronic A genes will not be expressed, there will be no CI repressor to prevent the transcription of the polycistronic B genes. Thus, butyrate will be produced and the LacI repressor will repress the polycistronic A genes.

READ MORE

References


  • [1] Levskaya A, Chevalier AA, Tabor JJ, Simpson ZB, Lavery LA, Levy M, Davidson EA, Scouras A, Ellington AD, Marcotte EM & Voigt CA (2005) Synthetic biology: Engineering Escherichia coli to see light. Nature 438: 441–442
  • [2] Lee JM, Lee J, Kim T & Lee SK (2013) Switchable Gene Expression in Escherichia coli Using a Miniaturized Photobioreactor. PLoS ONE 8: e52382
  • [3] Gambetta GA & Lagarias JC (2001) Genetic engineering of phytochrome biosynthesis in bacteria. PNAS 98: 10566–10571