Team:Evry/Sensor

From 2013.igem.org

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<b>Fig. 1</b> Construction of an iron-responsive genetic element by fusing a Fur-regulated promoter with a reporter gene.
<b>Fig. 1</b> Construction of an iron-responsive genetic element by fusing a Fur-regulated promoter with a reporter gene.
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These biosensors respond to ambient iron by using the <a href="https://2013.igem.org/Team:Evry/Project_FUR">Fur system</a> to repress the reporter gene placed downstream the promoter.
 
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<h2>Caracterisation of the iron-responsive biosensors</h2>
 
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As shown in figure 2, in our construction, sfGFP is placed downstream the Fur binding site. It means that in iron starvation sfGFP should be expressed and in high concentration of iron it should be repressed.
 
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<div align='center'><img src="https://static.igem.org/mediawiki/2013/1/12/ColiSensor.png" width="75%"/></div>
 
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<b>Fig. 2</b> Diagram of our genetic iron sensor. Iron binds the Ferric Uptake Regulator (Fur) to form a complex with high affinity for the Fur box in the promoter, here shown as the aceB promoter. Once the iron-Fur complex is bound to the promoter, it represses transcription of the target gene GFP. GFP expression is thus negatively correlated with iron availability.
 
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<div align='center'><img src="https://static.igem.org/mediawiki/2013/7/7b/P1-GFP.png" width="75%"/></div>
 
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<b>Fig 2</b> Construction of an iron-responsive genetic element by fusing a Fur-regulated promoter with a reporter gene. Promoter-reporter fusions were made with flanking restriction sites that are compatible with Biobrick-based cloning.
 
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<b>Table 1</b> Genetic elements used to make iron-responsive sensors.
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<b>Table I</b> Genetic elements used to make iron-responsive sensors.
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These biosensors respond to ambient iron by using the <a href="https://2013.igem.org/Team:Evry/Project_FUR">Fur system</a> to repress the reporter gene placed downstream the promoter.
</p>
</p>
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<h2>Caracterisation of the iron-responsive biosensors</h2>
 +
 +
<p>
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As shown in figure 2, in our construction, sfGFP is placed downstream the Fur binding site. It means that in iron starvation sfGFP should be expressed and in high concentration of iron it should be repressed.
 +
</p>
 +
 +
<br/>
 +
 +
<div align='center'><img src="https://static.igem.org/mediawiki/2013/1/12/ColiSensor.png" width="75%"/></div>
 +
<p align="center">
 +
<b>Fig. 2</b> Diagram of our genetic iron sensor. Iron binds the Ferric Uptake Regulator (Fur) to form a complex with high affinity for the Fur box in the promoter, here shown as the aceB promoter. Once the iron-Fur complex is bound to the promoter, it represses transcription of the target gene GFP. GFP expression is thus negatively correlated with iron availability.
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</p>
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<br>
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<br>
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<div align='center'><img src="https://static.igem.org/mediawiki/2013/7/7b/P1-GFP.png" width="75%"/></div>
 +
 +
<div align='center'><img src="https://static.igem.org/mediawiki/2013/f/f3/P1-Nat_Prom.png" width="58%"/></div>
 +
 +
<p>
 +
<b>Fig 2</b> Construction of an iron-responsive genetic element by fusing a Fur-regulated promoter with a reporter gene. Promoter-reporter fusions were made with flanking restriction sites that are compatible with Biobrick-based cloning.
 +
</p>
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 +

Revision as of 19:47, 28 October 2013

Iron coli project

Iron Sensor


Construction of the iron-responsive biosensors

E. coli's genome is composed of many Fur binding site. Based on a genome study, we identified 4 promoters which are controled by the FUR protein.

Using PCR on E. coli genome, we extracted these four promoters. We constructed iron-responsive biosensors by combining 3 genetic parts: an E. coli promoter with a Ferric Uptake Regulator (Fur) binding site, a fluorescent reporter (sfGFP), and a transcriptional terminator (see Figure 1 below). Promoter-reporter fusions were made with flanking restriction sites that are compatible with Biobrick-based cloning.

Fig. 1 Construction of an iron-responsive genetic element by fusing a Fur-regulated promoter with a reporter gene.

Name Figure Description

E. coli promoter with Fur binding site

iron-Fur complex binds promoter to repress expression

sfGFP

Fluorescent reporter gene

Terminator

terminator to stop transcription

Plasmid

Biobrick-compatible plasmid backbone

Table 1 Genetic elements used to make iron-responsive sensors.

These biosensors respond to ambient iron by using the Fur system to repress the reporter gene placed downstream the promoter.

Caracterisation of the iron-responsive biosensors

As shown in figure 2, in our construction, sfGFP is placed downstream the Fur binding site. It means that in iron starvation sfGFP should be expressed and in high concentration of iron it should be repressed.


Fig. 2 Diagram of our genetic iron sensor. Iron binds the Ferric Uptake Regulator (Fur) to form a complex with high affinity for the Fur box in the promoter, here shown as the aceB promoter. Once the iron-Fur complex is bound to the promoter, it represses transcription of the target gene GFP. GFP expression is thus negatively correlated with iron availability.



Fig 2 Construction of an iron-responsive genetic element by fusing a Fur-regulated promoter with a reporter gene. Promoter-reporter fusions were made with flanking restriction sites that are compatible with Biobrick-based cloning.