Team:Evry/Inverter

From 2013.igem.org

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Normally, the Ferric Uptake Regulator (Fur) binds iron to repress transcription of its target genes. However, we needed a system that activates gene expression in response to iron. We thus constructed a "genetic invertor" system that reverses the Fur-regulatory system so that it indirectly activates gene expression in response to iron. This genetic invertor basically consists of a Fur-regulated lacI gene and a lacI regulated reporter gene.
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Normally, the Ferric Uptake Regulator (Fur) binds iron to repress transcription of its target genes. However, we needed a system that activates gene expression in response to iron. We thus constructed a "genetic invertor" system that reverses the Fur-regulatory system so that it indirectly activates gene expression in response to iron. This genetic invertor basically consists of a Fur-regulated lacI gene and a lacI regulated gene of interest. For our project, the goal is to cloned as a lacI regulated gene, the enterobactin operon.
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Our inverter system is based on the interaction of two different plasmids. The first plasmid carries a lacI gene under the control of our AceB promoter that we have proved to be iron sensitive (cf Sensor part). The second plasmid carries the RFP reporter gene under the control of a lac promoter. We used the already existing PL-LacO-RFP biobrick from the registry.
 
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With that construct, when Fur binds iron, it represses expression of the LacI repressor which, in turn, permits expression of the reporter gene. Thus, reporter expression is positively correlated with iron concentration.
 
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x How does it work ?
 
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x Characterisation
 
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<div align='center'><img src="https://static.igem.org/mediawiki/2013/a/a2/FurInverter.png" width="75%"/></div>
 
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<b>Fig 1.</b> Iron-responsive genetic inverter. The iron-Fur complex binds to the Fur site, here in the aceB promoter, to repress transcription of the lacI gene. In the absence of LacI, the RFP reporter is expressed.
 
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As shown in the figure 1, we used the PL-LacO-RFP from the registry (<a href="http://parts.igem.org/Part:BBa_J04450">BBa_J04450</a>) to characterized our pAceB-LacI(<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1163103">BBa_K1163103</a>). <a href="https://2013.igem.org/Team:Evry/Results">See our results</a>
 
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<h3>Control of lacI expression with a Fur-regulated promoter</h3>
<h3>Control of lacI expression with a Fur-regulated promoter</h3>
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<b>Table 1.</b> Genetic elements used to make an invertor system reversing the Fur-regulatory mecanism to activate gene expression in response to iron
<b>Table 1.</b> Genetic elements used to make an invertor system reversing the Fur-regulatory mecanism to activate gene expression in response to iron
</p>
</p>
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Our inverter system is based on the interaction of two different plasmids. The first plasmid carries a lacI gene under the control of our AceB promoter that we have proved to be iron sensitive ((<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1163103">BBa_K1163103</a>)). The second plasmid carries the RFP reporter gene under the control of a lac promoter. We used the already existing PL-LacO-RFP biobrick from the registry (<a href="http://parts.igem.org/Part:BBa_J04450">BBa_J04450</a>).
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With that construct, when Fur binds iron, it represses expression of the LacI repressor which, in turn, permits expression of the reporter gene. Thus, reporter expression is positively correlated with iron concentration.
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</p>
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x Characterisation
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</p> 
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<div align='center'><img src="https://static.igem.org/mediawiki/2013/a/a2/FurInverter.png" width="75%"/></div>
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<p>
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<b>Fig 1.</b> Iron-responsive genetic inverter. The iron-Fur complex binds to the Fur site, here in the aceB promoter, to repress transcription of the lacI gene. In the absence of LacI, the RFP reporter is expressed.
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</p>
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<p>
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As shown in the figure 1, we used the PL-LacO-RFP from the registry (<a href="http://parts.igem.org/Part:BBa_J04450">BBa_J04450</a>) to characterized our pAceB-LacI(<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1163103">BBa_K1163103</a>). <a href="https://2013.igem.org/Team:Evry/Results">See our results</a>
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</div>
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Revision as of 20:59, 28 October 2013

Iron coli project

Fur Inverter System



Normally, the Ferric Uptake Regulator (Fur) binds iron to repress transcription of its target genes. However, we needed a system that activates gene expression in response to iron. We thus constructed a "genetic invertor" system that reverses the Fur-regulatory system so that it indirectly activates gene expression in response to iron. This genetic invertor basically consists of a Fur-regulated lacI gene and a lacI regulated gene of interest. For our project, the goal is to cloned as a lacI regulated gene, the enterobactin operon.

Inverter construction

Control of lacI expression with a Fur-regulated promoter

Our iron sensor results showed that the Fur-regulated promoter of aceB represses expression of its target gene in response to iron. To create a fur inverter that activates expression in response to iron, we first linked the aceB promoter to the lacI gene.

NAME FIGURE Description

Promoter

Fur-regulated aceB promoter

LacI LVA

LacI repressor

Terminator

transcription stop signal

Plasmid

Backbone with ampicillin resistance

Table 1. Genetic elements used to make an invertor system reversing the Fur-regulatory mecanism to activate gene expression in response to iron

Our inverter system is based on the interaction of two different plasmids. The first plasmid carries a lacI gene under the control of our AceB promoter that we have proved to be iron sensitive ((BBa_K1163103)). The second plasmid carries the RFP reporter gene under the control of a lac promoter. We used the already existing PL-LacO-RFP biobrick from the registry (BBa_J04450). With that construct, when Fur binds iron, it represses expression of the LacI repressor which, in turn, permits expression of the reporter gene. Thus, reporter expression is positively correlated with iron concentration.

x Characterisation

Fig 1. Iron-responsive genetic inverter. The iron-Fur complex binds to the Fur site, here in the aceB promoter, to repress transcription of the lacI gene. In the absence of LacI, the RFP reporter is expressed.

As shown in the figure 1, we used the PL-LacO-RFP from the registry (BBa_J04450) to characterized our pAceB-LacI(BBa_K1163103). See our results


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