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.  
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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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Our inverter system consists of a Fur-regulated lacI gene and a lacI regulated reporter gene, here the RFP.
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Our inverter system is based on two plasmids. The
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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.
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 Characterisation
x Characterisation
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This system consists of a Fur-regulated lacI gene and a LacI regulated reporter gene. 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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Revision as of 20:41, 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 reporter gene.

Inverter construction

Our inverter system is based on two plasmids. The 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 How does it work ? 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

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