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Team:Evry/Inverter
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| - | Our inverter system is based on two plasmids. The | + | 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. | 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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Revision as of 20:52, 28 October 2013
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 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. 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
