"
Team:Evry/Inverter
From 2013.igem.org
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.
Inverter construction
Our inverter system consists of a Fur-regulated lacI gene and a lacI regulated reporter gene, here the RFP. 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 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.
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
