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

Our inverter system is based on the interaction of two different plasmids. To create a fur inverter that activates expression in response to iron, we first cloned by golden gate assembly the lacI gene under the control of our AceB promoter that we have proved to repress expression of the downstream gene in response of iron (BBa_K1163103).

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

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).

Inverter characterisation

With these two plasmids, when Fur binds iron, it represses expression of the LacI repressor which, in turn, permits expression of the reporter gene - as shown Fig 1. Thus, reporter expression is positively correlated with iron concentration.

Fig 1. 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 a first step in the inverter system characterisation, we checked that the bacterial growth was not affected by the iron concentration variations. As shown in Fig 2, bacterial growth was weakly affected by the iron variations.

Fig 2. O.D. measurement of bacteria transformed by our pAceB-lacI + pLac-RFP genetic construction, in different concentration of iron.

Then, as we did for our iron sensing device, we concretely characterised our inverter system by growing bacteria in different iron concentration (0.1 µM, 1 µM and 10 µM). Using 96-wells plate reader, we measured O.D. (600 nm) and GFP intensity (530 nm) each 10 minutes of the bacterial growth. The results are shown Fig 3.

Fig 3. RFP expression of the pLac-RFP biobrick(BBa_J04450) is activated with the increase of iron concentration.

We obtained the results we expected for our inverter system, as the expression of our reporter gene is now upregulated by the increase of iron concentration. We thus characterised our second biobrick which is the pAceB-LacI (BBa_K1163103) and in the mean time improved the characterisation of an already existing biobrick of the registry (BBa_J04450).