Team:Evry/Sensor

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Iron coli project

Iron Sensor


Construction of the iron-responsive biosensors

E. coli's genome is composed of many Fur binding site. Based on a genome study, we identified 4 promoters which are controled by the FUR protein.

Using PCR on E. coli genome, we extracted these four promoters. We constructed iron-responsive biosensors by combining 3 genetic parts: an E. coli promoter with a Ferric Uptake Regulator (Fur) binding site, a fluorescent reporter (sfGFP), and a transcriptional terminator (see Figure 1 below). Promoter-reporter fusions were made with flanking restriction sites that are compatible with Biobrick-based cloning.

Fig. 1 Construction of an iron-responsive genetic element by fusing a Fur-regulated promoter with a reporter gene.


We put all the parts together using Golden Gate assembly method. In order to combine our part in the right way, we designed specific overhang as shown in the table 1.


Name Figure Description

E. coli promoter with Fur binding site

iron-Fur complex binds promoter to repress expression

sfGFP

Fluorescent reporter gene

Terminator

terminator to stop transcription

Plasmid

Biobrick-compatible plasmid backbone

Table 1 Genetic elements used to make iron-responsive sensors.

These biosensors respond to ambient iron by using the Fur system to repress the reporter gene placed downstream the promoter.

Caracterisation of the iron-responsive biosensors

As shown in figure 2, in our construction, sfGFP is placed downstream the Fur binding site. It means that in iron starvation sfGFP should be expressed and in high concentration of iron it should be repressed.


Fig. 2 Diagram of our genetic iron sensor. Iron binds the Ferric Uptake Regulator (Fur) to form a complex with high affinity for the Fur box in the promoter, here shown as the aceB promoter. Once the iron-Fur complex is bound to the promoter, it represses transcription of the target gene GFP. GFP expression is thus negatively correlated with iron availability.


We characterised our construction growing our bacteria in different iron concentrations (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.

Fig. 3 The blank is used to calibrate the O.D. measurement. pAceB-LacI is used as a growth control and as a standard for the GFP measurement.

1st step of the analysis

In a first step we checked that our growth was not affected by the difference in iron concentration. As shown in the figure 4, bacterial growth was weakly affected by the iron variation.

Fig. 4 O.D. measurement of bacteria transformed by our system pAceB-GFP, in different concentration of iron.