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.

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

We constructed 4 differents iron sensor using promoters regions from aceB (BBa_K1163102), fes (BBa_K1163108), fepA (BBa_K1163105) and yncE (BBa_K1163111). Finally, pAceB appears to be the best candidate to build our sensor system. See our results



Fig 1 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.



Fig 2 Construction of an iron-responsive genetic element by fusing a Fur-regulated promoter with a reporter gene. Promoter-reporter fusions were made with flanking restriction sites that are compatible with Biobrick-based cloning.

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 I Genetic elements used to make iron-responsive sensors.