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.

• AceB promoter - (BBa_K1163102)
• Fes promoter - (BBa_K1163108)
• FepA promoter - (BBa_K1163105)
• yncE promoter - (BBa_K1163111)

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.