Results

As our goal was to manipulate gene expression in response to ambient iron, we first needed to investigate how changing the iron concentration in the medium affected E. coli growth. We quantified growth of E. coli strains containing either the aceB-GFP iron sensor or the aceB-lacI+PL_lacO-RFP Fur inverter at a range of iron supplementations: 0.1, 1, 10, 100 uM iron. The growth results show a statistically insignificant decrease in growth at higher iron concentrations for the sensor strain (Fig 1A) and no affect for the inverter strain (Fig 1B). Although we concluded that changes in iron concentration did not alter growth, we still normalized reporter gene expression to culture density in the subsequent experiments.

Fig 1 Growth of E. coli strains containing either the A the aceB-GFP iron sensor or the B aceB-lacI+PL_lacO-RFP Fur inverter with the iron supplementations of either 0.1, 1, 10, 100 uM.

Construction of a iron-responsive biosensor also required identification of genetic elements that respond to iron. The Ferric Uptake Regulator (Fur) is a transcription factor that respresses expression of its target genes at elevated iron concentration. Previous studies (Zhang et al, 2005, Chen et al, 2007) have defined a putative Fur regulon in E. coli. Based on these studies, we cloned the promoter regions of 4 genes putatively regulated by Fur (aceB, fes, fepA, yncE) upstream of a GFP reporter to examine if fluorescence changed as a function of iron concentration.

Fig 2 Reporter gene expression of GFP fused to putative Fur-regulated promoters aceB, fes, fepA, and yncE.

Based on our characterization of how Fur-regulated promoters alters GFP expression as a function of iron, we selected the aceB-GFP construct for more detailed analysis. We grew E. coli expressing the aceB-GFP construct at 4 iron concentrations (0.1, 1, 10, 100 uM iron) and quantified GFP expression in late log phase. We found that GFP expression significantly decreased at higher iron concentrations (Fig 3). These results support that aceB-GFP functions as an iron-responsive biosensor that represses GFP expression at higher iron concentrations.

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Figure 5: Inverter caracterization on M9 medium with different concentrationof iron and in 3 conditions:

1. AceB-sfGFP/plLacO-RFP on M9 medium with IPTG
2. AceB-LacI/plLacO-RFP on M9 medium with IPTG
3. AceB-LacI/plLacO-RFP on M9 medium without IPTG