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 pAceB-GFP iron sensor or the pAceB-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). Our microfluidic characterization further supported the idea that cells grow well in both low and high concentration of iron. Although we concluded that changes in iron concentration used in these experiments 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 pAceB-GFP iron sensor or the B pAceB-lacI+PL_lacO-RFP Fur inverter with the iron supplementations of either 0.1, 1, 10, 100 uM.

Construction of a iron-responsive biosensor 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 changes as a function of iron concentration.

Based on our initial tests of how various Fur-regulated promoters repress GFP expression as a function of iron, we selected the pAceB-GFP construct for more detailed analysis. We grew E. coli expressing the pAceB-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 2). These results support that pAceB-GFP (submitted as BioBrick BBa_K1163102) functions as an iron-responsive biosensor that represses GFP expression at elevated iron concentrations.

Fig 2 GFP expression of the pAceB-GFP Biobrick BBa_K1163102 is repressed at higher iron concentrations. This construct thus functions as an iron-responsive biosensor to repress expression of the reporter gene GFP.

We were now able to repress gene expression in response to iron using Fur, but our ultimate goal was to activate expression of iron chelator genes at high iron levels. This required the construction of a Fur inverter. To this end, we replaced the GFP gene downstream of pAceB with lacI to yield an pAceB-lacI construct. Next we needed a reporter gene under the control of a lacI-responsive promoter. Luckily, a plLacO-RFP biobrick BBa_J04450 already existed in the registry. We ensured that the BBa_J04450 brick indeed contained a Lac-controlled RFP system by examining RFP expression in the presence and absence of IPTG. Our results showed that, as expected, IPTG relieved LacI-mediated repression of RFP (Fig 3) in BBa_J04450.

Fig 3 RFP expression of E. coli transformed with plLacO-RFP (Biobrick BBa_J04450) in presence (plates 1 and 2) and absence (plate 3) of IPTG. Adding IPTG increases RFP expression, confirming that Biobrick BBa_J04450 enables LacI-mediated RFP repression.

We co-transformed the plLacO-RFP (BBa_J04450) and the pAceB-LacI (BBa_k1163102) constructs into E. coli and again measured how reporter gene expression (now RFP) responded to changes in iron concentration. Our results show that the pAceB-LacI plus plLacO-RFP system indeed acts as a Fur inverter (Fig 4) where RFP expression is now positively correlated with iron concentration.

Fig 4 RFP expression as a function of iron concentration for E. coli expressing the Fur inverter. Cells were co-transformed with constructs bearing the pAceB-LacI and plLacO-RFP genetic elements. Together, these elements enable gene expression to be activated by elevated iron levels.

Next Steps: Now that we have built and characterized a Fur inverter, the final step is to replace the RFP gene with an operon of the 6 genes required for enterobactin synthesis. We have cloned the enterobactin genes entA-F and our current work focuses on inserting them into the Fur inverter.