The Theory

Our Fur repressor/RyhB reporter system is based upon the way that the cell normally detects, and then controls, its intracellular iron levels. This homeostasis is necessary as, although iron is vital for the cell to function, it is also potentially dangerous due to the Fenton reaction, which produces dangerous reactive oxygen species (ROS) under aerobic conditions (Andrews et al., 2003).

Clonning Diagrams

Our reporter system contains a RyhB binding sequence in the 5’ UTR of sfGFP and is present under the control of two promoters of different strengths, a medium and a strong. These constructs were cloned into both pBluescript KS(+) and pSB1C3 plasmids and then transformed into our siderophore production mutant, JC28, along with our wild type W3110 for use as a control (figure 1).

Fluorescence change when grown at different iron concentrations

As an initial test, we wanted to see if fluorescence changed in response to growth in different iron concentrations. We grew our mutant and wild type strains containing pBluescript KS(+)-RyhB-GFP construct in defined medium with either 5μM or 100μM FeCl3 for low or high iron conditions respectively. We then measured the OD600 and the fluorescence of our cells at 535nm, which we adjusted according to each samples respective OD (figure 2). We were unable to detect any significant correlation between fluorescence of the sample and iron concentration in the growth medium, suggesting that our RyhB-GFP reporter system is not affected by changes in extracellular iron concentrations.

Fluorescence change in response to addition of iron