Part:BBa_K2610018
pKatG-GFP
This reporter part features the basic part promoter KatG (BBa_K2610004) and the fluorescent reporter protein GFP (BBa_E0040).
The KatG gene encodes for hydroperoxidase I. It protects aerobic, phosphate-starved cells from oxidative damage. The expression of KatG is activated by the global regulator OxyR. This enzyme is specifically upregulated by oxidative agents, as demonstrated by previous research. It has been shown that the promoter of KatG fused to the lux operon can be strongly induced by hydrogen peroxide in a dose-dependent response. (Zhang et al, 2015)(Mitchell et al, 2004)
For more information please refer to our [http://2018.igem.org/Team:Leiden/Results Results page]
pKatG-GFP as a specific indicator for oxidative damage to E.coli
We, iGEM Leiden 2018, have designed this composite part as part of our project Fifty Shades of Stress. This reporter part allowed us to detect stress-induced changes in KatG transcription.
Figure 1. Median Fluorescence Intensity (MFI) in AU after 4 hour incubation with antimicrobial agents in various concentrations.
We have observed a specific increased fluorescence in response to hydrogen peroxide using the pKatG-GFP reporter (Figure 1). This supports our hypothesis that the promoter of KatG can be used to specifically detect stressful compounds that target the oxidative stress response in Escherichia coli. Our findings support previous research stating that KatG responds in a dose-response manner to hydrogen peroxide. Moreover, we have tested our pKat-GFP construct for response to ascorbic acid. We have previously established that upon co-administration of ascorbic acid less antibiotic is required to have the desired bactericidal effect. As can be seen in Figure 2, KatG is activated following treatment with ascorbic acid. Our flow cytometry data suggests that increasing concentrations of ascorbic acid lead to an increased median fluorescence intensity. This indicates a role of ascorbic acid as a possible oxidative agent.
Figure 2. Median Fluorescence Intensity after treatment with ascorbic acid (vitamin C) including pH controls.
References
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 50
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 793
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