Reporter

Part:BBa_K2610034

Designed by: Charlotte de Ceuninck van Capelle, Maaike de Jong   Group: iGEM18_Leiden   (2018-10-10)
Revision as of 02:09, 18 October 2018 by JazzyDeWaard (Talk | contribs)


pSoxS-GFP-pSoxS-GFP

This composite part features twice the regulatory part promoter pSoxS (BBa_K2610030) and the fluorescent protein GFP (BBa_E0040). It can be used to visualize upregulation of SoxS as a result of superoxide stress.

Regulatory protein SoxS is involved in the oxidative stress signaling pathway in Escherichia coli . Intracellular superoxide-generating compounds cause the SoxR to activate transcription of SoxS, which then triggers a set of defense and repair genes that form the oxidative response system.

Usage and Biology

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 SoxS transcription. We have created this part in order to amplify the fluorescent signal measured after pSoxS-GFP activation.

As can be observed in Figure 1 the pSoxS-GFP-pSoxS-GFP construct leads to an increased mean fluorescence intensity compared to pSoxS-GFP. This activation was demonstrated through treatment of the transformed E.coli cells with nalidixic acid. We successfully thus amplified the pSoxS-GFP stress reporter strain by increasing the number of promoters and GFP genes in the plasmid. This enables detection of even lower concentrations of stressful substances, allows signals to be detected by less sensitive detection devices and facilitates faster signal detection.

In addition, we assessed the dose-dependency of this reporter. We found that an increase in nalidixic acid concentration of nalidixic acid leads to an increasing mean fluorescence intensity (MFI). A decrease in detected GFP expression at higher concentrations can be attributed to the lethality of the stressor.

T--Leiden--amplificA.png T--Leiden--amplificB.jpeg

Figure 1. Median fluorescence intensity (MFI) as a result of treatment with nalidixic acid for pSoxS-GFP compared to pSoxS-GFP-pSoxS-GFP. It can also be seen that there is a dose response curve to nalidixic acid. The decrease in detected GFP expression at higher concentrations can be attributed to lethality of the stressor



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 947
    Illegal BsaI.rc site found at 1978


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