Difference between revisions of "Part:BBa K2610034"

Revision as of 01:07, 18 October 2018

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 median 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 amount 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.

Figure 1. Median fluorescence intensity (MFI) as a result of treatment with nalidixic acid for pSoxS-GFP compared to pSoxS-GFP-pSoxS-GFP.

Dose-dependency studies

After determination of the specific response to nalidixic acid, 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.

Figure 2. Dose-response curve as a result of treatment with nalidixic acid. A decrease in detected GFP expression at higher concentrations can be attributed to lethality of the stressor.

Sequence and Features