Difference between revisions of "Part:BBa K2610031"

Revision as of 00:56, 17 October 2018

pSoxS-GFP

This composite part features 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

iGEM Leiden 2018 has created a screening system for the detection of new antibiotics. This composite part which allowed us to detect stress-induced changes in SoxS transcription, signaling that a certain compound causes superoxide stress.

Screening for stress with flow cytometry

We have tested of these several stress-activated promoters against a repertoire of known antibiotics using flow cytometry. As can be seen in the heatmap below, treatment with both nalidixic acid and hydrogen peroxide causes a significant increase in fluorescent signal of pSoxS-GFP.

Figure 1. Heatmap with stress-activated promoters. Values for each promoter are normalised to its respective negative control (no stressor, not shown).

Confocal microscopy validation

The pSoxS-GFP BioBrick was also validated using confocal microscopy. Incubation of our reporter strain with nalidixic acid can be seen to increase GFP expression compared to basal GFP expression levels (see Figure 2).

Figure 2.Visualisation of GFP expression of the pSoxS-GFP strain after nalidixic acid treatment using confocal microscopy. Wild-type DH5α and the pSoxS-GFP (BBa_K2610031) strain were incubated with nalidixic acid (50 ng/mL) for two hours. A/B) Wild-type DH5α. C/D) pSoxS-GFP without stressor. Basal GFP expression can be observed. E/F) pSoxS-GFP with 50 ng/mL nalidixic acid. An increase in expression compared to the negative control can be observed. Pictures were obtained by using a confocal microscope. The scale bar represents 10 µm.

Dose-response to nalidixic acid

After determination of the specific response of pSoxS-GFP 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 3. Mean Fluorescence Intensity (MFI) in AU after 4 hour incubation with nalidixic acid in various concentrations.

Signal amplification

We successfully amplified the pSoxS-GFP stress reporter strain by increasing the amount of promoters and GFP genes in the plasmid. We created pSoxS-GFP-pSoxS-GFP (BBa_K2610034) and pSoxS-GFP-GFP-pSoxS-GFP-GFP (BBa_K2610035). 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. More details of these constructs can be found on their pages.

Sequence and Features