Revision as of 12:34, 21 October 2021

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.

Use in screening for oxidative stress

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).

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-dependency studies

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.

Characterization by team Leiden 2019

Signal response in various growth media

In addition to the data obtained by Leiden 2018, the Leiden 2019 team decided to test the effects of the SPG minimal medium on the expression of the H₂O₂-reactive pSoxS-GFP. To limit background fluorescence from the medium, the use of minimal medium is required in some experiments. The translation machinery can differ between media when using promoters activated by environmental cues. To determine whether this was the case for this construct, it was decided to focus on the activity of the composite part in DH5α under rich conditions (LB) and minimal conditions (SPG). A dilution series of varying H₂O₂ concentrations were prepared in a 96-well plate starting at 3%. The wells were inoculated with DH5α bacteria carrying the pSoxS-GFP plasmid with a starting OD₆₀₀ of approximately 0.05. Next, the plate was placed in a fluorescence spectrometer (Spark 10M), allowing for kinetic cycles. Fluorescence data and OD₆₀₀ data were obtained in triplicates every 20 minutes for 1000 min. (LB) or 3000 min. (SPG) at 37°C. This was done due to the difference in growth and expression delay between the two media. The data was corrected for the OD, averaged and standardized using the Fluorescein standardization as described in the iGEM measurement hub. With the settings used for fluorescence measurements, we obtained a concentration of 4.3277E-07 µM Fluorescein equivalent per unit fluorescence.

Figures 4 and 5 show a higher lethality of H₂O₂ in SPG than in LB, since in SPG the bacteria only showed growth at an H₂O₂ concentration of 3*2^-9% versus the highest possible concentration of 3*2^-5% in LB. Of note, in SPG medium some data points within the ‘lethal’ range of H₂O₂ were practically equal to the blank wells. This resulted in data points giving meaningless values upon normalization. Thus, the data points for the media with H₂O₂ concentrations higher than 3*2^-9% were omitted. In contrast to the survival rate, the GFP response in LB medium seemed to be significantly lower compared to the response in SPG medium. At a concentration of 3*2^-9% H₂O₂, the response in SPG was around three times higher as the response in LB. Figure 5 shows that the SoxS-GFP cassette in LB medium has a maximal GFP response of 0.015 µM Fluorescein equivalent/OD600, whereas in SPG medium a response higher than 0.02 µM Fluorescein equivalent/OD600 was observed (Fig. 4).

Figure 4. Expression of pSoxS-GFP cassette in DH5α grown in SPG medium with different H₂O₂ concentrations over a time of 3000 minutes. The data was corrected for the OD, averaged and standardized using the Fluorescein standardization as described in the iGEM measurement hub.

Figure 5. Expression of pSoxS-GFP cassette in DH5α grown in LB medium with different H₂O₂ concentrations over a time of 1000 minutes. The data was corrected for the OD, averaged and standardized using the Fluorescein standardization as described in the iGEM measurement hub.

In conclusion, distinct differences both in timing and maximum expression were observed when using two different media. When using a very simple medium, such as SPG, the cells were affected more severely by H₂O₂ supplementation. When using a different medium than the two described here, it is advised to find an optimal H₂O₂ concentration for both expression and growth maximization.

Characterization by team NCKU 2021

Oxidative Stress Assay

Hydrogen peroxide (H₂O₂) was changed to paraquat (PQ) as the inducer for the oxidative stress sensing system for the following reasons:

Paraquat: a better inducer for the oxidative stress sensing system

1. PQ produces superoxide radical (O₂^¯·) catalyzed by NADPH-cytochrome P450 reductase^{[<a href="#ref1">1</a>]}. Then, O₂^¯· is converted into hydrogen peroxide (H₂O₂) by the SOD enzyme system^{[<a href="#ref2">2</a>]} or into hydroxyl radical (OH^¯·) by the HWR enzyme system^{[<a href="#ref2">2</a>,<a href="#ref3">3</a>]}. The pathway is shown in (Fig.1).

Figure 6. Paraquat and its pathway to produce reactive oxygen species (ROS).

2. Because chronic stress-induced depression (CSID) is related to a variety of abnormal changes in oxidative stress^{[<a href="#ref4">4</a>]}, a single kind of oxidative stress molecule cannot specifically mimic the changes in the human body. Therefore, PQ was chosen as the inducer in our project to more closely simulate oxidative stress in the intestine.

3. In our oxidative stress assay, we compared the strength of the oxidative stress sensing system when induced by PQ compared to when it is induced by hydrogen peroxide (H₂O₂). As shown in figure 2, sfGFP expression is greater when induced by PQ .(Fig.2)

Figure 7. The oxidative stress assay result of two systems (soxR-PsoxS -sfgfp and PsoxS -sfgfp) individually induced through H2O2 and PQ.

Sequence and Features