Difference between revisions of "Part:BBa K2610034"
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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. | 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. | ||
| − | [[File:T--Leiden--amplificA.png| | + | [[File:T--Leiden--amplificA.png|300px]] [[File:T--Leiden--amplificB.jpeg|300px]]<br><br> |
<span style="font-size:1em"><b>Figure 1.</b> Median fluorescence intensity (MFI) as a result of treatment with nalidixic acid for pSoxS-GFP compared to pSoxS-GFP-pSoxS-GFP. </span> | <span style="font-size:1em"><b>Figure 1.</b> Median fluorescence intensity (MFI) as a result of treatment with nalidixic acid for pSoxS-GFP compared to pSoxS-GFP-pSoxS-GFP. </span> | ||
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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. | 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. | ||
| − | [[File:T--Leiden--amplification2.png| | + | [[File:T--Leiden--amplification2.png|600px]] <br> |
<span style="font-size:1em"><b>Figure 2.</b> Dose-response curve as a result of treatment with nalidixic acid. </span> | <span style="font-size:1em"><b>Figure 2.</b> Dose-response curve as a result of treatment with nalidixic acid. </span> | ||
Revision as of 01:02, 18 October 2018
pSoxS-GFP-pSoxS-GFP
This composite part features twice the regulatory part promoter SoxS (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 superoxide pathway in Escherichia coli. It acts as a superactivator of downstream stress genes.
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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 947
Illegal BsaI.rc site found at 1978