Difference between revisions of "Part:BBa K2610033"

Latest revision as of 02:29, 18 October 2018

pSoxS-AmilCP

This composite part features the regulatory part promoter SoxS (BBa_K2610030) and the chromoprotein AmilCP (BBa_K592009). It can be used to visualize to the naked eye 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.

For more information please refer to our [http://2018.igem.org/Team:Leiden/Results results page].

Usage and Biology

Use in screening for stressful compounds

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.

From interviews with experts, we gathered that this construct could be a useful addition to visual screenings for antibiotics. In search for new antibiotics, bacterial overlays and disk diffusion assays are common. These are experiments in which potential antibiotic-producing strains or disks containing antibiotics are placed on a plate, after which a bacterial test strain is seeded on top. Compounds diffuse through the agar and - if lethal - prevent bacterial growth surrounding the strain or disk, thereby creating a visible “zone of inhibition". Use of pSoxS-AmilCP would allow for detection of not only lethal compounds but also for stressful, non-lethal compounds. We have therefore performed a traditional disk diffusion assay in order to further validate our part.

In Figure 1 a plate of pSoxS-AmilCP cells are shown. Three disks containing nalidixic acid were placed on the petri dish which contained a layer of transformed Dh5a with pSoxS-AmilCP. After 48 hours, a death halo with blueish E. coli bacteria on the edge is visible. This shows that as a result of the upregulation of pSoxS in these cells, AmilCP is being produced, which is indicative of a stressful process occurring in these cells. Hereby, we have shown that non-lethal concentrations of nalidixic acid can be detected using disk diffusion with pSoxS-AmilCP.

Figure 1. Disk diffusion assay performed with E. coli DH5α expressing pSoxS-AmilCP, showing a typical death halo but also with a stress induced blue/greyish chromoprotein halo.

In addition to disk diffusion assays, we performed experiments with pSoxS-AmilCP in liquid culture. For this, we cultured pSoxS-AmilCP cells in LB overnight. Consequently, the saturated overnight culture was used to inoculate fresh medium in a 1:10 ratio. To this newly inoculated strain nalidixic acid was added in order to stress the bacterial cells after which cells were grown overnight at 37 degrees Celsius under continuous shaking.

It can be observed in Figure 2 that pSoxS-AmilCP results in coloured E.coli cells after addition of a stressful compound.

Figure 2. Expression of pSoxs-AmilCP on the left compared to untransformed DH5α on the right.

Dose-dependency studies

After validation of a pSoxS-AmilCP in liquid culture, we assessed the dose response in liquid cultures by incubating bacteria expressing the pSoxS-AmilCP BioBrick with increasing concentrations of nalidixic acid, results of which are shown in figure 3. However, we found no discernible difference in color for the stressed vs. unstressed strains. The stress caused by nalidixic acid at our used concentrations is not enough to create a difference in expression visible to the naked eye.

Figure 2. Attempted dose-response experiments in chromoprotein liquid cultures. pSoxS-AmilCP strains were stressed overnight with four concentrations of nalidixic acid. No dose-dependent response was observed.

Sequence and Features