Difference between revisions of "Part:BBa K2610033"

Revision as of 08:37, 11 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 superoxide pathway in Escherichia coli. It acts as a superactivator of downstream stress genes. We have coupled this promoter to chromoprotein AmilCP, a blue protein.

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Usage and Biology

iGEM Leiden has 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. Again cells were grown overnight at 37 degrees celsius under continuous shaking.

It can be observed in Figure 1 that pSoxS-AmilCP results in coloured E.coli cells after addition of a stressful compound.

Figure 1. Expression of pSoxs-AmilCP on the left compared to untransformed DH5α on the right.

From interviews with experts we have gathered that this particular construct would be a useful addition to visual screenings for antibiotics as it 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 validate our part further. Disk diffusions are commonly used to assess the bactericidal effect of antibiotics. In Figure 2 a plate of pSoxS-AmilCP cells are shown. In the middle of the plate three disks with nalidixic acid were placed. After 48 hours it is visible that the high nalidixic acid concentrations create a death halo around the disks. However, moving further away from the disk shows a coloured ring. These bacteria are alive but stressed. In response to the stress these bacterial cells are experiencing they are produced the blue chromoprotein AmilCP.

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

Sequence and Features