Part:BBa_K387003

PfdhF: fdhF promoter, a hypoxia inducible promoter

Hypoxia-inducible promoter. Under hypoxia environment it has a much higher efficiency of starting transcription than under normal 20% oxygen pressure.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

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Characterization of PfdhF, the hypoxia-inducible promoter

Yupeng He from [http://2010.igem.org/Team:SJTU-BioX-Shanghai iGEM2010_SJTU-BioX-Shanghai] built and characterized the hypoxia-inducible promoter, fdhF promoter (PfdfF, Part: BBa_K387003). In this test experiment, we expected to see that, at the same OD₆₀₀, the activity of PfdhF is higher in hypoxia than in normal oxygen pressure (20%). At the beginning, both the hypoxia group and control (20% oxygen partial pressure, i.e. in air) group were incubated with 200 microlitre suspension of bacteria with the testing part (PfdhF followed by firefly luciferase). Then, we sampled both group and measured OD₆₀₀ and value of luciferase every 30 minutes (control group) or 1 hour (hypoxia). The figure was the result of this test. Our result showed that this part worked as we expected: under hypoxia, the efficiency of this promoter was much higher than that under the condition of 20% oxygen at the same OD₆₀₀. (One thing that needs to be noticed is that at 0.4 OD₆₀₀, the bacteria are in log phase and the PfdhF begins to work.)

Then we went to measure the difference of PfdhF activity under these two conditions. We plotted another figure with OD₆₀₀ as x axis and the ratio of PfdhF activity under hypoxia to that under normal oxygen pressure as y axis. This curve showed that, during and after the log phase (OD₆₀₀>0.4), the activity of PfdhF is over 1.5 times more under hypoxia than under normal 20% oxygen pressure. Particularly, this ratio is over 3 when OD₆₀₀>0.5. Furthermore, in the normal 20% oxygen group, as the OD₆₀₀ increases, the oxygen supplies may be insufficient for E. coli which may lead to the increase of PfdhF activity. In this case, the activity of PfdhF under normal condition is probably overestimated. With all these data and analysis, it is evident that PfdhF is a rather good hypoxia-inducible promoter with much higher activity under hypoxia than under 20% oxygen pressure.

Further Characterization of the fdhF Promoter

[http://2017.igem.org/Team:McMaster_II McMaster_II] further characterized the hypoxia-inducible promoter by measuring the activity of the promoter under varying oxygen concentrations. We placed overnight cultures in syringes and added different volumes of air. By doing so, we simulated different oxygen concentrations in each syringe. Upon incubation, the fluorescence of the culture was measured. The results we obtained are displayed in the following figure.

Our results showed that the hypoxia-inducible promoter is maximally activated when there is no air present in the syringe (as seen by the first bar on the graph). Moreover, the activity of the promoter showed an inverse relationship to the volume of the volume of the air in the syringe. As the air volume increased, the fluorescence measured decreased.

Overall, [http://2017.igem.org/Team:McMaster_II McMaster_II] was able to improve the characterization of the fdhF promoter by testing its activity under various oxygen concentrations.