Part:BBa_K2913009

PfnrF8

This is a hypoxia-inducible promoter to restrict gene expression specifically in the hypoxic environment of tumors. The transcriptional activation promoted by the PfnrF8 needs a specific transcription factor called FNR, which can bind to oxygen. When the oxygen level is at a normal range in the internal environment, the transcription factor FNR will associate with oxygen, leading to its impeded binding to the PfnrF8 promoter, and reduce the transcription of its downstream gene. However, in the hypoxic environment of tumors, FNR will disassociate with oxygen and activate its downstream gene.

result

The series of concentrations of sodium sulfite (0, 0.25, 0.5, 0.75 and 1 g/l Na2SO3) can make hypoxia environment (Fig.2a). Under the PfnrF8 [6] promoter, the activity of LacZ can be seen to rise markedly from 0 g/l to 1 g/l Na2SO3 (Fig.2c). This promoter can respond our hypoxia environment regularly. Comparing with the FF+20 (mutation) promoter, its downstream gene had much higher expression (Fig.2d). We will debug hypoxic sensitivity of the PfnrF8 promoter in the future.

Fig.2(a) Dissolved oxygen change curve under a series of concentrations of sodium sulfite (0, 0.25, 0.5, 0.75 and 1 g/l Na2SO3). (b) Hypoxic induction of FF+20 (mutation) promoter in E. coli Nissle 1917. Expression of lacZ vary in different sodium sulfite concentrations (0, 0.25, 0.75 and 1 g/l Na2SO3). β-gal activity was measured as described in texts above. Values are expressed as percentage of promoter activity at 1 g/l Na2SO3. (c) Hypoxic inductions of PfnrF8 promoter in E. coli Nissle 1917. Expression of lacZ shows the Na2SO3 concentration dependence in increasing sodium sulfite concentrations (0, 0.25, 0.5, 0.75 and 1g/l Na2SO3). β-gal activity was measured as above. Values are expressed as percentage of promoter activity at positive (The mutant of PfnrF8, with strong expressing and not induced by hypoxia). (d) The comparison of Hypoxic inductions of PfnrF8 promoter and FF+20 (mutation) promoter in E. coli Nissle 1917. Expression of lacZ was compared in increasing sodium sulfite concentrations (0, 0.25, 0.75 and 1 g/l Na2SO3). β-gal activity was measured as above. Values are expressed as percentage of promoter activity at positive (The mutant of PfnrF8, with strong expressing and not induced by hypoxia). Error bars represent standard deviations.

References

[1] Ryan, R.M., et al., Bacterial delivery of a novel cytolysin to hypoxic areas of solid tumors. Gene Ther, 2009. 16(3): p. 329-39. [2] Bell, A. and S. Busby, Location and orientation of an activating region in the Escherichia coli transcription factor, FNR. Mol Microbiol, 1994. 11(2): p. 383-90. [3] Moser, F., et al., Dynamic control of endogenous metabolism with combinatorial logic circuits. Mol Syst Biol, 2018. 14(11): p. e8605.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 21
  Illegal NheI site found at 44
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]