Part:BBa_K4634000

pPepT: hypoxia-inducible promoter

pPepT, which is induced by hypoxia, is regulated by transcriptional activator: fumarate and nitrate reduction regulatory protein (FNR)(Fig. 1).[1]Under anaerobic conditions, FNR associates with [4Fe-4S]2+ clusters to form transcriptionally active homodimers, the dimer can bind DNA site-specifically and regulate transcription from target promoters. However, when the clusters are disrupted in the presence of oxygen, the FNR dimer dissociates into nonDNA-binding monomers. [2, 3]

Figure 1. Regulatory mechanism of hypoxia-inducible promoter pPepT. Under normoxic conditions, FNR monomer cannot dimerize and cannot promote the regulatory function of pPepT. It can be regarded that oxygen inhibits the function of pPepT. Under hypoxic conditions, FNR monomers can interact to form dimers, which can specifically bind to pPepT and promote the transcription of its downstream genes.

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]

Characterization

Functional Verification

To prove the normal function of pPepT, we designed an experiment to verify the expression strength of the pPepT promoter.

We inserted mCherry as a reporter gene downstream of pPepT to form an experimental group reporter system (Please refer BBa_K4634003) (Fig. 2). This experimental group was transferred into BL21(DE3) competent cells. During conditional induction for functional verification phase, we utilized a monoclonal bacterial culture for plate inoculation, followed by incubation at 37°C for 12 hours. Subsequently, the experimental was induced in separate normoxic and hypoxic environments for 48 hours. The normoxic environment was characterized by ambient atmospheric oxygen concentration, approximately 21%, while the hypoxic environment maintained an oxygen concentration of less than 1%.

Figure 2. The construction of experimental group reporter system.

Then, we used a microplate reader to measure the fluorescence intensity (Ex. 587 nm, Em. 612 nm) and OD600 of the experimental group. Next, we calculated the ratio of fluorescence intensity to OD600: F/OD. The experimental group showed the expected results: the F/OD under hypoxia and normoxia conditions were 45.72 and 30.09 respectively, and the former was 1.51 times that of the latter (Fig. 3).

Figure 3 Functional verification results of the experimental group (pPepT) in hypoxic and normoxic environments. After 48 hours of conditioned induction, we used a microplate reader to measure the fluorescence intensity and OD600 of the experimental group. Then calculate the ratio of fluorescence intensity to OD600: F/OD.

Conclusion

The above results show that the pPepT promoter can indeed be induced by anaerobic conditions, thus providing controllable input conditions for the AND gate of our TabcT. What needs to be clarified is that Av. F/OD(Hypoxia): Av. F/OD(Normoxia) = 1.51. This induction efficiency result can be further optimized through dry experiments to improve its expression efficiency.

References

[1]Yu, Bin, et al. "Explicit hypoxia targeting with tumor suppression by creating an “obligate” anaerobic Salmonella Typhimurium strain." Scientific reports 2.1 (2012): 436.
[2]Crack, Jason C., et al. "Signal perception by FNR: the role of the iron sulfur cluster1." (2008): 1144-1148.
[3] CRACK J C, GASKELL A A, GREEN J, et al. Influence of the Environment on the [4Fe−4S]2+ to [2Fe−2S]2+ Cluster Switch in the Transcriptional Regulator FNR [J]. Journal of the American Chemical Society, 2008, 130(5): 1749-58.