Part:BBa_K4392009

Hypoxia inducible PfnrF8 promoter with UnaG reporter

This part consists of a hypoxia-inducible Pfnr8 promoter followed by a strong RBS with the UnaG reporter downstream followed by a double terminator. Azospirillum brasilense has nitrogenase genes[a] that are regulated by the FNR/CRP complex[b], so it was hypothesized that this promoter would show regulated activity in Azospirillum.

To demonstrate and validate the activity of any hypoxia-inducible promoter, it is necessary to use a reporter gene that is functional under hypoxic conditions. The requirement of oxygen for forming the chromophore in fluorescent proteins like GFP and RFP renders them futile for experiments under hypoxia. The fluorescence of the UnaG protein, on the other hand, is not limited to aerobic conditions suggesting that it can be utilized for the vast expanse of experiments that require hypoxia. However, the presence of unconjugated bilirubin is a must for UnaG fluorescence. UnaG is a protein originally extracted from the muscle fibers of the Japanese eel, Unagi, that utilizes bilirubin as a ligand for fluorescing. Hence, bilirubin has to be exogenously provided to the cultures [c].

Constructs like these can prove to be useful in synthetic biomedical engineering and would have wide uses in targeting specific tumor microenvironments. These can also be used for an environmental project like ours that deals with low oxygen conditions.

[a] Zhang Y, Burris RH, Ludden PW, Roberts GP. Regulation of nitrogen fixation in Azospirillum brasilense. FEMS Microbiol Lett. 1997 Jul 15;152(2):195-204. DOI: 10.1111/j.1574-6968.1997.tb10428.x. PMID: 9231412.

[b] https://www.uniprot.org/uniprotkb/A0A2K1G750/entry

[c] https://www.cell.com/action/showPdf?pii=S0092-8674%2813%2900644-2

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 300
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 300
  Illegal NheI site found at 21
  Illegal NheI site found at 44
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 300
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 300
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 300
  
• 1000
  COMPATIBLE WITH RFC[1000]