Part:BBa_M50440

bioluminescent "pHlash" reporter for intracellular pH

This is a genetically encodable probe that will report cytosolic pH in vivo of mammalian, yeast, or bacterial cells, through Bioluminescence Resonance Energy Transfer (BRET) data. The device utilizes a Rluc8 luciferase-cpVenus fluorophore fusion, where the Rluc8 luciferase protein exhibits pH-dependent activity. The luciferase cleaves a luciferin substrate, specifically coelenterazine, emitting a photon to excite the fluorophore, causing emission of light. Since luciferase activity rate depends on the pH of the environment, the emission by the fluorophore is an indicator of pH, which can be calibrated or relatively measured by calculating the BRET ratio (475nm: 525nm recommended).

This is the full plasmid construct of this part, viable in S. cerevisiae and E. coli.

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
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 150
  Illegal AgeI site found at 759
  Illegal AgeI site found at 1648
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1500
  Illegal BsaI.rc site found at 1194
  Illegal SapI.rc site found at 1513