Measurement

Part:BBa_K4462125

Designed by: Kredai Raaman   Group: iGEM22_IISc-Bengaluru   (2022-09-30)


pPEM7-sfGFP

Construct for hypoxic promoter characterisation: PEM7-T7RBS-sfGFP-6xHis-Lt0_Ter

Usage and Biology

We used this part to determine the basal change in fluorescence intensity that resulted from exposure to hypoxic conditions due to factors like global changes in protein expression and reduced folding efficiency that are independent of promoter activity. Prior iGEM teams have reported issues with fluorescent proteins folding incorrectly under low oxygen tension, but there was no strong evidence for this with superfolder GFP in literature. Nevertheless, we could control for differences in fluorescence intensity that resulted from inherent differences in sfGFP folding or by global changes in transcription under hypoxia by using a strong constitutive promoter with no oxygen-sensitivity to drive sfGFP expression, and measuring the change in expression, if any, between hypoxic and normoxic conditions for this. Activity would be quantified by imaging single cells and measuring fluorescence intensity across a large number of cells in the presence and absence of oxygen.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 712
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
None