Part:BBa_K4462125
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 712
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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