Part:BBa_K5159025
pTEFin-GFP-LIP2t
This part is constructed to measure the fluorescence of a common GFP under the strong constitutive promoter pTEF with intron in Yarrowia lipolytica. The fluorescence intensity serves as an indicator of the expression level and thus the promoter strength. It is constructed as a standard for comparing relative fluorescence intensity when expressing GFP under different Yarrowia lipolytica promoters. This construct can also bridge the expression level of functional proteins in our project with the GFP intensity, by comparing quantitative Western blot results and fluorescence measured under the same condition. To ensure the comparability, an identical promoter, spacer and terminator settings as the PUFA synthase expression cassettes are applied.
During testing, the average fluorescence was measured, blanked with untransformed groups and normalized with OD600 to minimize influence from other factors and the differences between individual cells transformed.
The reporter gene in the construct can be changed to hrGFP, luciferase, eGFP or other fluorescent proteins to achieve a similar goal, each having its own advantage.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1392
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 2
Illegal BsaI.rc site found at 1175
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