Part:BBa_K2364002
rtTA
reverse tetracycline-controlled transactivator) was created by fusing rTetR with VP16.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 439
Improvement from iGEM 2019 Team BM-AMU
In iGEM 2017, the team Greece submitted a basic part Part:BBa_K2364002 called rtTA. The effect of doxycycline as an inducer was investigated by the expression of GFP in FACS. Optimal control of gene expression requires a Tet-On system with low background activity in the absence of dox and high activity in the presence of the effector. Unfortunately, The results of the FACS illustrates that without induction with doxycycline, GFP is still expressed.
In iGEM2019, the team BM-AMU improved this part:Part:BBa_K3120013 called tetR-krab. KRAB is a 75 aa domain found in about one third of the several hundreds of human zinc finger proteins and is located in the amino terminal end of proteins that contain Krüppel-class zinc fingers in their carboxy termini. A chimeric transrepressor protein was engineered by fusing the KRAB domain of human Kox1 to the TetR.
To investigate DOX-dependent efficiency of the combined activator strategy, TetO-GFP cell line was constructed in 293-FT cells. Finally, relative fluorescence expression was detected by flow cytometry. Experimental details can be found in our protocol.
Equal amount of HEK293T cells were seeded in a 6-well plate and transfected with TetO-GFP. Flow cytometry was used to detect GFP expression on 5, 7, 9 days after transfection. On the day 9, DOX (1 μg/μL) was added to initiate downstream gene expression, and GFP expression was detected 5 days later. WT293FT cells were used as blank controls at each stage.
The results of the FACS illustrates that without induction with doxycycline, GFP in the original system is still expressed. However, our improved part prevents the leakage of fluorescent proteins. And according to the slope of figure 1, the improved part turned on the expression of fluorescent protein faster than the original part after adding DOX.
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