Difference between revisions of "Part:BBa K3120013"
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| + | ===Biology and Usage=== | ||
| + | |||
| + | Systems that modulate (trans)gene expression in mammalian cells and tissues are important for a wide variety of basic and applied biological research areas, including functional genomics, gene therapy, animal models for human diseases and biopharmaceutical protein production. All these applications require that the expression of the gene(s) of interest can be strictly controlled in a quantitative and temporal manner. The Tet system, in which gene expression is stringently controlled by tetracycline (Tc) or its derivative doxycycline (dox), is the most widely used regulatory circuit. This system is based on the sequence-specific, high-affinity binding of the Escherichia coli Tet repressor protein (TetR) to the tet operator (tetO) DNA sequence. Tc or dox binds to TetR and triggers a conformational change that prevents the repressor protein from binding to tetO. | ||
| + | |||
| + | ===Improvement on a previous part=== | ||
| + | |||
| + | In iGEM 2017, the team Greece submitted a basic part Part:[https://parts.igem.org/Part:BBa_K2364002 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:[https://parts.igem.org/Part:BBa_K3120013 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 [https://2019.igem.org/Team:BM-AMU/Protocols 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. | ||
| + | [[Image:T--BM-AMU--part5.png|center|thumb|660px|]] | ||
Latest revision as of 17:14, 12 October 2019
tetR-krab
This part belong to the tet-on induction expression system. Under the action of the CAG promoter, tetR-Rrab fusion protein is highly expressed. In the presence of DOX,tetR-Rrab binds with TRE to promote snail1 transcription, promote EMT process and achieve quantitative control.
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 922
Biology and Usage
Systems that modulate (trans)gene expression in mammalian cells and tissues are important for a wide variety of basic and applied biological research areas, including functional genomics, gene therapy, animal models for human diseases and biopharmaceutical protein production. All these applications require that the expression of the gene(s) of interest can be strictly controlled in a quantitative and temporal manner. The Tet system, in which gene expression is stringently controlled by tetracycline (Tc) or its derivative doxycycline (dox), is the most widely used regulatory circuit. This system is based on the sequence-specific, high-affinity binding of the Escherichia coli Tet repressor protein (TetR) to the tet operator (tetO) DNA sequence. Tc or dox binds to TetR and triggers a conformational change that prevents the repressor protein from binding to tetO.
Improvement on a previous part
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.
