Difference between revisions of "Part:BBa K1431301"
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Revision as of 02:14, 18 October 2014

TRE-3G promoter+SV40 PolyA, an ideal controller of mammalian gene expression with Tet-On 3G protein

TRE-3G promoter(PTRE3G) is a kind of eukaryocyte cell promoters that will be switched on after combine with the compound of Tet-On 3G(BBa_K1431101) protein and doxycycline(Dox) mix.

The inducible promoter TRE3G provides for very low basal expression and high maximal expression after induction (Loew et. al., 2010). It consists of 7 repeats of a 19 bp tet operator sequence located upstream of a minimal CMV promoter. In the presence of Dox, Tet-On 3G binds specifically to PTRE3G and activates transcription of the downstream gene of interest (GOI). PTRE3G lacks binding sites for endogenous mammalian transcription factors, so it is virtually silent in the absence of induction.(Source: Clontech)

SUSTC-Shenzhen_Tet_On_3G.png
Figure 1.The Tet-On 3G systems allow inducible gene expression only in the presence of doxycycline. When Dox binds, the
transactivator undergoes a conformational change allowing it to bind tet operator (tetO) repeats within the
TREG Promoter. The transactivator activates expression through transcription activation domain repeats.

Click here to learn more details about Tet-On and PTRE3G system.

Data

The plasmid transfected into Hela Cell shows below.

Plasmid_transfected.jpg
pBX-093 PB5-HS4-SV40-puro-2A-tetON3G-pA-HS4-TRE-AzaminGreen-2A-T


Transfect Hela Cell with different doxycycline(Dox) concentration by lipofectamine3000 and get the data after 30hours.

fluorescence microscope

30h_0_BF.jpg
30h_0_GF.jpg
0 ng/mL doxycycline after 30h
0 ng/mL doxycycline after 30h
30h_100_BF.jpg
30h_100_gf.jpg
100 ng/mL doxycycline after 30h
100 ng/mL doxycycline after 30h


Flow Cytometry

Transfected_cells_induced_in_0_ngmL_doxycycline.png
Transfected_cells_induced_in_0.1_ngmL_doxycycline.png
Transfected_cells_induced_in_1.0_ngmL_doxycycline.png
0 ng/mL doxycycline
0.1 ng/mL doxycycline
1.0_ng/mL_doxycycline
Transfected_cells_induced_in_10_ngmL_doxycycline.png
Transfected_cells_induced_in_100_ngmL_doxycycline.png
Transfected_cells_induced_in_1000_ngmL_doxycycline.png
10_ng/mL_doxycycline
100_ng/mL_doxycycline
1000_ng/mL_doxycycline

We could find that without Dox, TRE-3G promoter hardly be activated because the fluorescent protein do not expressed. The higher concentration of Dox,the higher fluorescent protein expressed. But if the concentration reached 1000 ng/ml, the expression become low, that may be toxic to Hela cell for too high concentration.



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]