Difference between revisions of "Part:BBa K1431301"

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==Improvement:NUDT_CHINA 2021===
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Our improved part:TCE ([[BBa_K4016011]]) is validated to have stronger effect to initiate downstream gene expression with the same dose of doxycycline induction than this part.
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===Method===
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We used SEAP assay in vitro to validate the improvement.
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SEAP reporter gene was constructed respectively downstream of TRE3G promoter and TCE promoter into two plasmids. ( BBa_K143130-SEAP and BBa_K4016011-SEAP)Meanwhile, we constructed tetR-3G into pcDNA3.1 vector to obtain pcDNA3.1-tetR-3G. TetR-3G can specifically bind to tetO and turn on the transcription of its downstream genes SEAP. The existence of SEAP and therefore the activity of the promotor can be measured by the addition of para-Nitrophenylphosphate (pNPP). The SEAP enzyme catalyzes the reaction from pNPP to para-Nitrophenol, this new product absorbs light at 405 nm and can be measured via photometry which can served as the reporter.
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BBa_K143130-SEAP -containing plasmid and BBa_K4016011-SEAP-containing plasmid are simultaneously co-transfected with tetR-3G -expressing plasmid 293T cells. While cells in control group were co-transfected with tetR-3G -expressing plasmid and empty plasmid. Cells were cultured for 24h before adding 2ng/ml doxycycline. SEAP activity was detected 24/48 hours after transfection.
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===Result===
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HEK-293T cells co-transfected with BBa_K4016011-SEAP and tetR-3G expressing plasmids showed significantly increased SEAP production upon 24h or 48 h of doxycycline induction comparing to the control group, whose SEAP activity is below detection level. This proves that TCE does initiates the expression of downstream gene. Meanwhile, HEK-293T cells co-transfected with BBa_K4016011-SEAP and tetR-3G expressing plasmids shows ~30 times higher SEAP activity compared to cells transfected with the same dose of BBa_K143130-SEAP and tetR-3G plasmids, demonstrating the successful improvement of BBa_K4016011 in our project.
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<img src="" class="figure-img img-fluid rounded"  height="350px">
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Figure1. (A)SEAP activity in the culture medium collect from cells 24 hours after 2ng/ml doxycycline induction. (B)SEAP activity in the culture medium collect from cells 48 hours after 2ng/ml doxycycline induction. BDL stands for below detection level.

Revision as of 21:41, 21 October 2021

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 number of 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]



Improvement:NUDT_CHINA 2021=

Our improved part:TCE (BBa_K4016011) is validated to have stronger effect to initiate downstream gene expression with the same dose of doxycycline induction than this part.

Method

We used SEAP assay in vitro to validate the improvement.

SEAP reporter gene was constructed respectively downstream of TRE3G promoter and TCE promoter into two plasmids. ( BBa_K143130-SEAP and BBa_K4016011-SEAP)Meanwhile, we constructed tetR-3G into pcDNA3.1 vector to obtain pcDNA3.1-tetR-3G. TetR-3G can specifically bind to tetO and turn on the transcription of its downstream genes SEAP. The existence of SEAP and therefore the activity of the promotor can be measured by the addition of para-Nitrophenylphosphate (pNPP). The SEAP enzyme catalyzes the reaction from pNPP to para-Nitrophenol, this new product absorbs light at 405 nm and can be measured via photometry which can served as the reporter.

BBa_K143130-SEAP -containing plasmid and BBa_K4016011-SEAP-containing plasmid are simultaneously co-transfected with tetR-3G -expressing plasmid 293T cells. While cells in control group were co-transfected with tetR-3G -expressing plasmid and empty plasmid. Cells were cultured for 24h before adding 2ng/ml doxycycline. SEAP activity was detected 24/48 hours after transfection.


Result

HEK-293T cells co-transfected with BBa_K4016011-SEAP and tetR-3G expressing plasmids showed significantly increased SEAP production upon 24h or 48 h of doxycycline induction comparing to the control group, whose SEAP activity is below detection level. This proves that TCE does initiates the expression of downstream gene. Meanwhile, HEK-293T cells co-transfected with BBa_K4016011-SEAP and tetR-3G expressing plasmids shows ~30 times higher SEAP activity compared to cells transfected with the same dose of BBa_K143130-SEAP and tetR-3G plasmids, demonstrating the successful improvement of BBa_K4016011 in our project.


Figure1. (A)SEAP activity in the culture medium collect from cells 24 hours after 2ng/ml doxycycline induction. (B)SEAP activity in the culture medium collect from cells 48 hours after 2ng/ml doxycycline induction. BDL stands for below detection level.