Difference between revisions of "Part:BBa K1431301"
 
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==Improvement:NUDT_CHINA 2021==
 
==Improvement:NUDT_CHINA 2021==
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Our improved part:TCE ([[Part:BBa_K4016011]]) is validated to have stronger effect to initiate downstream gene expression with the same dose of doxycycline induction than this part.
+
Our improved part is TCE promoter ([[Part:BBa_K4016011]]), a tetO7 promoter consists of seven direct 19-bp tet operator sequence (tetO) repeats upstream of a minimal CMV promoter (PminCMV). tetOs were separated by a 17bp flanking sequence, which was essential to the dox responsiveness of this promoter. The tetO constitutively binds tetR, it also binds to transcriptional activator tet-on 3G under the presence ofdoxycyline This promoter should be constructed just upstream of a Kozark consensus ribosome binding site to enhance the expression level of GOI.
 +
 
 +
The improved TCE promoter shares a similar nucleic acid sequence to the original promoter in BBa_K1431301, with only a few nucleotides different on the flanking regions of tetO. However, these differences were sufficient to generate folds of changes on dox responsiveness.
 +
 
  
 
===Method===
 
===Method===
−
We used SEAP assay in vitro to validate the improvement.
+
We used SEAP assay 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.
+
SEAP reporter gene was constructed downstream of original TRE3G promoter and TCE promoter as BBa_K143130-SEAP and BBa_K4016011-SEAP.. Therefore the SEAP activity in the culture medium could be used to report the activation of the promoters.
 +
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. SEAP activity could be measured witha para-Nitrophenylphosphate (pNPP) based assay, as SEAP catalyzes the reaction from pNPP to para-Nitrophenol, this new product absorbs light at 405 nm and can be measured via photometry.
  
−
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.
+
Either BBa_K143130-SEAP encoding plasmid or BBa_K4016011-SEAPencoding plasmid were co-transfected with tetR-3G encoding plasmid into HEK-293 cells. Cells were cultured for 6h before adding 500 ng/ml doxycycline, cells transfected with the same plasmids without doxycycline treatment were used as control. SEAP activity was detected 24/48 hours after transfection.
  
  
 
===Result===
 
===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.
+
HEK-293T cells co-transfected with tetR-3G expressing plasmid and either BBa_K4016011-SEAP or BBa_K143130-SEAP plasmid showed significantly boosted SEAP production upon 24h or 48 h of doxycycline induction comparing to their respective control group, in which SEAP activities were below detection limit. This proves that both promoters did initiate the transcription of downstream gene. Intriguingly, under doxycycline induction, HEK-293T cells co-transfected with BBa_K4016011-SEAP and tetR-3G expressing plasmids showed ~30 times higher SEAP activity comparing to cells transfected with same dose of BBa_K143130-SEAP and tetR-3G plasmids, showing a significantly improved performance of [[Part:BBa_K4016011]] comparing to Part:BBa_K143130.
  
 
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<figure class="figure">
 
<figure class="figure">
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<img src="https://static.igem.org/mediawiki/parts/d/dd/T--NUDT_CHINA--Part_improvement_11.png" class="figure-img img-fluid rounded"  height="350px">
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<img src="https://static.igem.org/mediawiki/parts/d/dd/T--NUDT_CHINA--Part_improvement_11.png" class="figure-img img-fluid rounded"  height="400px">
  
 
</figure>
 
</figure>
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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.
+
Figure1. Comparation of doxycycline responsiveness between the improved version and the original version of tetO7 promoters. (A)SEAP activity in the culture medium collect from cells 24 hours after 500 ng/ml doxycycline induction. (B)SEAP activity in the culture medium collect from cells 48 hours after 500 ng/ml doxycycline induction. BDL stands for below detection limit. Data represent mean ± s.e.m. (n=3 biological replicates)

Latest revision as of 23:19, 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 is TCE promoter (Part:BBa_K4016011), a tetO7 promoter consists of seven direct 19-bp tet operator sequence (tetO) repeats upstream of a minimal CMV promoter (PminCMV). tetOs were separated by a 17bp flanking sequence, which was essential to the dox responsiveness of this promoter. The tetO constitutively binds tetR, it also binds to transcriptional activator tet-on 3G under the presence ofdoxycyline This promoter should be constructed just upstream of a Kozark consensus ribosome binding site to enhance the expression level of GOI.

The improved TCE promoter shares a similar nucleic acid sequence to the original promoter in BBa_K1431301, with only a few nucleotides different on the flanking regions of tetO. However, these differences were sufficient to generate folds of changes on dox responsiveness.


Method

We used SEAP assay to validate the improvement.

SEAP reporter gene was constructed downstream of original TRE3G promoter and TCE promoter as BBa_K143130-SEAP and BBa_K4016011-SEAP.. Therefore the SEAP activity in the culture medium could be used to report the activation of the promoters. 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. SEAP activity could be measured witha para-Nitrophenylphosphate (pNPP) based assay, as SEAP catalyzes the reaction from pNPP to para-Nitrophenol, this new product absorbs light at 405 nm and can be measured via photometry.

Either BBa_K143130-SEAP encoding plasmid or BBa_K4016011-SEAPencoding plasmid were co-transfected with tetR-3G encoding plasmid into HEK-293 cells. Cells were cultured for 6h before adding 500 ng/ml doxycycline, cells transfected with the same plasmids without doxycycline treatment were used as control. SEAP activity was detected 24/48 hours after transfection.


Result

HEK-293T cells co-transfected with tetR-3G expressing plasmid and either BBa_K4016011-SEAP or BBa_K143130-SEAP plasmid showed significantly boosted SEAP production upon 24h or 48 h of doxycycline induction comparing to their respective control group, in which SEAP activities were below detection limit. This proves that both promoters did initiate the transcription of downstream gene. Intriguingly, under doxycycline induction, HEK-293T cells co-transfected with BBa_K4016011-SEAP and tetR-3G expressing plasmids showed ~30 times higher SEAP activity comparing to cells transfected with same dose of BBa_K143130-SEAP and tetR-3G plasmids, showing a significantly improved performance of Part:BBa_K4016011 comparing to Part:BBa_K143130.


Figure1. Comparation of doxycycline responsiveness between the improved version and the original version of tetO7 promoters. (A)SEAP activity in the culture medium collect from cells 24 hours after 500 ng/ml doxycycline induction. (B)SEAP activity in the culture medium collect from cells 48 hours after 500 ng/ml doxycycline induction. BDL stands for below detection limit. Data represent mean ± s.e.m. (n=3 biological replicates)