Difference between revisions of "Part:BBa K4414041"
 
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<partinfo>BBa_K4414041 short</partinfo>
 
<partinfo>BBa_K4414041 short</partinfo>
  
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This composite part consists of a TCE promoter(BBa_K4016011) and a coding sequence of SEAP(BBa-K1470004). This is a reporter part that is used to make a response to stimulation of upstream genes.
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This composite part consists of a TCE promoter([[Part:BBa_K4016011]]) and a coding sequence of SEAP([[Part:BBa_K1470004]]). This is a reporter part that is used to make a response to stimulation of upstream genes.
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==Usage and Biology==
 
==Usage and Biology==
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TCE 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 TCE and activates transcription of the downstream gene of interest (GOI). TCE is the improvement of TRE (BBa_K1431301), The improved TCE promoter shares a similar nucleic acid sequence to the original promoter in TRE, with only a few nucleotides different on the flanking regions of tetO. However, these differences were sufficient to enhance the expression level of GOI.
 
  
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SEAP (Secreted Alkaline Phosphatase) is a hydrolyase which derives from the human placental alkaline phosphatase and is secreted into the medium by the cell. SEAP is an ideal reporter protein and a significant tool for eucaryotic promotor studies and part of many commercial kits. The big advantage is its self-excretion into the medium. It can be easily measured with para-nitrophenylphosphate(pNPP) as a substrate. Cleavaeging the phosphate group leads to the formation of para-nitrophenol (pNP), which can be detected with light of 405 nm wave length.
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TCE 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 TCE and activates transcription of the downstream gene of interest (GOI). TCE is the improvement of TRE ([[Part:BBa_K1431301]]), The improved TCE promoter shares a similar nucleic acid sequence to the original promoter in TRE, with only a few nucleotides different on the flanking regions of tetO. However, these differences were sufficient to enhance the expression level of GOI.
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In this expression system , SEAP is equivalent to downstream GOI . When Tet-On 3G binds specifically to TCE, it activates transcription of the SEAP. In our project, we used it as a downstream part of the response to cortisol stimulation. With the increase of cortisol concentration, using pNPP as a substrate, the activity of SEAP can be easily detected by detecting pNP formed by phosphate group cleavage under 405 nm wavelength light, so as to know the response of upstream genes to cortisol.
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 +
SEAP (Secreted Alkaline Phosphatase) is a hydrolyase which derives from the human placental alkaline phosphatase and is secreted into the medium by the cell. SEAP is an ideal reporter protein and a significant tool for eucaryotic promotor studies and part of many commercial kits. The big advantage is its self-excretion into the medium. It can be easily measured with para-nitrophenylphosphate(pNPP) as a substrate. Cleavaeging the phosphate group leads to the formation of para-nitrophenol (pNP), which can be detected with light of 405 nm wave length(Berger, Hauber, Hauber, Geiger, & Cullen, 1988).
 +
In this expression system , SEAP is equivalent to downstream GOI . When Tet-On 3G binds specifically to TCE, it activates transcription of the SEAP. (Figure 1)
 +
In our project, we used it as a downstream part of the response to cortisol stimulation. With the increase of cortisol concentration, using pNPP as a substrate, the activity of SEAP can be easily detected by detecting pNP formed by phosphate group cleavage under 405 nm wavelength light, so as to know the response of upstream genes to cortisol(Shao, Qiu, & Xie, 2021).
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<img src="https://static.igem.org/mediawiki/parts/1/17/T--NUDT_CHINA--Part_PixD-PixE_Schematic.png" class="figure-img img-fluid rounded"  height="250px">
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<img src="https://static.igem.wiki/teams/4414/wiki/041-1.png" class="figure-img img-fluid rounded"  height="300px">
  
 
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Figure1. Schematic figure of PixE-PixD interaction under blue light stimulation
 
  
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Figure 1: This is a model for the major functions of TCE-SEAP.When Tet-On 3G binds specifically to TCE, it activates transcription and translation of SEAP.
  
  
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<span class='h3bb'>Sequence and Features</span>
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===Sequence and Features===
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<partinfo>BBa_K4016001 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K4414041 SequenceAndFeatures</partinfo>
  
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K4016001 parameters</partinfo>
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<partinfo>BBa_K4414041 parameters</partinfo>
 
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==Functional Validation==
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To test the feasibility and specific effect of this part for constructing the gene pathway of our project, we cotransfected a plasmid with the upstream gene ([[Part:BBa_K4414044]]) and a plasmid with TCE-SEAP into HEK-293T cells.
  
 
===Method===
 
===Method===
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To test the feasibility and specific effect of this part for constructing the gene pathway of our project, we cotransfected a plasmid with the upstream gene ([[Part:BBa_K4414044]]) and a plasmid with TCE-SEAP into HEK-293T cells. Cells were treated with 2,5,10,15,20,30,40 or 50 nM Glucocorticoids 6 h post-transfection. Cells without glucocorticoid treatment were used as control. Culture medium was collected at 24 h or 48 h post glucocorticoids treatment. SEAP activity was measured according to a published protocol as above (Shao, Qiu, & Xie, 2021).
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<figure class="figure">
 
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<img src="https://static.igem.org/mediawiki/parts/6/65/T--NUDT_CHINA--Part_Validation_SEAP_PixE-PixD.png
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<img src="https://static.igem.wiki/teams/4414/wiki/41-22.png" class="figure-img img-fluid rounded"  height="350px">
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" class="figure-img img-fluid rounded"  height="350px">
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Figure 2: Cotransfected our upstream plasmid with the upstream gene and a plasmid with TCE-SEAP into cells
  
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To test the feasibility and specific effect of this part for constructing the gene pathway of our project, we cotransfected a plasmid with the upstream gene (BBa_K4414044) and a plasmid with TCE-SEAP into HEK-293T cells. Cells were treated with 2,5,10,15,20,30,40 or 50 nM Glucocorticoids 6 h post-transfection. Cells without glucocorticoid treatment were used as control. Culture medium was collected at 24 h or 48 h post glucocorticoids treatment. SEAP activity was measured according to a published protocol as above .
 
  
 
===Result===
 
===Result===
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<img src="https://2021.igem.org/wiki/images/d/d6/T--NUDT_CHINA--Part_Result_00-01_.png
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<img src="https://static.igem.wiki/teams/4414/wiki/041-3.png" class="figure-img img-fluid rounded"  height="350px">
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Figure 2 Result of SEAP test. The SEAP activity was calculated at 24h and 48h after transfection.
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Figure 3. Result of SEAP test. The SEAP activity was calculated at 24h after transfection.
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<img src="https://static.igem.wiki/teams/4414/wiki/041-4.png" class="figure-img img-fluid rounded"  height="350px">
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Figure 4. Result of SEAP test. The SEAP activity was calculated at 24h after transfection
  
 
The results showed that SEAP expression increased significantly with increasing glucocorticoid concentration in treated cells. A dose dependence was observed within 0-50 nM of glucocorticoid.
 
The results showed that SEAP expression increased significantly with increasing glucocorticoid concentration in treated cells. A dose dependence was observed within 0-50 nM of glucocorticoid.
  
 
As it turns out, this part works .Future iGEM teams can use this part by simply replace the upstream part to transform the abstract into concrete work as we did.
 
As it turns out, this part works .Future iGEM teams can use this part by simply replace the upstream part to transform the abstract into concrete work as we did.
 +
  
 
===Reference===
 
===Reference===
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[1] Weikum ER, Knuesel MT, Ortlund EA, Yamamoto KR. Glucocorticoid receptor control of transcription: precision and plasticity via allostery. Nat Rev Mol Cell Biol. 2017 Mar;18(3):159-174. doi: 10.1038/nrm.2016.152. Epub 2017 Jan 5. PMID: 28053348; PMCID: PMC6257982.
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1. Berger, J., Hauber, J., Hauber, R., Geiger, R., & Cullen, B. R. (1988). Secreted placental alkaline phosphatase: a powerful new quantitative indicator of gene expression in eukaryotic cells. Gene, 66(1), 1-10. doi:10.1016/0378-1119(88)90219-3
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[2] Secreted placental alkaline phosphatase: a powerful new quantitative indicator of gene expression in eukaryotic cells., J. Berger, J. Hauber, R. Hauber, R. Geiger, B. R. Cullen, Gene. 1988 June 15; 66(1): 1–10.
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2. Shao, J., Qiu, X., & Xie, M. (2021). Engineering Mammalian Cells to Control Glucose Homeostasis. Methods Mol Biol, 2312, 35-57. doi:10.1007/978-1-0716-1441-9_3

Latest revision as of 18:01, 11 October 2022


TCE-SEAP

This composite part consists of a TCE promoter(Part:BBa_K4016011) and a coding sequence of SEAP(Part:BBa_K1470004). This is a reporter part that is used to make a response to stimulation of upstream genes.

Usage and Biology

TCE 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 TCE and activates transcription of the downstream gene of interest (GOI). TCE is the improvement of TRE (Part:BBa_K1431301), The improved TCE promoter shares a similar nucleic acid sequence to the original promoter in TRE, with only a few nucleotides different on the flanking regions of tetO. However, these differences were sufficient to enhance the expression level of GOI.

SEAP (Secreted Alkaline Phosphatase) is a hydrolyase which derives from the human placental alkaline phosphatase and is secreted into the medium by the cell. SEAP is an ideal reporter protein and a significant tool for eucaryotic promotor studies and part of many commercial kits. The big advantage is its self-excretion into the medium. It can be easily measured with para-nitrophenylphosphate(pNPP) as a substrate. Cleavaeging the phosphate group leads to the formation of para-nitrophenol (pNP), which can be detected with light of 405 nm wave length(Berger, Hauber, Hauber, Geiger, & Cullen, 1988). In this expression system , SEAP is equivalent to downstream GOI . When Tet-On 3G binds specifically to TCE, it activates transcription of the SEAP. (Figure 1) In our project, we used it as a downstream part of the response to cortisol stimulation. With the increase of cortisol concentration, using pNPP as a substrate, the activity of SEAP can be easily detected by detecting pNP formed by phosphate group cleavage under 405 nm wavelength light, so as to know the response of upstream genes to cortisol(Shao, Qiu, & Xie, 2021).


Figure 1: This is a model for the major functions of TCE-SEAP.When Tet-On 3G binds specifically to TCE, it activates transcription and translation of SEAP.



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 597
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 908
    Illegal NgoMIV site found at 1877
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1238
    Illegal BsaI.rc site found at 1727



Functional Validation

To test the feasibility and specific effect of this part for constructing the gene pathway of our project, we cotransfected a plasmid with the upstream gene (Part:BBa_K4414044) and a plasmid with TCE-SEAP into HEK-293T cells.

Method

To test the feasibility and specific effect of this part for constructing the gene pathway of our project, we cotransfected a plasmid with the upstream gene (Part:BBa_K4414044) and a plasmid with TCE-SEAP into HEK-293T cells. Cells were treated with 2,5,10,15,20,30,40 or 50 nM Glucocorticoids 6 h post-transfection. Cells without glucocorticoid treatment were used as control. Culture medium was collected at 24 h or 48 h post glucocorticoids treatment. SEAP activity was measured according to a published protocol as above (Shao, Qiu, & Xie, 2021).


Figure 2: Cotransfected our upstream plasmid with the upstream gene and a plasmid with TCE-SEAP into cells


Result

The test results are as follows:

Figure 3. Result of SEAP test. The SEAP activity was calculated at 24h after transfection.

Figure 4. Result of SEAP test. The SEAP activity was calculated at 24h after transfection

The results showed that SEAP expression increased significantly with increasing glucocorticoid concentration in treated cells. A dose dependence was observed within 0-50 nM of glucocorticoid.

As it turns out, this part works .Future iGEM teams can use this part by simply replace the upstream part to transform the abstract into concrete work as we did.


Reference

1. Berger, J., Hauber, J., Hauber, R., Geiger, R., & Cullen, B. R. (1988). Secreted placental alkaline phosphatase: a powerful new quantitative indicator of gene expression in eukaryotic cells. Gene, 66(1), 1-10. doi:10.1016/0378-1119(88)90219-3

2. Shao, J., Qiu, X., & Xie, M. (2021). Engineering Mammalian Cells to Control Glucose Homeostasis. Methods Mol Biol, 2312, 35-57. doi:10.1007/978-1-0716-1441-9_3