Part:BBa_K4414041
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 597
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 908
Illegal NgoMIV site found at 1877 - 1000INCOMPATIBLE 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).
Result
The test results are as follows:
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
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