Part:BBa_K4414027

tetR-vp64-GGGSG-LBD

This part is an integrated tool for the perception of cortisol stimulation and activates the transcription of the reporter gene.

Usage and Biology

As a glucocorticoid sensor, this part is designed to enter the nucleus upon glucocorticoid stimulation and bind to the TCE promoter to activate downstream transcription. Tet R on the N terminus in our design provides DNA binding domain tightly binding to the downstream gene, which binds to the TCE promoter (BBa_K4016011) consisting of seven direct 19-bp Tet operator sequence (Teto) repeats. VP64 is a transcriptional activator composed of four tandem copies of VP16 connected with glycine-serine (GS) linkers. GGGSG linker, owning some flexibility and allowing the proteins on both sides to complete their own independent functions. The NR3C1 LBD domain on the C terminus is the ligand binding domain of the glucocorticoid receptor (GR). This LBD domain can translocate the fusion protein into the nucleus upon glucocorticoid stimulation. It also has a transactivating domain 2 (τ2) and an activation function domain 2 (AF2) which activates downstream gene expression.[1]

Fugure1.Schematic figure of BBa_K4414027 and BBa_K4414041.

Sequecing

The plasmid was sequenced correct.

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
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 166
  
• 1000
  COMPATIBLE WITH RFC[1000]

Functional test

To test the ability of this part to respond to glucocorticoids, HEK-293T cells were co-transfected with plasmids encoding both Tet R-VP64-1*GS linker-LBD(BBa_K4414027) and TCE-SEAP(BBa_K4414041).

Method

Cells were treated with 0 or 100 nm Glucocorticoids 6h post-transfection. Cells without glucocorticoid treatment were used as control. Culture medium was collected at 24 h post glucocorticoids treatment. SEAP activity was measured according to a published protocol.[2]

Figure2.Schematic representation of the experimental process of validation for BBa_K4414027 and BBa_K4414041.

Result

Results showed similar SEAP expression in glucocorticoid-treated cells compared to the non-treated control (1.15 folds).

Figure3.Glucocorticoid-stimulated transcriptional activation of SEAP mediated by BBa_K4414027.

Reference

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.

2.Shao J, Qiu X, Xie M. Engineering Mammalian Cells to Control Glucose Homeostasis. Methods Mol Biol. 2021; 2312:35-57. doi: 10.1007/978-1-0716-1441-9_3. PMID: 34228283.