Regulatory

Part:BBa_K3755010

Designed by: Kaijun Wang   Group: iGEM21_ShanghaiTech_China   (2021-09-30)
Revision as of 15:29, 9 October 2021 by Arisaema (Talk | contribs) (→‎Fluorescence colocalization)


NFAT response element

NFAT can bind to this part to activate downstream gene transicription. NFAT is related to calcium signal.


Usage and Biology

NFAT response element

NFATs are transcription factors involved in immune system regulation, development, cancer progression, and apoptosis. NFATs were found in T cells at first, and they are expressed by a variety of cells. Among the five NFAT paralogues (NFAT1–5), NFAT1–4 are present in the cytoplasm as hyperphosphorylated forms in the basal state(low intracellular calcium concentration). When the calcium concentration improves, NFATs will be dephosphorylated through the calmodulin/calcineurin pathway. The dephosphorylated NFAT will expose its NLS and then transform into the nucleus. In the nucleus, NFATs can combine with a specific DNA sequence, which is what we called NFAT- response element.

Figure1:Mechanism of NFATs


So NFAT response element is a cis-acting element. This part includes three copies of the sequence which is the cole region of IL-2 promoter. This part is on a commercial plasmid pGL4.30[luc2P/NFAT-RE/Hygro].

Experiment and result

We replaced the luciferase in pGL4.30 with EGFP, because our lab doesn't have luciferin. The cole region of this constructed plasmid is handed in as Part:BBa_K3755014.

Figure2:Linearized pGL4.30 and EGFP                                                       Map of pGL4-EGFP


Fluorescence colocalization

We cotransfected NFAT-RE+minP+EGFP(Part:BBa_K3755014) and Piezo1.1(Part:BBa_K3755006) into HEK 293 cells. Piezo1.1 is a mechanosensitive calcium channel, which can provide the calcium signal to activate NFAT. We applied mechanical stimulation by placing it on a horizontal shaker for 10min, 200rpm. After 24 hours, we observed the cells again and noticed that some cells had produced EGFP successfully.

Figure2a.Cells with no stimulation and no fluorescent signal is detected by microscope.   b.Green channel of the cells 24h after shaking, identifying the expression of EGFP, 40X objective    c.Red channel of the cells to identify the Piezo1.1, 40X objective    d.Merge green and red channel to colocalize the cells


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]


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Categories
Parameters
None