Part:BBa_K3755014

NFAT-RE+minP+EGFP

This composite part is designed to prove that the calcium signal activates downstream gene expression.

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].

minP(Part:BBa_M50098)

minP is a mammalian constitutive promoter with low activity. It has been used as a standard promoter in some commercial plasmids, like pGL4. You can insert cis-acting elements in front of it to construct a conditionally active promoter. For example, NFAT response element was inserted in front of minP, which constitutes a calcium response promoter in this composite part.

Experiment and result

We used Gibson assembly to replace the luciferase with EGFP in pGL4.30[luc2P/NFAT-RE/Hygro].

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

Fluorescence colocalization

We cotransfected NFAT-RE+minP+EGFP 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 as we expected.

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]