Part:BBa_K3755010
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.
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.
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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
2022 NMU_China Improvement
Improvement background
In our project, we designed a kill-switch circuit which involved NFAT response element.
NFAT serves as a block signal, which can be ignited by antigen stimulation. When the stimulation occurs and the concentration of NFAT meets the requirements, Gal4-KRAB should be produced quickly to block the promoter of the suicide gene, inhibiting the suicide process. So we require our NFAT response element to be of high response strength.
We searched in the registry for existing part. BBa_K3244013 designed by iGEM19_AFCM-Egypt and BBa_K3755010 designed by iGEM21_ShanghaiTech_China were available for us to improve.
In Part:BBa_K3244013, they generally introduced the biological background of NFAT response element, but with no experiment results.
In Part:BBa_K3755010, they cotransfected NFAT-RE+minP+EGFP(Part:BBa_K3755014) and Piezo1.1(Part:BBa_K3755006,calcium signal provider) into HEK 293 cells and through fluorescence colocalization, they observed some cells had produced EGFP successfully, which meant NFAT and NFAT response element functioned well.
Design
We performed 33 random mutations on BBa_K3755010.
Note: They replaced the luciferase in pGL4.30 with EGFP for lack of luciferin; while we took the original pGL4.30 to carry out the experiments.
Result
Taking BBa_K3755010 as control, from the figure, we can clearly see that the response strength of mutation 13 is significantly higher than the control and other mutants, promising to achieve ideal results in our experiment.
>BBa_K3755010
ggaggaaaaa ctgtttcata cagaaggcgt ggaggaaaaa ctgtttcata cagaaggcgt ggaggaaaaa ctgtttcata cagaaggcgt
>mutation 13 (26: g→c)
ggaggaaaaa ctgtttcata cagaacgcgt ggaggaaaaa ctgtttcata cagaaggcgt ggaggaaaaa ctgtttcata cagaaggcgt
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