Difference between revisions of "Part:BBa K3755010"

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NFAT can bind to this part to activate downstream gene transicription. NFAT is related to calcium signal.
 
NFAT can bind to this part to activate downstream gene transicription. NFAT is related to calcium signal.
  
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<!-- Add more about the biology of this part here
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===Usage and Biology===
 
===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'''.
 +
[[File:NFAT.png|thumb|500px|left|'''Figure1:'''Mechanism of NFATs]]
 +
<br style="clear: both" />
 +
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]].
 +
[[File:PGL4-EGFP.png|thumb|600px|left|'''Figure2:'''Linearized pGL4.30 and EGFP &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; Map of pGL4-EGFP]]
 +
<br style="clear: both" />
  
 +
==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.
 +
[[File:NFAT imaging.png|thumb|800px|left|'''Figure2'''a.Cells with no stimulation and no fluorescent signal is detected by microscope.&nbsp;&nbsp; b.Green channel of the cells 24h after shaking, identifying the expression of EGFP, 40X objective &nbsp;&nbsp; c.Red channel of the cells to identify the Piezo1.1, 40X objective &nbsp;&nbsp; d.Merge green and red channel to colocalize the cells]]
 +
<br style="clear: both" />
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K3755010 parameters</partinfo>
 
<partinfo>BBa_K3755010 parameters</partinfo>
 
<!-- -->
 
<!-- -->
 +
 +
=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.
 +
<div><ul>
 +
<center>
 +
  <li style="display: inline-block;"> [[File:Kill switch circuit-NMU.png|thumb|none|400px|<b></b> kill switch circuit]] </li>
 +
</center>
 +
    </ul></div>
 +
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.
 +
<div><ul>
 +
<center>
 +
  <li style="display: inline-block;"> [[File:Improve design.png|thumb|none|400px|<b></b> ]] </li>
 +
</center>
 +
    </ul></div>
 +
 +
==Result==
 +
<div><ul>
 +
<center>
 +
  <li style="display: inline-block;"> [[File:Improve result.png|thumb|none|600px|<b></b>]] </li>
 +
</center>
 +
    </ul></div>
 +
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

Latest revision as of 16:39, 3 October 2022


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]


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.

  • kill switch circuit

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