Difference between revisions of "Part:BBa K3244013"
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==Improvement background== | ==Improvement background== | ||
In our project, we designed a kill-switch circuit which involved NFAT response element. | 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. | 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> | <div><ul> | ||
<center> | <center> | ||
| − | <li style="display: inline-block;"> [[File:Kill switch circuit-NMU.png|thumb|none| | + | <li style="display: inline-block;"> [[File:Kill switch circuit-NMU.png|thumb|none|400px|<b></b> kill switch circuit]] </li> |
</center> | </center> | ||
</ul></div> | </ul></div> | ||
| − | We searched in the registry for existing part. | + | 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. |
| − | 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_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. | 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== | ==Design== | ||
We performed 33 random mutations on BBa_K3755010. | 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. | 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> | <div><ul> | ||
<center> | <center> | ||
| − | <li style="display: inline-block;"> [[File:Improve design.png|thumb|none| | + | <li style="display: inline-block;"> [[File:Improve design.png|thumb|none|400px|<b></b> ]] </li> |
</center> | </center> | ||
</ul></div> | </ul></div> | ||
| Line 56: | Line 59: | ||
<div><ul> | <div><ul> | ||
<center> | <center> | ||
| − | <li style="display: inline-block;"> [[File:Improve result.png|thumb|none| | + | <li style="display: inline-block;"> [[File:Improve result.png|thumb|none|600px|<b></b>]] </li> |
</center> | </center> | ||
</ul></div> | </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 | >BBa_K3755010 | ||
| + | |||
ggaggaaaaa ctgtttcata cagaaggcgt ggaggaaaaa ctgtttcata cagaaggcgt ggaggaaaaa ctgtttcata cagaaggcgt | ggaggaaaaa ctgtttcata cagaaggcgt ggaggaaaaa ctgtttcata cagaaggcgt ggaggaaaaa ctgtttcata cagaaggcgt | ||
| + | |||
>mutation 13 (26: g→c) | >mutation 13 (26: g→c) | ||
| + | |||
ggaggaaaaa ctgtttcata cagaacgcgt ggaggaaaaa ctgtttcata cagaaggcgt ggaggaaaaa ctgtttcata cagaaggcgt | ggaggaaaaa ctgtttcata cagaacgcgt ggaggaaaaa ctgtttcata cagaaggcgt ggaggaaaaa ctgtttcata cagaaggcgt | ||
Latest revision as of 16:36, 3 October 2022
NFAT-Response Ellement
NFAT RE for cytokine expression regulation
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 6
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
MIT_MAHE 2020
Summary
Nuclear factor of activated T-cells (NFAT) is a family of transcription factors whose activation is controlled by Ca and the Ca/calmodulin-dependent serine phosphatase, calcineurin. It is shown to be important in immune response. NFAT is also involved in the development of cardiac, skeletal muscle, and nervous systems.The molecular functions of this gene include chromatin binding, DNA-binding transcription factor activity, RNA polymerase II cis-regulatory region sequence-specific DNA binding and transcription factor binding while the molecular functions include negative regulation of synaptic vesicle exocytosis, positive regulation of transcription by RNA polymerase II and response to salt stress.
References
1. Freeman, A., Franciscovich, A., Bowers, M., Sandstrom, D. J., & Sanyal, S. (2011). NFAT regulates pre-synaptic development and activity-dependent plasticity in Drosophila. Molecular and cellular neurosciences, 46(2), 535–547. https://doi.org/10.1007/s10930-019-09833-8
2. Keyser, P., Borge-Renberg, K., & Hultmark, D. (2007). The Drosophila NFAT homolog is involved in salt stress tolerance. Insect biochemistry and molecular biology, 37(4), 356–362. https://doi.org/10.1016/j.ibmb.2006.12.009
3. Pascale Gaudet, Michael S. Livstone, Suzanna E. Lewis, Paul D. Thomas, Phylogenetic-based propagation of functional annotations within the Gene Ontology consortium, Briefings in Bioinformatics, Volume 12, Issue 5, September 2011, Pages 449–462, https://doi.org/10.1093/bib/bbr042
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