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.

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

>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