Coding

Part:BBa_K5317016

Designed by: Vanessa Bruhn   Group: iGEM24_Hannover   (2024-09-22)
Revision as of 15:50, 30 September 2024 by Vanessa09 (Talk | contribs) (→‎Characterization)


ATF2

Usage and Biology

ATF2 belongs to the ATF/CREB family and regulates genes involved in cell growth, stress responses and apoptosis. The ATF-2 protein is DNA-binding that binds to cyclic AMP-response elements (CREs), thereby forming a homodimer or heterodimer with c-Jun. It then stimulates CRE-dependent transcription (Kawasaki et al. , 2000)

We employed ATF2 as a transcription factor in our cell-based beta-lactam-sensor, which in contrast to CcpA (K5317014) or GraR (K5317015) originates from an eukaryotic background, to transfer the PknB-detected signal into reporter gene expression.

Cloning

Theoretical Part Design

The ATF2 gene was synthesized, and the gene sequence was explicity chose from its cDNA to exclude intons and shorten the gene sequence inserted into the final plasmid for characterization (K5317021).

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 21
    Illegal EcoRI site found at 283
    Illegal XbaI site found at 324
    Illegal PstI site found at 731
    Illegal PstI site found at 1180
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 21
    Illegal EcoRI site found at 283
    Illegal PstI site found at 731
    Illegal PstI site found at 1180
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 21
    Illegal EcoRI site found at 283
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 21
    Illegal EcoRI site found at 283
    Illegal XbaI site found at 324
    Illegal PstI site found at 731
    Illegal PstI site found at 1180
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 21
    Illegal EcoRI site found at 283
    Illegal XbaI site found at 324
    Illegal PstI site found at 731
    Illegal PstI site found at 1180
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1031

Characterization

The functionality of ATF2 in our cell-based sensor was assessed by analysing its general expression after transfection in HEK293T cells and assessing its localization by linking ATF2 with the reporter gene mRuby2 (K5317001). For results please visit the registry entry K5317021.

References

Kirsch, K., Zeke, A., Tőke, O., Sok, P., Sethi, A., Sebő, A., Kumar, G. S., Egri, P., Póti, Á. L., Gooley, P., Peti, W., Bento, I., Alexa, A., & Reményi, A. (2020). Co-regulation of the transcription controlling ATF2 phosphoswitch by JNK and p38. Nature Communications, 11(1), 5769. https://doi.org/10.1038/s41467-020-19582-3

Kawasaki, H., Schiltz, L., Chiu, R., Itakura, K., Taira, K., Nakatani, Y., & Yokoyama, K. K. (2000). ATF-2 has intrinsic histone acetyltransferase activity which is modulated by phosphorylation. 'Nature', 405(6783), 195–200. https://doi.org/10.1038/35012097


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