Composite

Part:BBa_K5317021

Designed by: Vanessa Bruhn   Group: iGEM24_Hannover   (2024-09-22)
Revision as of 16:55, 23 September 2024 by Vanessa09 (Talk | contribs)


CMV-ATF2-mRuby2

Usage and Biology

ATF2 belongs to the ATF/CREB family and regulates genes involved in cell growth, stress responses and apoptosis. With the addition of the ATF2 gene, this plasmid enables the study of transcriptional regulation of ATF2 (Kirsch et al., 2020) and its phosphorylation by PknB, making it important for research into signaling pathways related to cell stress and survival. It also could play a role in modulating bacterial virulence by altering key regulatory proteins, affecting their role in infection or antibiotic resistance. (Zhang et al., 2020) while mRuby2 provides a fluorescent marker for visualization. In our cell-based & #946;-lactam ring-containing antibiotics sensor, ATF2 serves as a translator of changes in PknB activity at the level of gene regulation, in particular the activity of the ATF2-3xCre2xAP1 promoter.

Cloning

Furthermore, the CMV promoter ensures robust expression in mammalian systems (Radhakrishnan et al., 2008) that can be easily detected and analysed. This composite part was engineered with NEBBuilder® HIFI assembly method. We linearized eGFP-C2 with BamHI and AseI inserting ATF2 and mRuby.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1398
    Illegal EcoRI site found at 1660
    Illegal XbaI site found at 1373
    Illegal XbaI site found at 1701
    Illegal PstI site found at 2108
    Illegal PstI site found at 2557
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1398
    Illegal EcoRI site found at 1660
    Illegal PstI site found at 2108
    Illegal PstI site found at 2557
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1398
    Illegal EcoRI site found at 1660
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1398
    Illegal EcoRI site found at 1660
    Illegal XbaI site found at 1373
    Illegal XbaI site found at 1701
    Illegal PstI site found at 2108
    Illegal PstI site found at 2557
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1398
    Illegal EcoRI site found at 1660
    Illegal XbaI site found at 1373
    Illegal XbaI site found at 1701
    Illegal PstI site found at 2108
    Illegal PstI site found at 2557
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 670
    Illegal SapI.rc site found at 2408

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

Radhakrishnan, P., Basma, H., Klinkebiel, D., Christman, J., & Cheng, P.-W. (2008). Cell type-specific activation of the cytomegalovirus promoter by dimethylsulfoxide and 5-Aza-2’-deoxycytidine. The International Journal of Biochemistry & Cell Biology, 40(9), 1944–1955. https://doi.org/10.1016/j.biocel.2008.02.014

Zhang, C., Zhang, R., Dai, X., Cao, X., Wang, K., Huang, X., & Ren, Q. (2020). Activating transcription factor 2 (ATF2) negatively regulates the expression of antimicrobial peptide genes through tumor necrosis factor (TNF) in Macrobrachium nipponense. Fish & Shellfish Immunology, 107, 26–35. https://doi.org/10.1016/j.fsi.2020.09.043


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