Composite

Part:BBa_K3978050

Designed by: Minqi Hu   Group: iGEM21_HiZJU-China   (2021-10-21)


reget-kill switch

This part acts as a killing switch that is induced by estrogen like EE2 and designed for E.coli. The engineered bacteria with this switch will die automatically when the EE2 is completely degraded. CⅠDN is a mutant of CI repressor bearing a mutation in the DNA binding surface which eliminated the binding affinity of the cⅠ mutant to operator sites but increased the affinity to non-specific DNA binding site. When the transcription starts, with a stronger RBS, many more CⅠDN molecules are present in the cell compared to CI molecules. Therefore, almost all of the monomers will form heterodimers with CⅠDN, and there will be no CI2 to activate PRM promoter. After the induction period, because of the degradation tag added with CⅠDN to ensure quick degradation of the monomeric proteins, CⅠDN molecules degrade quickly giving CI molecules a chance to form dimers and activate PRM promoter leading to the expression of mazF. It consists of four parts. The first one(BBa_K3978049) is a detection part, an estrogen receptor to detect estrogen and bind to the promoter. The second(BBa_K3978048) one is the controlling part. The third one(BBa_K3978047) is the killing part which express the toxic protein MazF leading to cell death. The fourth one is the degradation part. This part codes the protease ClpX which can degrade protein with ssrA tail, CIDN-ssrA in this switch.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 112
    Illegal EcoRI site found at 5428
    Illegal EcoRI site found at 8740
    Illegal EcoRI site found at 15123
    Illegal EcoRI site found at 21537
    Illegal EcoRI site found at 21882
    Illegal EcoRI site found at 22232
    Illegal XbaI site found at 30
    Illegal XbaI site found at 7054
    Illegal XbaI site found at 8110
    Illegal XbaI site found at 8479
    Illegal XbaI site found at 8658
    Illegal XbaI site found at 14085
    Illegal XbaI site found at 15041
    Illegal PstI site found at 131
    Illegal PstI site found at 8759
    Illegal PstI site found at 15142
    Illegal PstI site found at 22251
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 112
    Illegal EcoRI site found at 5428
    Illegal EcoRI site found at 8740
    Illegal EcoRI site found at 15123
    Illegal EcoRI site found at 21537
    Illegal EcoRI site found at 21882
    Illegal EcoRI site found at 22232
    Illegal PstI site found at 131
    Illegal PstI site found at 8759
    Illegal PstI site found at 15142
    Illegal PstI site found at 22251
    Illegal NotI site found at 149
    Illegal NotI site found at 8777
    Illegal NotI site found at 15160
    Illegal NotI site found at 22269
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 112
    Illegal EcoRI site found at 5428
    Illegal EcoRI site found at 8740
    Illegal EcoRI site found at 15123
    Illegal EcoRI site found at 21537
    Illegal EcoRI site found at 21882
    Illegal EcoRI site found at 22232
    Illegal BglII site found at 305
    Illegal BglII site found at 5704
    Illegal BglII site found at 7789
    Illegal BglII site found at 7834
    Illegal BglII site found at 7882
    Illegal BglII site found at 8065
    Illegal BglII site found at 8101
    Illegal BamHI site found at 106
    Illegal BamHI site found at 8734
    Illegal BamHI site found at 15117
    Illegal BamHI site found at 22226
    Illegal XhoI site found at 354
    Illegal XhoI site found at 8982
    Illegal XhoI site found at 15365
    Illegal XhoI site found at 22474
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 112
    Illegal EcoRI site found at 5428
    Illegal EcoRI site found at 8740
    Illegal EcoRI site found at 15123
    Illegal EcoRI site found at 21537
    Illegal EcoRI site found at 21882
    Illegal EcoRI site found at 22232
    Illegal XbaI site found at 30
    Illegal XbaI site found at 7054
    Illegal XbaI site found at 8110
    Illegal XbaI site found at 8479
    Illegal XbaI site found at 8658
    Illegal XbaI site found at 14085
    Illegal XbaI site found at 15041
    Illegal PstI site found at 131
    Illegal PstI site found at 8759
    Illegal PstI site found at 15142
    Illegal PstI site found at 22251
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 112
    Illegal EcoRI site found at 5428
    Illegal EcoRI site found at 8740
    Illegal EcoRI site found at 15123
    Illegal EcoRI site found at 21537
    Illegal EcoRI site found at 21882
    Illegal EcoRI site found at 22232
    Illegal XbaI site found at 30
    Illegal XbaI site found at 7054
    Illegal XbaI site found at 8110
    Illegal XbaI site found at 8479
    Illegal XbaI site found at 8658
    Illegal XbaI site found at 14085
    Illegal XbaI site found at 15041
    Illegal PstI site found at 131
    Illegal PstI site found at 8759
    Illegal PstI site found at 15142
    Illegal PstI site found at 22251
    Illegal NgoMIV site found at 324
    Illegal NgoMIV site found at 671
    Illegal NgoMIV site found at 5348
    Illegal NgoMIV site found at 8952
    Illegal NgoMIV site found at 9299
    Illegal NgoMIV site found at 13976
    Illegal NgoMIV site found at 15335
    Illegal AgeI site found at 21198
    Illegal AgeI site found at 21936
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 7949
    Illegal BsaI.rc site found at 1256
    Illegal BsaI.rc site found at 9884
    Illegal BsaI.rc site found at 16267
    Illegal BsaI.rc site found at 23376
    Illegal SapI.rc site found at 2916
    Illegal SapI.rc site found at 11544
    Illegal SapI.rc site found at 17927
    Illegal SapI.rc site found at 25036


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