Part:BBa_K3978050
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
- 10INCOMPATIBLE 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 - 12INCOMPATIBLE 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 - 21INCOMPATIBLE 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 - 23INCOMPATIBLE 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 - 25INCOMPATIBLE 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 - 1000INCOMPATIBLE 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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