Part:BBa_K3505037

pAndersonJ23115:RBS-TetR-terminator- pAndersonJ23115:tetO:RBS-LacI-terminator

AndersonJ23115:TetR:Double Terminator BBa_K3505033 and AndersonJ23115:TetO:RBS-LacI-Double Terminator BBa_K3505034.

Usage and Biology

This parts consists of AndersonJ23115:TetR:Double Terminator BBa_K3505033 and AndersonJ23115:TetO:RBS-LacI-Double Terminator BBa_K3505034. AndersonJ23115 is a strong constitutive promoter and TetR is constitutively expressed. TetR binds to tet operator, and inhibits the expression of LacI. In the presence of tetracycline or strong chemical repressor as anhydrotetracycline (aTC), TetR inhibition is blocked and the transcription of LacI is allowed. In absence of aTC, TetR binds to the tetracycline response element-TRE and inhibits LacI expression. (Gossen and Bujard, 1993).

Design Notes

The coding sequence was domesticated . We removed BsmBI ,BsaI , BtgZI, BpiI sites in order to be compatible with GoldenBraid and MoClo. The sequence is cloned in omega1 vector BBa_K3505010 and has overhangs compatible for GoldenBraid cloning.

Figure 2. The level B module of Tet-Off system : a1R:AndersonJ23115:RBS-TetR-Double terminator-a2:AndersonJ23115:TetO:RBS- LacI-Double terminator

Verification of Cloning

Fig.1:: (U=Uncut , C= Cut) Restriction digestion of omega1R-TetR-LacI (C1-C 4) with :EcoRV +EcoRI(C1-C4) , Expected bands : 4823 + 465bp ,Positive result: C1,C2,C3,C4

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 76
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 11
  Illegal NheI site found at 34
  Illegal NheI site found at 838
  Illegal NheI site found at 861
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 76
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 76
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

• Gossen, M. and Bujard, H., 1993. Anhydrotetracycline, a novel effector for tetracycline controlled gene expression systems in eukaryotic cells. Nucleic Acids Research, 21(18), pp.4411-4412.