Regulatory

Part:BBa_K3505014

Designed by: Asteria Tsapadikou   Group: iGEM20_Thessaly   (2020-10-20)
Revision as of 16:36, 27 October 2020 by Asteriat (Talk | contribs)


AndersonJ23115:TetO:RBS GB compatible with A1-B2

Constitutive Anderson BBa_J23115with Tet Operator and RBS BBa_B0034

Usage and Biology

This part is a constitutive promoter from Andesron family , J23115 , having downstream with a Τet Operator and RBS. The activity of this part is regulated by TetR BBa_K3505003. TetR binds to TRE -tetracycline response element, and inhibits the expression of the desirable transcription unit. This part can be used in a Tet-off system, expression of Tet-regulated genes can be inhibited by tetracycline and its chemical derivatives , such as anhydrotetracycline aTc-a strong chemical repressor , that binds to TRE and the desirable transcription unit is blocked.




Design Notes

The sequence was domesticated . We removed BsmBI ,BsaI , BtgZI, BpiI sites in order to be compatible with GoldenBraid and MoClo. The sequence is cloned in pUPD2 BBa_K3505007 and has overhangs compatible for Golden Braid cloning. This has position A1-B3.

Fig.1:The overhangs of this part in the Golden Braid Grammar
Figure 2. The level 0 module : pupd2- AndersonJ23115:TetO:RBS (illustration from SnapGene)

Verification of cloning

Fig.1:Figure 7: (U=Uncut , C= Cut) Restriction digestion of TetO (C1-C4 ) with :Not-HF(C1-C4) , Expected bands : 2046 + 129 bp, Positive result: C1,C2,C4

Experimental Use and Experience

This part is used in BBa_K3505034

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 11
    Illegal NheI site found at 34
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]
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Categories
Parameters
None