Composite

Part:BBa_K1132022:Design

Designed by: iGEM Toulouse   Group: iGEM13_INSA_Toulouse   (2013-09-20)

BBa_J23116-TetR-pTet-RFP


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1526
    Illegal AgeI site found at 1638
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The couple TetR-pTet system was already described in the litterature. This construction is a first step to characterize the entire system. A constitutive promoter (BBa_J23116) was assemble to tetR and then assemble to pTet-rbs-RFP-term. pTet is a constitutive promoter. TetR is an inducer that binds to pTet promoter and thus, represses expression the downstream system. Supply of tetracycline or its analog aTc (anhydrotetracycline) is known to bind to tetR and invert the operation (inhibits expression of mRFP in this case).



After 18 hours, clones containing the plasmid (BBa_J23116-TetR-pTet-RFP) show a leaky basal expression of mRFP. We suppose that the promoter was too weak to express TetR in large quantity. Assembly of a stronger promoter could improve the system and lock the response to an ON/OFF response.



Besides, an experience was done to analyze the effect of the aTc inducer. Result show a visible induction of the red fluorescent protein expression by addition of aTC (60 ng/mL).


Source

BBa_J23116
BBa_P0440
BBa_I13521

References