Intermediate

Part:BBa_S03595:Design

Designed by: Karmella Haynes   Group: iGEM06_Davidson   (2006-09-14)
Revision as of 18:43, 16 September 2006 by Kahaynes (Talk | contribs) (→‎Design Notes)


TT : RBS-TetF


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 302
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 448
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 474
    Illegal NgoMIV site found at 842
    Illegal NgoMIV site found at 1002
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Read-through transcription is extremely problematic when the utility of a device requires an off state. This part was constructed to test the capacity of the Double Forward Terminator BBa_B0015 to insulate a coding region from read-through transcription.


Part BBa_S03562, which contains a tetracycline resistance coding region (forward orientation) with a ribosomal binding site (RBS-TetF), is sufficient to convey tetracycline resistance in the absence of a promoter. We suspect that Tet might be expressed via forward read-through transcription from the carrier vector (pSB1A2 and pSB1A3).


Consistent with this observation, the following parts, which are predicted to not show expression, do show expression

  • BBa_S03532 - contains a tetracycline resistance coding region (backward orientation) with a backwards ribosomal binding site (TetB-RBS)
  • BBa_J3106 - contains the pBad promoter in the forward orientation followed by TetB-RBS in the reverse orientation (pBad-hixC-TetB-RBS-hixC-TT-RE)
  • BBa_S03562 - contains the pBad promoter in the reverse orientation followed by RBS-TetF (pBadreverse-RBS-TetF)
  • RFP (red flourescent protein) - contains RBS-RFP with no promoter

Note that backwards Tet is also expressed without a promoter. This suggests that there is also reverse read-through from the carrier vector.


Once the Double Forward Terminator BBa_B0015 is placed upstream of RBS-TetR, the cells are no longer tetracycline resistant. We conclude that the Double Forward Terminator is sufficient to block read-through transcription.


Construction of an "insulator vector" where the double terminator is placed before the BioBrick prefix and after the BioBrick suffix (in the reverse orientation) is currently under way. This vector should be able to insulate devices from read-through allowing total control over expression within the device.

Source

References