Intermediate

Part:BBa_S03595:Design

Designed by: Karmella Haynes   Group: iGEM06_Davidson   (2006-09-14)
Revision as of 17:07, 19 October 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 usefulness of a device depends upon 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_J44004 - contains the pBad promoter in the reverse orientation followed by RBS-TetF (hixC-pBadrev-hixC-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. Thus, the Double Forward Terminator was used to contruct a new cloning vector pSB1A4 (BBa_J31009).

Source

References