Intermediate

Part:BBa_S03595:Design

Designed by: Karmella Haynes   Group: iGEM06_Davidson   (2006-09-14)
Revision as of 22:29, 14 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

This part was constructed to test the capacity of the Double Forward Terminator [BBa_B0015] to block read-through transcription.

Part [BBa_S03562], which contains TetR with a ribosomal binding site, is sufficient to convey tetracycline resistance in the absence of a promoter. We suspected that TetR might be expressed via read-through transcription from the carrier vector.

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

  • pBad promoter-Hix C-TetR Backward-Backwards RBS-Hix C-Double Forward Terminator- Recombinational Enhancer: BBA_J3106

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, allowing control over the expression of the downstream coding region.

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.

Source

References