Intermediate

Part:BBa_S03595:Design

Designed by: Karmella Haynes   Group: iGEM06_Davidson   (2006-09-14)
Revision as of 22:52, 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

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 TetR with a ribosomal binding site (RBS-TetR), is sufficient to convey tetracycline resistance in the absence of a promoter. We suspect that TetR might be expressed via 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 RBS-TetR in the reverse orientation with no promoter
  • [BBaJ3106] - contains the pBad promoter in the forward orientation followed by RBS-TetR in the reverse orientation
  • [BBa_S03562] - contains the pBad promoter in the reverse orientation followed by RBS-TetR in the forward orientation
  • RFP (red flourescent protein) - contains RBS-RFP with no promoter

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