Plasmid_Backbone
pSB1A7

Part:pSB1A7:Design

Designed by: Karmella Haynes   Group: iGEM06_Davidson   (2006-10-19)
Revision as of 20:14, 19 October 2006 by Kahaynes (Talk | contribs) (→‎Design Notes)


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Design Notes

Purpose
Read-through transcription is extremely problematic when the usefulness of a device depends upon an off state. This cloning vector was constructed to insulate BioBrick parts from read-through transcription coming from the backbone.

The following parts show expression in cloning vectors pSB1A2 and pSB1A3 even in the absence of a promoter

  • BBa_S03562 - Promoterless tetracycline resistance coding region (forward orientation) with a ribosomal binding site (RBS-TetF) is expressed (tet resistant) in pSB1A2 and pSB1A3
  • BBa_S03532 - Promoterless tetracycline resistance coding region (backward orientation) with a backwards ribosomal binding site (TetB-RBS) is expressed (tet resistant) in pSB1A2 and pSB1A3
  • BBa_J3106 - pBad promoter in the forward orientation followed by TetB-RBS in the reverse orientation (pBad-hixC-TetB-RBS-hixC-TT-RE) is expressed (tet resistant) in pSB1A2
  • 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

These observations suggest that there is forward and reverse read-through coming from the backbone of the carrier vector into the BioBrick parts.

Double Forward and Backwards Terminator Assembly
The double forward terminator BBa_B0015 successfully blocks RBS-TetF from read through in pB1AK3 and pSB1A2 (no tet resistance) (see The double terminators were assembled from smaller overlapping ssDNA oligos. The outer-most EcoRI and PstI sites were mutated while the inner-most BioBrick cut sites were restored.

Source

Based upon pSB1A3. Includes two double terminators assembled from DNA oligos based upon the sequence of BBa_B0015.

References