Difference between revisions of "Part:BBa S03595:Design"

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<partinfo>BBa_S03595 short</partinfo>
 
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===Design Notes===
 
===Design Notes===
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This part was constructed to test the capacity of the Double Forward Terminator [[https://parts.igem.org/wiki/index.php/Part:BBa_B0015 BBa_B0015]] to block read-through transcription.
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The tetracycline resistance coding region (forward orientation) with a ribosomal binding site (RBS-TetF) is expressed (tet resistant) in pSB1A2 and pSB1A3 without a promoter.This, along with other observations, suggests that BioBrick parts cloned in pSB1A2 and pSB1A3 are subject to constitutive read-through transcription coming from the vector backbone (see [https://parts.igem.org/wiki/index.php/Part:BBa_J31009:Design pSB1A4 Part Design] for details). '''BBa_S03539''' was constructed to test the capacity of the double forward terminator [https://parts.igem.org/wiki/index.php/Part:BBa_B0015 BBa_B0015] to insulate a BioBrick part from read-through transcription.  
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Part [[https://parts.igem.org/wiki/index.php/Part:BBa_S03562 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.
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Consistent with this observation, the following parts (which are predicted to not show expression) show expression
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* pBad promoter-Hix C-TetR Backward-Backwards RBS-Hix C-Double Forward Terminator- Recombinational Enhancer: BBA_J3106
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Once the Double Forward Terminator [[https://parts.igem.org/wiki/index.php/Part:BBa_B0015 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.
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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.
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RBS-TetF [https://parts.igem.org/wiki/index.php/Part:BBa_S03567 BBa_S03567] was placed downstream of the double forward terminator [https://parts.igem.org/wiki/index.php/Part:BBa_B0015 BBa_B0015] (TT) in pSB1AK3. Then, TT-RBS-TetF (EcoRI/ PstI) was cloned into pSB1A2. When the TT is upstream of RBS-TetF, the cells are no longer tetracycline resistant. We conclude that the double forward terminator is sufficient to block read-through transcription coming from the vector backbone. Thus, the double forward terminator was used to construct a new cloning vector (pSB1A7 [https://parts.igem.org/wiki/index.php/Part:BBa_J31009 BBa_J31009]) to insulate BioBrick parts from read-through transcription.
  
 
===Source===
 
===Source===

Latest revision as of 18:40, 22 October 2006

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

The tetracycline resistance coding region (forward orientation) with a ribosomal binding site (RBS-TetF) is expressed (tet resistant) in pSB1A2 and pSB1A3 without a promoter.This, along with other observations, suggests that BioBrick parts cloned in pSB1A2 and pSB1A3 are subject to constitutive read-through transcription coming from the vector backbone (see pSB1A4 Part Design for details). BBa_S03539 was constructed to test the capacity of the double forward terminator BBa_B0015 to insulate a BioBrick part from read-through transcription.

RBS-TetF BBa_S03567 was placed downstream of the double forward terminator BBa_B0015 (TT) in pSB1AK3. Then, TT-RBS-TetF (EcoRI/ PstI) was cloned into pSB1A2. When the TT is upstream of RBS-TetF, the cells are no longer tetracycline resistant. We conclude that the double forward terminator is sufficient to block read-through transcription coming from the vector backbone. Thus, the double forward terminator was used to construct a new cloning vector (pSB1A7 BBa_J31009) to insulate BioBrick parts from read-through transcription.

Source

References