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

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<partinfo>BBa_S03595 short</partinfo>
 
<partinfo>BBa_S03595 short</partinfo>
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===Design Notes===
 
===Design Notes===
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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''' [https://parts.igem.org/wiki/index.php/Part:BBa_B0015 BBa_B0015] to insulate a coding region from 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 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
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* [https://parts.igem.org/wiki/index.php/Part:BBa_S03532 BBa_S03532] - contains a tetracycline resistance coding region (backward orientation) with a backwards ribosomal binding site ('''TetB-RBS''')
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* [https://parts.igem.org/wiki/index.php/Part:BBa_J3106 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)
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* [https://parts.igem.org/wiki/index.php/Part:BBa_J44004 BBa_J44004] - contains the pBad promoter in the reverse orientation followed by RBS-TetF (hixC-'''pBad<sub>rev</sub>'''-hixC-'''RBS-TetF''')
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* RFP (red flourescent protein) - contains '''RBS-RFP''' with no promoter
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Note that backwards Tet is also expressed without a promoter. This  suggests that there is also reverse read-through from the carrier vector.
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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. Thus, the '''Double Forward Terminator''' was used to contruct a new cloning vector '''pSB1A4''' ([https://parts.igem.org/wiki/index.php/Part:BBa_J31009 BBa_J31009]).
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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