Difference between revisions of "Part:pSB1A7:Design"
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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. | 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 | + | The following parts show expression in cloning vectors pSB1A2 and pSB1A3, even in the absence of a promoter (or where the promoter is not in the proper orientation) |
* [https://parts.igem.org/wiki/index.php/Part:BBa_S03562 BBa_S03562] - Promoterless tetracycline resistance coding region (forward orientation) with a ribosomal binding site ('''RBS-TetF''') is expressed (tet resistant) in pSB1A2 and pSB1A3 | * [https://parts.igem.org/wiki/index.php/Part:BBa_S03562 BBa_S03562] - Promoterless tetracycline resistance coding region (forward orientation) with a ribosomal binding site ('''RBS-TetF''') is expressed (tet resistant) in pSB1A2 and pSB1A3 | ||
* [https://parts.igem.org/wiki/index.php/Part:BBa_S03532 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 | * [https://parts.igem.org/wiki/index.php/Part:BBa_S03532 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 | ||
* [https://parts.igem.org/wiki/index.php/Part:BBa_J3106 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 | * [https://parts.igem.org/wiki/index.php/Part:BBa_J3106 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 | ||
| − | * [https://parts.igem.org/wiki/index.php/Part:BBa_J44004 BBa_J44004] - | + | * [https://parts.igem.org/wiki/index.php/Part:BBa_J44004 BBa_J44004] - pBad promoter in the reverse orientation followed by RBS-TetF (hixC-'''pBad<sub>rev</sub>'''-hixC-'''RBS-TetF''') |
| − | + | ||
These observations suggest that there is forward and reverse read-through coming from the backbone of the carrier vector into the BioBrick parts. | 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'''<br> | '''Double Forward and Backwards Terminator Assembly'''<br> | ||
| − | The double forward terminator BBa_B0015 successfully blocks RBS-TetF from read through in | + | The double forward terminator BBa_B0015 successfully blocks RBS-TetF from read through in 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=== | ===Source=== | ||
Revision as of 20:27, 19 October 2006
No part name specified with partinfo tag.
No part name specified with partinfo tag.
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 (or where the promoter is not in the proper orientation)
- 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 - pBad promoter in the reverse orientation followed by RBS-TetF (hixC-pBadrev-hixC-RBS-TetF)
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 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.