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

(→‎Design Notes)
 
(11 intermediate revisions by the same user not shown)
Line 1: Line 1:
−
 
 
__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_S03595 short</partinfo>
 
<partinfo>BBa_S03595 short</partinfo>
Line 7: Line 6:
  
 
===Design Notes===
 
===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''' [https://parts.igem.org/wiki/index.php/Part:BBa_B0015 BBa_B0015] to insulate a coding region from read-through transcription.
+
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.  
−
 
+
−
 
+
−
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
+
−
* [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''')
+
−
* [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)
+
−
* [https://parts.igem.org/wiki/index.php/Part:BBa_S03562 BBa_S03562] - contains the pBad promoter in the reverse orientation followed by RBS-TetF ('''pBad<sub>reverse</sub>-RBS-TetF''')
+
−
* RFP (red flourescent protein) - contains '''RBS-RFP''' with no promoter
+
−
Note that backwards Tet is also expressed without a promoter. This  suggests that there is also reverse read-through from the carrier vector.
+
−
 
+
−
 
+
−
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.
+
−
 
+
  
−
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.
+
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