Difference between revisions of "Part:BBa K598001:Design"
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===Design Notes===
 
===Design Notes===
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The RBS of this device is altered into AGGAGGU. When changing the RBS, the anti-RBS sequence should be changed as well.
 
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Theophylline riboswich is composed of a synthesized theophylline aptamer and a regulatory platform.
 
  
  
 +
We optimized original RBS sequence of P1G1 to AGGAGGU, the consensus RBS sequence predicted to initiate high rate of translation according to the RBS calculator. Simultaneously, as we positioned RNA controller 1G1 upstream of GFP coding sequence by standard assembly, the spacing between RBS and AUG strat codon is ACUAG (scar generated during standard assembly). The modification of RBS sequence and spacing between RBS and AUG start codon had inappreciable influence on the core secondary stem-loop structure of functional original riboswitch and almost preserves the quondam base-pairing region.
  
 
===Source===
 
===Source===
 
Use mutagenesis PCR of [https://parts.igem.org/wiki/index.php?title=Part:BBa_K598007 BBa_K598007] to alter RBS sequence.
 
Use mutagenesis PCR of [https://parts.igem.org/wiki/index.php?title=Part:BBa_K598007 BBa_K598007] to alter RBS sequence.
 +
Engineered RBS is selected from constitutive library based on RBS calculator.
  
 
===References===
 
===References===
 
Beatrix Suess, Barbara Fink, Christian Berens, Régis Stentz and Wolfgang Hillen. (2004) A theophylline responsive riboswitch based on helix slipping controls gene expression in vivo. Nucleic Acids Research, 32, 1610-1614.
 
Beatrix Suess, Barbara Fink, Christian Berens, Régis Stentz and Wolfgang Hillen. (2004) A theophylline responsive riboswitch based on helix slipping controls gene expression in vivo. Nucleic Acids Research, 32, 1610-1614.

Latest revision as of 19:29, 5 October 2011

Theophylline Responsive Riboswitch 1G1 with Engineered RBS+GFP generator


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 71
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 730


Design Notes

We optimized original RBS sequence of P1G1 to AGGAGGU, the consensus RBS sequence predicted to initiate high rate of translation according to the RBS calculator. Simultaneously, as we positioned RNA controller 1G1 upstream of GFP coding sequence by standard assembly, the spacing between RBS and AUG strat codon is ACUAG (scar generated during standard assembly). The modification of RBS sequence and spacing between RBS and AUG start codon had inappreciable influence on the core secondary stem-loop structure of functional original riboswitch and almost preserves the quondam base-pairing region.

Source

Use mutagenesis PCR of BBa_K598007 to alter RBS sequence. Engineered RBS is selected from constitutive library based on RBS calculator.

References

Beatrix Suess, Barbara Fink, Christian Berens, Régis Stentz and Wolfgang Hillen. (2004) A theophylline responsive riboswitch based on helix slipping controls gene expression in vivo. Nucleic Acids Research, 32, 1610-1614.