Difference between revisions of "Part:BBa K187005:Design"

 
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===Design Notes===
 
===Design Notes===
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The 25% promoter produces ~25% of the expression level from a consensus promoter. The pAB plasmid adapts the promoter for assembly with other parts using the BioBytes assembly system. See the BioBytes RFC for details on this method.
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The 25% promoter produces ~25% of the expression level from a consensus promoter. Two extra nucleotides were included past the -10 region to make the first A in the Pst restriction site the start site of transcription (This had to be done or a T would have been bp +1). The pAB plasmid adapts the promoter for assembly with other parts using the BioBytes assembly system. See the BioBytes RFC for details on this method.  
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The Biobytes assembly method requires that the DNA fragments to be assembled have long sticky ends of particular sequence. DNA fragments with these sticky ends can be prepared using genes in pBA or pAB as a template. The structure of pAB and pBA will result in a ribosome binding site being included 7bp upstream of the start codon of the gene. It is not necessary to include a promoter with the gene when cloning it into pAB or pBA. Instead, a promoter with long sticky ends can be prepared from separate pAB and pBA plasmids just containing promoters. The desired promoter can then be assembled upstream of a gene using the BioBytes method. This abilty to easily 'mix and match' promoters and genes allows gene expresison to be standardized and optimized.
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Unlikely standard cloning that would requires several days to insert a promoter into a plasmid upstream of a gene, the addition of one DNA segment with the BioBytes method takes only 20minutes.
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The Biobytes assembly method requires that the DNA fragments to be assembled have long sticky ends of a particular sequence. DNA fragments with these sticky ends can be prepared using genes in pBA or pAB as a template. If the DNA cloned into pAB begins with a start codon, the structure of pAB and pBA will result in a ribosome binding site being included 7bp upstream of the start codon. It is not necessary to include a promoter or ribosome binding site with the gene when cloning it into pAB or pBA. Instead, a promoter with long sticky ends can be prepared from separate pAB and pBA plasmids just containing promoters. The desired promoter can then be assembled upstream of a gene using the BioBytes method. This ability to easily 'mix and match' promoters and genes allows gene expression to be standardized and optimized.
  
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The addition of one DNA segment with the BioBytes method takes only 20minutes.
  
 
===Source===
 
===Source===

Latest revision as of 21:57, 18 October 2009

Sigma 70 25% promoter in pAB, BioBytes plasmid


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 10
    Illegal PstI site found at 47
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 47
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 10
    Illegal PstI site found at 47
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 10
    Illegal PstI site found at 47
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The 25% promoter produces ~25% of the expression level from a consensus promoter. Two extra nucleotides were included past the -10 region to make the first A in the Pst restriction site the start site of transcription (This had to be done or a T would have been bp +1). The pAB plasmid adapts the promoter for assembly with other parts using the BioBytes assembly system. See the BioBytes RFC for details on this method.

The Biobytes assembly method requires that the DNA fragments to be assembled have long sticky ends of a particular sequence. DNA fragments with these sticky ends can be prepared using genes in pBA or pAB as a template. If the DNA cloned into pAB begins with a start codon, the structure of pAB and pBA will result in a ribosome binding site being included 7bp upstream of the start codon. It is not necessary to include a promoter or ribosome binding site with the gene when cloning it into pAB or pBA. Instead, a promoter with long sticky ends can be prepared from separate pAB and pBA plasmids just containing promoters. The desired promoter can then be assembled upstream of a gene using the BioBytes method. This ability to easily 'mix and match' promoters and genes allows gene expression to be standardized and optimized.

The addition of one DNA segment with the BioBytes method takes only 20minutes.

Source

The 25% promoter was modified from Anderson collection of promoters, part BBa_J23105. Plasmid pAB was derived from pUC19, and is entered as part BBa_K187000.

References