Difference between revisions of "Part:BBa K187008:Design"

 
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===Design Notes===
 
===Design Notes===
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The 100% promoter has a sigma 70 consensus sequence, and should produce maximal expression. 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 100% promoter has consensus -35 and -10 sequences. 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 NheI and AvrII sites were also removed by changing two base pairs inside the promoter sequence.
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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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Unlike 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 pAB plasmid adapts the promoter for assembly with other parts using the BioBytes assembly system. See the BioBytes RFC for details. 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 20 minutes.
  
 
===Source===
 
===Source===
  
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100% promoter was modified from Anderson collection of promoters, part BBa_J23119. Plasmid pAB was derived from pUC19, and is entered as part BBa_K187000.
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The 100% promoter was modified from Anderson collection of promoters, part BBa_J23119. Plasmid pAB was derived from pUC19, and is entered as part BBa_K187000.
  
 
===References===
 
===References===

Latest revision as of 21:55, 18 October 2009

Sigma 70 100% 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 100% promoter has consensus -35 and -10 sequences. 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 NheI and AvrII sites were also removed by changing two base pairs inside the promoter sequence.

The pAB plasmid adapts the promoter for assembly with other parts using the BioBytes assembly system. See the BioBytes RFC for details. 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 20 minutes.

Source

The 100% promoter was modified from Anderson collection of promoters, part BBa_J23119. Plasmid pAB was derived from pUC19, and is entered as part BBa_K187000.

References