Difference between revisions of "Part:BBa J119375"
 
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<partinfo>BBa_J119375 short</partinfo>
 
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Mutations were introduced into the Ptac promoter using NNNNNN in place of the TTGACA of the -35 region. The strength of each of 83 mutant promoters was measured by cloning them into pClone Red (J119137) and measuring RFP expression with fluorometry.
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Mutations were introduced into the Ptac promoter using NNNNNN in place of the TTGACA of the -35 region. Synthetic oligonucleotides carry the 4096 possible sequences were cloned via Golden Gate Assembly into [https://parts.igem.org/Part:BBa_J119137 pClone Red]. A total of 81 different promoters were picked from clone library plates like the one shown below.  The DNA sequence of the 81 mutant promoters was determined and their strength was measured by fluorometry.
  
 
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[[File:81 Ptac promoters.png|950px|]]
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[[File:Ptac_-35_NNNNNN_plate.png|350px|]]
 
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Below is the DNA sequence of the 81 mutant promoters.  The strength of the promoters was measured using the RFP reporter gene in pClone Red. In the table below, strength is reported in the column labeled RFP as the ratio of the red fluorescence of each clone to that of Ptac.
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[[File:Table_of_81_clones_4-28-15.png|950px|]]
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Consensus sequences from data sets such as the one shown are usually determined by analyzing the top 5-20% of the promoters. In order to use the information collected for all of the 81 promoter sequences, a weighted method of consensus building was developed that assigned a score to each base in each -35 region based on the RFP expression of the mutant promoter that contained it. The consensus produced is shown below.
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[[File:Ptac_-35_Consensus.png|500px|]]
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Latest revision as of 13:22, 28 April 2015

81 Mutated Ptac Promoters with Varying Strengths

Mutations were introduced into the Ptac promoter using NNNNNN in place of the TTGACA of the -35 region. Synthetic oligonucleotides carry the 4096 possible sequences were cloned via Golden Gate Assembly into pClone Red. A total of 81 different promoters were picked from clone library plates like the one shown below. The DNA sequence of the 81 mutant promoters was determined and their strength was measured by fluorometry.

Ptac -35 NNNNNN plate.png

Below is the DNA sequence of the 81 mutant promoters. The strength of the promoters was measured using the RFP reporter gene in pClone Red. In the table below, strength is reported in the column labeled RFP as the ratio of the red fluorescence of each clone to that of Ptac.

Table of 81 clones 4-28-15.png

Consensus sequences from data sets such as the one shown are usually determined by analyzing the top 5-20% of the promoters. In order to use the information collected for all of the 81 promoter sequences, a weighted method of consensus building was developed that assigned a score to each base in each -35 region based on the RFP expression of the mutant promoter that contained it. The consensus produced is shown below.

Ptac -35 Consensus.png


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]