Difference between revisions of "Part:BBa K3040009"
 
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<partinfo>BBa_K3040009 short</partinfo>
 
<partinfo>BBa_K3040009 short</partinfo>
  
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rrnD promoter is one of the bacteria rRNA promoter. Our team intended to replace the leaked fadBA promoter with this endogenous promoter in E. coli to get a lower baseline expression. Moreover, upstream promoter elements is reported to interact with &#945;-subunit of carboxy-terminal domain of RNA polymerase core enzyme to enhance the transcriptional activity of native promoter. Thus, we modified the rrnD promoter with TP24, an UP element from library built in the literature which is able to get a higher expression level than commonly used promoter OXB 15 to 9-fold.  
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rrnD promoter is one of the bacteria rRNA promoters. Our team intended to replace the leaked fadBA promoter with this endogenous promoter in E. coli to get a lower baseline expression. Moreover, upstream promoter elements are reported to interact with &#945;-subunit of the carboxy-terminal domain of RNA polymerase core enzyme to enhance the transcriptional activity of native promoter. Thus, we modified the rrnD promoter with TP24, a UP element from the library built in the literature which is able to get a higher expression level than commonly used promoter OXB 15 to 9-fold.  
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In order to regulate the expression of this promoter with fatty-acid, we inserted the fadR binding site between the Pribnow box to see whether the site has the greater impact toward the activity of promoter.
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In order to regulate the expression of this promoter with fatty-acid, we inserted the fadR binding site between the Pribnow box to see whether the site has a greater impact on the activity of the promoter.
  
 
==Result==
 
==Result==
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The result turned out to be frustrated that none of them function. We had proposed some reasons that might lead to the failure. UP element might have caused some conformational change of the promoter, there might be too much changes at a same time, or the fadR binding site might not function well with the rrnD promoter. Yet, we can’t figure out what has really happened inside the cell. (Fad BS1-BBa_K3040008, Fad BS2-BBa_K3040009, Fad BS3-BBa_K3040010)
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The result turned out to be frustrated that none of them function. We had proposed some reasons that might lead to failure. UP element might have caused some conformational change of the promoter, there might be too many changes at the same time, or the fadR binding site might not function well with the rrnD promoter. Yet, we can’t figure out what has really happened inside the cell. (Fad BS1-BBa_K3040008, Fad BS2-BBa_K3040009, Fad BS3-BBa_K3040010)
 
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                 <p style="color:gray; font-size:13px; text-align:center;">Figure 1.Protein expression of fatty acid promoter TP24-rrnD-fadRBS1, TP24-rrnD-fadRBS2, TP24-rrnD-fadRBS3 after 16 hours of induction under 5mM fatty acid (n=3)</p>
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                 <p style="color:gray; font-size:13px; text-align:center; padding-left:13%; padding-right:13%;">Figure 1.Protein expression of fatty acid promoter TP24-rrnD-fadRBS1, TP24-rrnD-fadRBS2, TP24-rrnD-fadRBS3 after 16 hours of induction under 5mM fatty acid (n=3)</p>
 
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Latest revision as of 16:25, 21 October 2019


rrnD promoter with TP24 UP element and a fadR binding site inserted between spacer region regulating

rrnD promoter is one of the bacteria rRNA promoters. Our team intended to replace the leaked fadBA promoter with this endogenous promoter in E. coli to get a lower baseline expression. Moreover, upstream promoter elements are reported to interact with α-subunit of the carboxy-terminal domain of RNA polymerase core enzyme to enhance the transcriptional activity of native promoter. Thus, we modified the rrnD promoter with TP24, a UP element from the library built in the literature which is able to get a higher expression level than commonly used promoter OXB 15 to 9-fold. In order to regulate the expression of this promoter with fatty-acid, we inserted the fadR binding site between the Pribnow box to see whether the site has a greater impact on the activity of the promoter.

Result

The result turned out to be frustrated that none of them function. We had proposed some reasons that might lead to failure. UP element might have caused some conformational change of the promoter, there might be too many changes at the same time, or the fadR binding site might not function well with the rrnD promoter. Yet, we can’t figure out what has really happened inside the cell. (Fad BS1-BBa_K3040008, Fad BS2-BBa_K3040009, Fad BS3-BBa_K3040010)

Figure 1.Protein expression of fatty acid promoter TP24-rrnD-fadRBS1, TP24-rrnD-fadRBS2, TP24-rrnD-fadRBS3 after 16 hours of induction under 5mM fatty acid (n=3)

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Unknown
  • 23
    INCOMPATIBLE WITH RFC[23]
    Unknown
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 705
    Illegal AgeI site found at 817
  • 1000
    COMPATIBLE WITH RFC[1000]