Difference between revisions of "Part:BBa K3040110"

 
 
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<partinfo>BBa_K3040110 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 α-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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In order to regulate the expression of this promoter with fatty-acid, we inserted the fadR binding site after the Pribnow box to see whether the site has the greater impact toward the activity of promoter.
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==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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    <style>
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        .fig_title{
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            color:gray;
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            text-align:center;
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            margin-bottom:20px;
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            <img src="https://static.igem.org/mediawiki/parts/b/b9/T--NTHU_Taiwan--BBaK3040008_9_10.png" style="margin:20px auto 5px auto;" width=50%>
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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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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 13:26, 20 October 2019


TP24-rrnD-fadR binding site-3

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 α-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. In order to regulate the expression of this promoter with fatty-acid, we inserted the fadR binding site after the Pribnow box to see whether the site has the greater impact toward the activity of promoter.

Result

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)

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]
    Illegal BamHI site found at 70
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]