Part:BBa_K3040110
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 70
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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