Part:BBa_K3040008
rrnD promoter with TP24 UP element and a fadR binding site inserted before 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 the 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 before 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 87
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 718
Illegal AgeI site found at 830 - 1000COMPATIBLE WITH RFC[1000]
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