Part:BBa_K4169030
Cre-reverse loxP system:working when you need to express genes in the other direction
In the presence of theophylline, subsequent expression of Cre is induced. In addition, the promoter and RBS between the two reverse loxP's will be flipped so that it expresses the gene in the other direction.
Usage and Biology
When our engineered bacteria sense theophylline, it expresses Cre, which recognizes the reverse repeat sequence at both ends of the loxP site and binds to form a dimer, which then binds to a dimer at another loxP site to form a tetramer. In this way, Cre mediates sequence flips between reverse loxPs.
Characterization
SDS-PAGE
First, we identified Cre by SDS-PAGE. We identified that Cre can be expressed after we add theophylline induction.
Through SDS-PAGE analysis, We found that DH5αwith the Cre plasmid transformed had two bands between 35 kDa and 40 kDa, but only one band in DH5α without the transformed plasmid. Cre's molecular mass size is 38 kDa，We identified that this part can produce Cre.
Functional Parameters
To verify the functionality of our part, we had the upstream gene of the promoter replaced with red fluorescence，and then transferred our plasmid into Escherichia coli DH5α and measured the intensity of red fluorescence with a microplate reader. Unfortunately, our bacteria did not produce red fluorescence, and upon analysis, it was conjectured that the transcriptional loxP formed a strong secondary structure and could not translate the red fluorescence protein behind it.
Sequence and Features
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NheI site found at 1292
Illegal NheI site found at 1315 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 454
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 39
Illegal AgeI site found at 140 - 1000COMPATIBLE WITH RFC[1000]
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