Part:BBa_K3007000
HucR binding site
It is a special DNA sequence which can interact with HucR protein and repress the expression of downstream gene when uric acid is absent. If uric acid presents, the formation of HucR-hucO complex can be reduced and restore downstream gene expression. In our design, we used the HucR-hucO system as a detector for uric acid.
Contribution: QHFZ-China 2019
Group: QHFZ-China
Authors: Cheng Li
Introduction: HucR is a novel uric acid-responsive member of the MarR Family from Deinococcus radiodurans [1]. When uric acid is absent, HucR can bind as a homodimer to a single operator site on DNA (hucO) with very high affinity (Kd = 0.29 ± 0.02 nM). We located this part in a modified lac promoter, which contains a HucR binding site (hucO) and a mutate lacO sequence (BBa_K3007001). Therefore, this promoter could be under the control of HucR, instead of LacI. If uric acid presents in high concentration, HucR will release from hucO and the expression of downstream gene will recover from the inhibition. In our design, HucR-hucO system works as a uric acid concentration detector.
DesignDocumentation:
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Figure 1. Schematic cartoon of the DNA construct of BBa_K3007000
We constructed a recombined plasmid composed of pR(BBa_R0051)+RBS(BBa_B0030)+sfGFP(BBa_I746916)+6xHis-tag to express the sfGFP. By the way, there was not repressor CI of pR in the expression system. After purification by Ni resin, we made it a series of gradient dilutions. Then we measured the sfGFP protein concentration by Bradford method and the fluorescence intensity.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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