Part:BBa_K4164017
Inductive expression of FXR-ddRFPA1-RXR-ddRFPB1
In order to find an appropriate expression intensity to achieve balance between metabolic burden and detection efficiency, we also tried T7lac promoter from pET-29a+(Novagen).
The composite part can be directly imported into plasmid and express FXR-ddRFPA1 and RXR-ddRFPB1 induced with IPTG.
We constructed FXR-ddRFPA1 and RXR-ddRFPB1 expression plasmids with different promoters(BBa_J23105, T7 promoter). And cultured them in LB medium with antibiotics and grew at 37°C to OD600 of about 0.6. Then IPTG was added to the final concentration of 0.5mM and induced protein expression at 28℃ for 3 hours. We tested fluorescence intensity at different concentrations of CDCA (0, 10, 25, 50, 100μM) in E.coli BL21(DE3). Samples were taken after incubation at room temperature for eight hours. Fig. 1 shows the effects of CDCA concentration and promoter intensity on different plasmids within the cell.
Fig.1 Effects of CDCA concentration and promoter intensity on report device within the cell.
It is clear that at a CDCA concentration of 25μM, the T7 lac promoter has the best results.
Therefore, we chose T7 lac promoter to form our final detection device.
Then, we simulated the binding efficiency of FXR and RXR in different CDCA concentrations and determined that the optimum concentration is 25 μM.
Fig.2 The curve of binding efficiency in different CDCA concentrations.
Having selected the appropriate part, we proceeded to evaluate the ability of our cell-free system and the protein to interact.Thus, we were the first to test the cell-free system. We added co-expression plasmid of FXR-ddRFPA1 and RXR-ddRFPB1(BBa_K4164017)to the cell-free system, and then incubated them to a sufficient concentration of proteins. Then we added different concentrations of CDCA(0, 10, 25, 50, 100μM) to test the cell-free system. After 8 hours, we could see a clear difference in fluorescence intensity under the corresponding wavelength of light excitation.
Fig.3 Impact of different concentrations of CDCA on report device fluorescence intensity. blank: cell-free system with no plasmid a. Direct observation of fluorescence. b. Fluorescence under specific wavelength excitation.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1964
Illegal NheI site found at 2232 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 3846
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 454
Illegal AgeI site found at 1481 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 836
Illegal SapI.rc site found at 3435
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