Difference between revisions of "Part:BBa K4164017"
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The composite part can be directly imported into plasmid and express FXR-ddRFPA1 and RXR-ddRFPB1 induced with IPTG.
 
The composite part can be directly imported into plasmid and express FXR-ddRFPA1 and RXR-ddRFPB1 induced with IPTG.
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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.
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<p style="text-align: center;"><img src="https://static.igem.wiki/teams/4164/wiki/result/figure-3.png"with="1000" height="" width="500" height=""/></p>
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<p style="text-align: center!important;"><b>Fig.1 Effects of CDCA concentration and promoter intensity on report device within the cell.</b></p>
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 12:43, 12 October 2022


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.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1964
    Illegal NheI site found at 2232
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 3846
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 454
    Illegal AgeI site found at 1481
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 836
    Illegal SapI.rc site found at 3435