DNA

Part:BBa_K3734001

Designed by: Xingjun Zhao   Group: iGEM21_CSU_CHINA   (2021-10-01)
Revision as of 13:11, 19 October 2021 by Shinichi (Talk | contribs)


CHREBP promoter

CHREBP promoter is a glucose-induced promoter, and its expression increases with the increase of glucose concentration.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 584
    Illegal BamHI site found at 346
    Illegal XhoI site found at 449
    Illegal XhoI site found at 2640
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 3317
    Illegal BsaI site found at 3385
    Illegal BsaI site found at 3632
    Illegal BsaI.rc site found at 452
    Illegal BsaI.rc site found at 475
    Illegal BsaI.rc site found at 2160
    Illegal BsaI.rc site found at 2270
    Illegal SapI.rc site found at 3150


                   CHREBP promoter
               </h2>

1.Pattern diagram


                   <img class="image" src="T--CSU_CHINA--tupian1.png">

Fig.1 The model diagram of CHREBP-LUC

2.Experiment

2.1 Method

We used LUC as report genes to reflect the level of expression through detecting luminescence value at 560nm wavelength and the error REN luminescence caused by the number of eliminated cells.

2.2 Result

CHERBP promoter is induced by glucose and the expression rises along with the raise of glucose concentration. 

                   <img class="image" src="T--CSU_CHINA--tupian2.png">

Fig.2 Electrophoretic diagram of CHREBP PCR product

                   <img class="image" src="T--CSU_CHINA--tupian3.png">

Fig.3 The expression level of inducible promoter CHREBP was respectively analyzed at 48h in 25mM, 5.6mm and 0mM glucose culture

To eliminate the effect of residual glucose in the medium during transmission, we have conducted experiments in a 72-hour group, the result fits our anticipations more. 

                   <img class="image" src="T--CSU_CHINA--tupian4.png">

Fig.4 The expression level of inducible promoter CHREBP was respectively analyzed at 72h in 25mM, 5.6mm and 0mM glucose culture

3.Caution

Due to the relatively long sequence of CHREBP promoter, DH5α receptor cells containing recombinant enzymes are difficult to meet the 
requirements during cloning, and receptor cells with recombinant enzymes may 
need to be removed.

Meanwhile, we hope that we can shorten the length of the sequence to improve it although it can work quite well.

Reference：

[1]Jian Meng, Ming Feng, Weibing Dong.Identification of HNF-4α as a key transcription factor to promote ChREBP expression in response to glucose[J].Sci Rep. 2016 Mar 31;6:23944.

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