Plasmid

Part:BBa_K3734023

Designed by: Xingjun Zhao   Group: iGEM21_CSU_CHINA   (2021-10-01)

CHREBP-LUC

CHREBP is a promoter that can be induced by glucose. Luciferin(LUC) is a protein that can activate fluorescein.We use it as reporter gene.

CHREBP-LUC

In our experiment, the change of glucose concentration is an important switch of the work loop . CHREBP is only studied as a part of lipid metabolism in current literature. We used it as a glucose-induced promoter and achieved ideal result in cell 293T

LUC is a reported gene in the luminescence system of fluorease. It comes from fireflies and characterizes the amount of gene expression in the target by detecting the luminescence value of its excited fluorescent hormone at a wavelength of 560nm.

1.Pattern diagram

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.

TRE-mCherry-miR21 works well.

Fig.2 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.

Fig.3 The expression level of inducible promoter CHREBP was respectively analyzed at 72h in 25mM, 5.6mm and 0mM glucose culture

3.Caution

Because the INSR sequence is very long, it is difficult for DH5α receptor cells containing recombinant enzymes to meet the requirements during cloning, and receptor cells of recombinant enzymes may need to be removed.

Meanwhile, we hope we can shorten the sequence to improve it although it is good enough. Try to convert REN at the same time to adjust the errors caused by other factors such as different cell numbers and cell states caused by different culture conditions when using LUC.

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.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 895
    Illegal NotI site found at 974
    Illegal NotI site found at 2798
    Illegal NotI site found at 2975
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 12
    Illegal BamHI site found at 2926
    Illegal XhoI site found at 2982
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1065
    Illegal NgoMIV site found at 2409
    Illegal NgoMIV site found at 2430
    Illegal NgoMIV site found at 2691
    Illegal NgoMIV site found at 3420
    Illegal NgoMIV site found at 4761
    Illegal NgoMIV site found at 5044
    Illegal AgeI site found at 2133
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 882
    Illegal BsaI.rc site found at 6569
    Illegal SapI.rc site found at 2315
    Illegal SapI.rc site found at 4610
    Illegal SapI.rc site found at 4820
    Illegal SapI site found at 5489


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