Designed by: Adlung L,Al Sabah J,Bayer P,Berrens R,Cristiano E,Flocke L,Kleinsorg S,Kolodziejczyk A,Macias Torre A,Mathur A,Mayilo D,Neumann S,Niopek D,Pisa R,Schad J,Schuhmacher L,Uhlig T,Wu X,Grimm D,Eils R   Group: iGEM10_Heidelberg   (2010-10-22)

pSMB_miTuner Plasmid HD3 (BGH(rc)/shRNA10(rc)/RSV(rc)/CMV/Luc2_sv40/CMV/Kozag_hRluc_BGH )

miTuner plasmid- scheme

The dual-luciferase miTuner construct is the basic measurement and expression construct (fig. 2). It contains three expression cassettes: the synthetic microRNA expression cassette in reverse complementary orientation for tuning of gene expression (RSV driven), as well as a measurement firefly luciferase (CMV driven) and a reference Renilla luciferease (CMV driven) in forward direction. HindIII and AflII sites allow for the exchange of the synthetic microRNA, XmaI and XhoI sites allow for introducing synthetic microRNA binding sites into the 3’UTR of the firefly luciferase gene. BamHI sites enable an easy cloning of the microRNA_cDNA (Luc2) fragment into a pTRUF3 single stranded AAV context for production of AAVs and efficient infection of target cells with the miTuner construct (BB-2, RFC12).
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Sequence and Features

Assembly Compatibility:
  • 10
  • 12
  • 21
    Illegal BamHI site found at 1
    Illegal BamHI site found at 3613
    Illegal XhoI site found at 3355
  • 23
  • 25
    Illegal NgoMIV site found at 1739
    Illegal NgoMIV site found at 3083
    Illegal NgoMIV site found at 3104
    Illegal NgoMIV site found at 3376
    Illegal AgeI site found at 2807
  • 1000
    Illegal BsaI.rc site found at 4792
    Illegal SapI.rc site found at 2989

Promoter efficiency of miRNA kit was checked in HEK T-Rex cells. Therefor 50ng of each construct with different promoter set-ups (table 1) were transfected into HEK 293 T-REx cells in 96-well plate format using FuGENE transfection reagent. As every construct is expressing firefly luciferase (luc2) and renilla luciferase (hRluc) at the same time the setup is unaffected by transfection efficiency and cell number variations. Each sample was transfected and measured by Dual luciferase assay in 8 replicates. As by this time no shRNA has been cloned into plasmid NO knock-down of luc2 is expected and the different expression efficiencies allow for characterization of the different promoters combinations. BBa_K337032 leads to a relative luciferase unit (RLU) of luc2 to hRluc expression of 6 RLU. BBa_K337035 and BBa_K337035 are showing a comparable expression of 12 - 13 RLU, which is in line with the knowledge that both luciferases are driven by the CMV promoter. Hek 293 T-Rex cells stably express the Tet repressor thus allows us to observe very efficient repression of Firefly luciferse expression if a CMV-TetO2 promoter is driving luc2 (BBa_K337038 and BBa_K337046). BBa_K337040 transfection into Hek T-Rex cells results in an expression of 15 RLU. BBa_K337042 and BBa_K337044 are constructed in a way that luc2 is driven by the CMV promoter and hRluc is driven by the RSV promoter and show a comparable expression of 17-20 RLU. This leads to the conclusion that the CMV promoter shows comparable expression to the RSV promoter in Hek T-Rex cell lines.

Table 1

part promoter driving luc2 (Firefly) promoter driving (Renilla) promoter driving shRNA expression
BBa_K337032 RSV CMV SV40
BBa_K337035 CMV CMV SV40
BBa_K337038 CMV TetO2 CMV RSV
BBa_K337040 RSV RSV SV40
BBa_K337042 CMV RSV SV40
BBa_K337046 CMV TetO2 RSV RSV

Promoter strength characterization of tuning constructs in HEK 293 T-REx cell line

Characterization of BBa_K337035 and BBa_K337036 in different cell lines

Promoter strength characterization in different cell lines

If transfecting BBa_K337035 and BBa_K337036 are transfected into different cell lines it is obvious that Hek293T cells are the easiest to transfect with both constructs an expression of 17-22 RLU is to be measured. Hek T-Rex cells are showing and expression level of 12 RLU of both constructs. Hela cells are also showing constant expression levels of 8 RLU with both constructs. A rather low expression of 2RLU is to be seen by transfecting the 2 constructs in Huh7 cells. This might be due to low transfection efficiency of this cell line in general. All together it is to say that BBa_K337035 and BBa_K337036 show comparable expression.