Measurement

Part:BBa_K337032

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)
Revision as of 03:28, 27 October 2010 by Rebecca (Talk | contribs)

pSMB_miTuner Plasmid HD1 (BGH(rc)/shRNA10(rc)/sv40(rc)/RSV/Luc2_sv40/CMV/Kozag_hRluc_BGH )

miTuner plasmid for microRNA binding site measurment and expression tuning.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1
    Illegal BamHI site found at 3421
    Illegal XhoI site found at 3163
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1547
    Illegal NgoMIV site found at 2891
    Illegal NgoMIV site found at 2912
    Illegal NgoMIV site found at 3184
    Illegal AgeI site found at 2615
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 4600
    Illegal SapI.rc site found at 2797


Characterization

In order to test for promoter efficiency and to check whether the miRNA kit assembly works fine 50ng of each construct with different promoter set-ups (table 1) were transfected into HEK 293 T-REx cells and other cell lines HEK, HeLa, Huh7 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 allows is unaffected by transfection efficiency and cell number. 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. 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_K337036 CMV CMV RSV
BBa_K337038 CMV TetO2 CMV RSV
BBa_K337040 RSV RSV SV40
BBa_K337042 CMV RSV SV40
BBa_K337044 CMV RSV RSV
BBa_K337046 CMV TetO2 RSV RSV
Promoter strength characterization of tuning constructs in HEK 293 T-REx cell line
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Categories
Parameters
None